The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19), which was rapidly declared a pandemic by the World Health Organization (WHO). Early clinical symptomatology focused mainly on respiratory illnesses. However, a variety of neurological manifestations in both adults and newborns are now well-documented. To experimentally determine whether SARS-CoV-2 could replicate in and affect human brain cells, we infected iPSC-derived human brain organoids. Here, we show that SARS-CoV-2 can productively replicate and promote death of neural cells, including cortical neurons. This phenotype was accompanied by loss of excitatory synapses in neurons. Notably, we found that the U.S. Food and Drug Administration (FDA)-approved antiviral Sofosbuvir was able to inhibit SARS-CoV-2 replication and rescued these neuronal alterations in infected brain organoids. Given the urgent need for readily available antivirals, these results provide a cellular basis supporting repurposed antivirals as a strategic treatment to alleviate neurocytological defects that may underlie COVID-19- related neurological symptoms. This study shows that SARS-CoV-2 virus can infect human brain cortical organoids, inducing cell death and synapse loss; application of Sofosbuvir, an FDA-approved antiviral, rescues these neuronal alterations in infected brain organoids. Coronavirus disease 2019 (COVID-19), caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), was declared a pandemic by the World Health Organization (WHO) on 11th March 2020. The outbreak quickly spread to 216 countries in a couple of months, with more than 626 million cases and more than 6.5 million confirmed deaths worldwide, as of this writing. Despite vaccines currently being distributed, new variants emerge rapidly, and thousands of new infections are still reported every day. Thus, fast-deployable and efficient antiviral therapies are urgently needed.postmortem tissues of COVID-19 patients found evidence that ciliated cells in the respiratory mucosa and that sustentacular cells  in the olfactory mucosa are the main target cell types for SARS-CoV-2; routes through which olfactory sensory neurons could become affected . Primary antibodies were diluted with blocking solution and incubated with cells overnight at 4°C: Cleaved caspase-3 , Anti-Nestin , Anti-MAP2 antibody , Anti-vGLUT1 [317D4] , Anti-Synapsin1, ; Anti-PSD-95, ; anti-CUX1 , anti-SATB2 , anti-TBR2 , anti-Vimentin , anti-Aquaporin-4 , Anti-GFAP , anti-SOX2, , influenza A virus nucleoprotein (NP) HT-103 mouse monoclonal antibody . SARS-CoV-2 (2019-nCoV) Nucleoprotein (N) Antibody  was incubated diluted with blocking solution and incubated with cells for 20 minutes at room temperature. Cells were then washed twice with 1xPBS and incubated with the secondary antibody for 30 minutes at room temperature. Secondary antibodies  were purchased from Life Technologies and used at a 1:1000 dilution. After the 30 minutes incubation, samples were washed twice (1xPBS), incubated for 5 minutes with fluorescent nuclear DAPI stain , and mounted with Slow fade gold antifade reagent (Life Technologies). Samples were imaged using an Axio Observer Z1 Microscope with ApoTome (Zeiss). For TUNEL analysis, samples were fixed with 4% paraformaldehyde and permeabilized with 0.25% Triton X-100 for 15 minutes and then stained for TUNEL following manufacturer’s instructions (Click-iT TUNEL assay kit from Life Technologies). Cells were blocked with 3% BSA for 1 hour and then incubated in primary and secondaries, as shown above. Images were blindly collected using an Axio Observer Z1 Microscope with ApoTome (Zeiss) and analyzed with ImageJ software.We quantified the number of fluorescent cells corresponding to each staining was measured automatically with Image J software by splitting the different channels. The data is represented as percentage of positive cells for a specific staining overall the total number of the cells (DAPI) in each region of interest (ROI) or as the number of cells per ROI. Two different batches of BCOs from two different iPSC lines were used, and 5 organoids were analyzed per condition. Quantifications was done manually with 6 ROIs per sample. The quantifications were performed blindly. To generate the videos, 10–12 Z-stack images were acquired per condition with Axio Observer Z1 Microscope with ApoTome (Zeiss) and 3D reconstructions were generated using Zeiss Zen Microscope Software and exported as.MOV.Total RNA was purified from infected cells using the NucleoSpin 96 RNA Kit (Takara) in accordance with the manufacturer’s instructions. The viral mRNA was quantified using TaqPath one-step RT-qPCR Master Mix (Thermofisher) using ActinB CTRL Mix (Thermofisher) as housekeeping gene, and the following primers and probe for qPCR measurements of viral genes, targeting SARS-CoV-2 Nucleoprotein:N-Fwd: 5’-TTACAAACATTGGCCGCAAA-3’; N-Rev: 5’-GCGCGACATTCCGAAGAA-3’; N-Probe: 5’-FAM-ACAATTTGCCCCCAGCGCTTCAG-BHQ-3’. ΔΔCT was used to calculate the fold changes relative to the controls.RNA from BCO infected at MOI 2.5 and treated with vehicle (Veh) or 20μM Sofosbuvir (SOF) was extracted 7 days post-infection. In brief, three samples per condition, each consisting of two pooled BCOs, were processed following RNeasy Mini Kit (Qiagen #74104) instructions to extract total RNA. RNA was assessed for quality using an Agilent Tapestation 4200, and samples with an RNA Integrity Number (RIN) greater than 8.0 were used to generate RNA sequencing libraries using the TruSeq Stranded mRNA Sample Prep Kit with TruSeq Unique Dual Indexes . Samples were processed following manufacturer’s instructions, modifying RNA shear time to five minutes. Resulting libraries were multiplexed and sequenced with 100 basepair (bp) paired end reads (PE100) to a depth of approximately 25 million reads per sample on an Illumina NovaSeq 6000. Samples were demultiplexed using bcl2fastq v2.20 Conversion Software .https://rosalind.onramp.bio/), with a HyperScale architecture developed by OnRamp BioInformatics, Inc. . Reads were trimmed using cutadapt [Data was analyzed by ROSALIND  [The functional network analyses were generated through the use of Ingenuity Pathway Analysis (IPA)   and Metanalysis) . Terms wSupernatants from SARS-CoV-2 infected brain organoids were collected at 16 h post-infection and stored at -80°C until used. 600,000 Vero E6 cells were seeded overnight at 37°C / 5% CO2 in 12-well plates. Confluent Vero E6 cells were then washed once with 1xPBS and infected with 10-fold serial dilutions of the collected supernatants. Cells were incubated with the virus for 1 h at room temperature, followed by inoculum removal and addition of 1ml overlay media  at a 1:1 ratio). 2xMEM contains 100 ml 10x MEM (Gibco), 10 ml 100x penicillin-streptomycin (Fisher Scientific), 10 ml 100x L-Glutamine, 6 ml 35% BSA, 10 ml 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 24 mL 5% NaHCO3 (Gibco) and 340 ml water. Plates were incubated 3 days at 37°C, 5%CO2, and then fixed and stained using 0.1% Crystal Violet and 5% PFA (Boston BioProducts) overnight at 4°C. Plaques were quantified and recorded as PFU/ml.BCO were treated with increasing doses of SOF or DMSO (vehicle control). Drugs were reapplied every second day for a total duration of 7 days. Media was then removed and replaced with Cell Titer Glo Luminescent Cell Viability Assay (Promega). Cells were incubated for 30 min at room temperature prior measurement of the relative light units (RLU). Data was normalized to the Vehicle condition.Results were analyzed using Prism Software . Each sample is shown on each graph as an individual dot. Statistical significance was determined using one- or two-way ANOVA tests followed by Tukey or Sidak multiple comparisons tests to compare different groups with one or two variables respectively using a p< 0.05. The reported values are means ± SEM, as mentioned in relevant figure captions. Individual data points are shown on graphs when the samples size was lower than n< 10. Sample sizes, n, reported in figure legends. The statistical significance of the structural similarities between different viral polymerases were detected by FATCAT. FATCAT is evaluated by a P-value that measures the chance of getting the same similarity in two random structures. This P-value is calculated based on the empirical fitting of the extreme value distribution (EVD) to the FATCAT similarity score. The smaller the P-value, the more statistically significant the similarity between corresponding structures.S1 Figa. GTEx Transcriptome expression profile  for the genes ACE2, DDP4, BSG, TMPRSS2 and NRP1 in different human tissues, including various brain regions. b. Various brain regions are color coded and correspond to quantification of each region in c. c. Expression of the genes ACE2, DDP4, BSG, TMPRSS2 and NRP1 in different brain regions and compartments from datasets of different repositories45–47. d. Gene expression levels of SARS-CoV-2 receptors in human BCO (in TPM). e. Receptor and cell surface marker gene expression in BCO (in TPM). Data was generated from bulk RNA sequencing data obtained from BCO. All data points are represented as individual points (n = 12 BCO). The raw data for the panels on this figure is located in (TIFF)Click here for additional data file.S2 Figa. BCO immunostaining shows progenitor cells (SOX2+) neurons (MAP2+) and astrocyte (GFAP+ and AQP4+) expression. Scale bar upper panel 50 μm, lower panel 20 μm. Two different batches of BCOs from two different iPSC lines (WT83 and CVB) were used, and 5 organoids were analyzed per condition. b. Percentage of cell fractions showing NPC, neuron, and astrocyte populations within BCO. c. Upon BCO infection with SARS-CoV-2, we noted 477 differentially expressed genes at 1.25-fold change (p<0.05). Upregulated and downregulated genes (red and blue respectively) are represented . Each sample represents three pooled organoids. d. Top 20 significant canonical pathways of the core analysis in IPA (Ingenuity) of most highly expressed genes in Mock vs SARS-CoV-2. p-values indicate the significance of enrichment for the most highly expressed genes from our dataset. Z-score > 0 in red, Z-score < 0 in blue, Z-score = 0 in white, Z-score unavailable in grey. e. The 477 differentially expressed genes at 1.25-fold change (p<0.05) were subjected to functional enrichment analyses using Metascape. Top 20 enriched pathways with their respective p-values are shown. f. Schematic of the experimental design; organoids BCO were first infected with SARS-CoV-2 and treated with Sofosbuvir (SOF) post-infection and analyzed for viral infection, synapse number and cell death 7 days after infection g. Quantification of SARS-CoV-2 intracellular mRNA by qPCR of BCO infected at MOI 2.5 and treated with vehicle (Veh) or increasing concentrations of SOF . RNA was collected 7 days post-infection. Bars represent mean. Error bars represent SEM p**<0.01, ***p<0.001, n = 4 biological replicates . Significance was assessed using one-way ANOVA and Dunnett’s post-hoc test. h. Viability of BCO treated with vehicle (Veh) or increasing concentrations of SOF . Viability was measured 7 days post-treatment. Relative light units (RLU) were normalized to VEH. Bars represent mean. Error bars represent SEM, n = 3 biological replicates (four pooled organoids per replicate). Significance was assessed using one-way ANOVA with Dunnett’s post-hoc test, no statistical difference was found. i. Western-blot of SARS-CoV-2 Nucleocapsid (N) infected BCO at MOI 2.5 treated with vehicle (Veh) or 20μM Sofosbuvir (SOF) 7 days post-infection. Beta actin was used as a loading control. n = 12 biological replicates (pooled into two protein lysates). The BCOs were fixed and analyzed 7 days post-infection. The raw data for the panels on this figure is located in (TIFF)Click here for additional data file.S3 Figa-d. Immunolabeling of mock, infected, and infected, and SOF-treated organoid sections stained for TUNEL (white), SARS-CoV-2 N protein (green), Nestin (red), SOX2 (green), MAP2 (red), GFAP (red), respectively by confocal microscopy. Arrows point to colocalization with different cell types. Scale bar, 20 μm. These are representative images from the WT83 iPSC line.(TIFF)Click here for additional data file.S4 FigImmunolabeling of mock and infected organoid sections stained for TUNEL (white) and excitatory upper , intermediate progenitors (TBR2), and lower cortical neuron markets (CTIP2). Images below each inset show split channels. Scale bar, 20 μm n = 5 biological replicates per condition. The BCOs were fixed and analyzed 7 days post-infection. These are representative images from the CVB iPSC line.(TIFF)Click here for additional data file.S5 Figa. Immunolabeling SARS-CoV-2 infected organoids with MAP2 (red), SARS-CoV-2 (green) and TUNEL (white). b. Immunolabeling mock and SARS-CoV-2 infected organoids with MAP2 (red), SYN1 (green) and TUNEL (white). c. Immunolabeling mock and SARS-CoV-2 infected organoids with astrocytic markers Aquaporin-4 , GFAP (in white and green) and Vimentin (VIM in white). Images below each inset show split channels. Scale bar, 20 μm n = 5 biological replicates per condition. d. The integrated density was measured for each marker and normalized to mock-infected conditions and the total number of cells. Bars represent mean. Error bars represent SEM, n = 3 biological replicates. Significance was assessed using Students’ t-test, n.s. not significant. Two different batches of BCOs from two different iPSC lines (WT83 and CVB) were used, and 3 ROI from each of the 5 organoids were analyzed per condition. The BCOs were fixed and analyzed 7 days post-infection. The raw data for the panels on this figure is located in (TIFF)Click here for additional data file.S6 Figa. The integrated density for SYN1 and PSD-95 was measured in mock-infected vehicle or SOF-treated organoids and normalized to mock-infected vehicle treated conditions and the total number of cells. Bars represent mean. Error bars represent SEM, n = 3 biological replicates. Significance was assessed using Students’ t-test, n.s. not significant. 3 ROIs per organoid, and 5 organoids per condition were assessed. b. Immunolabeling of vGLUT1-positive cells (yellow) within MAP2+ neurons (red) in BCO infected at MOI 2.5 and treated with vehicle (Veh) or 20μM Sofosbuvir (SOF). Scale bar, 20 μm. c. Immunolabeling of SYN1-positive cells (green) and PSD95 (red) within MAP2+ neurons (white) in BCO infected at MOI 2.5 and treated with vehicle (Veh) or 20μM Sofosbuvir (SOF). Scale bar, 20 μm. d. Immunolabeling of TUNEL (white), MAP2 (red), Influenza A virus nucleoprotein  in BCO infected at MOI 2.5 and treated with Vehicle (Veh) or Sofosbuvir (SOF). Scale bar, 100 μm. Two different batches of BCOs from two different iPSC lines (WT83 and CVB) were used, and 5 organoids were analyzed per condition. The BCOs were fixed and analyzed 7 days post-infection. The panels a, b, c, and d are lower magnification images that are part of the figures shown and quantified in Fig 3E. Structural superposition of SARS-CoV-2 RdRp (colored blue) and Influenza A RdRp (colored yellow) shows minimal structural overlap within the polymerase active site. Both structures are not statistically similar (p = 5.20e-02), calculated from raw FATCAT score73. A total of 453 equivalent positions with an RMSD of 6.81Å and 5 twists were found between these two structures. f-g. Pairwise alignment of SARS-CoV-2 RdRP (nsp12) and HCV (NS5B) (d) and SARS-CoV-2 RdRP and Influenza A PB2 (e). Residues that partake in SOF binding or catalytic activity are highlighted in red to signify a match between proteins, orange to signify a partial match, and yellow to signify a mismatch. “*” = Residue identity is conserved; “.” = Residues have similar properties. The raw data for the panels on this figure is located on (TIFF)Click here for additional data file.S1 VideoImmunolabeling of mock-infected organoid sections stained for TUNEL (white), SARS-CoV-2 N protein (green) and MAP2 (red) by confocal microscopy. Scale bar, 20 μm. The BCOs were fixed and analyzed 7 days post-infection.(MOV)Click here for additional data file.S2 VideoImmunolabeling of SARS-CoV-2 infected vehicle-treated organoid sections stained for TUNEL (white), SARS-CoV-2 N protein (green), and MAP2 (red) by confocal microscopy. Scale bar, 20 μm. The BCOs were fixed and analyzed 7 days post-infection.(MOV)Click here for additional data file.S3 VideoImmunolabeling of SARS-CoV-2 infected SOF-treated organoid sections stained for TUNEL (white), SARS-CoV-2 N protein (green), and MAP2 (red) by confocal microscopy. Scale bar, 20 μm. The BCOs were fixed and analyzed 7 days post-infection.(MOV)Click here for additional data file.S1 TableA total of 477 genes are differentially expressed between mock and SARS-CoV-2 infected BCO . The BCOs were analyzed 7 days post-infection.(XLSX)Click here for additional data file.S2 TableThe BCOs were analyzed 7 days post-infection.(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S1 Raw Images(PDF)Click here for additional data file.


Human astroviruses (HAstV), positive sense single-stranded RNA viruses, are one of the leading causes of diarrhea worldwide. Despite their high prevalence, the cellular mechanisms of astrovirus pathogenesis remain ill-defined. Previous studies showed HAstV increased epithelial barrier permeability by causing a re-localization of the tight junction protein, occludin. In these studies, we demonstrate that HAstV replication induces epithelial-mesenchymal transition (EMT), by upregulating the transcription of EMT-related genes within 8 hours post-infection (hpi), followed by the loss of cell-cell contacts and disruption of polarity by 24 hpi. While multiple classical HAstV serotypes, including clinical isolates, induce EMT, the non-classical genotype HAstV-VA1 and two strains of reovirus are incapable of inducing EMT. Unlike the re-localization of tight junction proteins, HAstV-induced EMT requires productive replication and is dependent transforming growth factor-β (TGF-β) activity. Finally, inhibiting TGF-β signaling and EMT reduces viral replication, highlighting its importance in the viral life cycle. This finding puts classical strains of HAstV-1 in an exclusive group of non-oncogenic viruses triggering EMT. While human astroviruses (HAstV) were discovered nearly 45 years ago, these small positive-sense RNA viruses remain critically understudied, leaving us with limited knowledge of its pathogenesis. This study fills the gap in knowledge demonstrating that HAstV are among a select group of viruses that induce epithelial-mesenchymal transition (EMT). Here we demonstrate that HAstV-1 replication, along with TGF-β signaling, induces transcriptional and phenotypic changes in intestinal epithelial cells associated with EMT. Additionally, we show inducing EMT is not common to enteric RNA viruses, as reovirus strains were found to be incapable of inducing EMT. Unlike the viruses that are known to induce EMT, astrovirus infection is not associated with cancer. We hypothesize that our findings will extend beyond astrovirus and may shed light on novel ways viruses can circumvent the barriers meant to protect against them. Epithelial and mesenchymal cells share inherent plasticity that allows for switching between the two cell states through a biological process known as epithelial-mesenchymal transition (EMT). EMT is essential in the development, differentiation, and repair of tissues and organs; however, EMT can negatively contribute to organ fibrosis and the initiation of cancerous metastases. EMT has been shown to induce stem cell properties ,2, prevein vivo and in vitro [Epithelial cells that line the intestinal lumen function as a barrier that absorbs nutrients and electrolytes while restricting entry of harmful substances or pathogens . Breachein vitro ,16. BecaIndeed, several viruses are known to induce EMT including hepatitis B virus (HBV) , hepatit2 Profiler system (2 Profiler findings by quantitating mRNA levels of EMT-related transcription factors as well as E-cadherin (CDH1), occludin (OCLN), Snail (SNAI1), and vimentin (VIM) by qRT-PCR. We observed a modest upregulation of the EMT-related transcription factors SNAI1, TWIST1, ZEB1, and ZEB2 around 4 hpi , and adherens junction proteins, E-cadherin and β-catenin, at 6, 12, 18, and 24 hpi. Mock-infected cells showed normal cell junction morphology, with a cobblestone-like staining pattern as protein localization was restricted to the cell periphery. However, HAstV-infected cells had disrupted junctional proteins. The disruption was as early as 6 hpi with occludin beginning to re-localize away from the cell periphery . Re-locaCellular junctions act as a physical barrier that prevent the movement of lipids and membrane proteins from migrating between the apical and basolateral cell membranes .When celCDH1 or upregulate SNAI1 or TWIST1 , SJ60.212 (HAstV-8), and SJ177.110 (HAstV-2), as well as lab adapted classical serotypes HAstV-8 and HAstV-2. All clinical isolates and lab-adapted serotypes disrupted the localization  and exprr TWIST1  despite r TWIST1 . The lacr TWIST1  or exprer TWIST1 . Finallyr TWIST1 . This shSNAI1 mRNA, decrease CDH1 leading to reduced E-cadherin expression, or disrupt cellular polarity . Cells were propagated in minimum essential medium  supplemented with 20% fetal bovine serum , GlutaMax-I (Gibco), and 1 mM sodium pyruvate (Gibco). The human colorectal adenocarcinoma cell line HT29 was obtained from ATCC (HTB-38). HT29 cells were propagated in Dulbecco’s Modified Eagle’s Medium  supplemented with 10% fetal bovine serum .2 with a UV cross-linker as described previously [HAstV-1, HAstV-2, and HAstV-8 lab adapted viral stocks were propagated in Caco-2 cells. Infectious titers were quantitated on Caco-2 cells by the fluorescent-focus assay as previously described . To UV ieviously . Inactiveviously .The reovirus T1l and T3SA+ strains were generous gifts from Dr. Terence Dermody’s lab at the University of Pittsburgh. HAstV-VA1 was a gift from Dr. David Wang’s lab at Washington University in St. Louis.Briefly, Caco-2 cells were seeded onto glass coverslips  or transwells (polarity and SMAD3 staining). Once confluent, the cells were infected with HAstV-1 (MOI 10) or mock infected. At various times post-infection, cells were fixed with 4% paraformaldehyde  or 100% ice cold methanol (polarity and SMAD3 staining), and then blocked with 5% normal goat serum (NGS) in PBS at room temperature for 1 hour. The cells were stained for E-cadherin , occludin , ZO-1 , sodium-potassium ATPase , ezrin , β-catenin , SMAD3 , HAstV capsid , and vimentin  for 1 hour followed by anti-mouse IgG-Alexa Fluor 488 or anti-rabbit IgG-Alexa Fluor 555 (Invitrogen) secondary antibodies and DAPI  in 1% NGS for 30 min at room temperature. Following staining, coverslips or transwells were mounted onto slides with Prolong Gold Antifade Mountant (Invitrogen) and sealed. Cells were imaged with a Nikon TE2000 inverted microscope Images were captured with a Nikon 60x objective lens using Nikon NIS Elements software.g for 5 min at 4°C. Protein concentrations were determined using the BCA Protein Assay Kit (Pierce). Equal protein concentrations of the soluble fraction were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (4–20%) under reducing conditions. Following transfer to nitrocellulose and probing for for E-cadherin , occludin , ZO-1 , β-catenin , vimentin , and β-actin . The blot was imaged on Licor Odyssey Fc and band densitometry was measured using Image Studio version 5.2 software.Caco-2 cells were mock- or HAstV-1 (MOI 10) infected or were treated with an equal amount of UV-inactivated virus. At the indicated times, monolayers were lysed in 100 μl of RIPA buffer (Abcam) and 1× protease inhibitor cocktail (Pierce) for 15 min at room temperature and centrifuged at 14,000 × https://www.gsea-msigdb.org/gsea/msigdb).Total RNA (100 ng) was converted to biotin-labeled cDNA using the Affymetrix WT Plus kit and hybridized to an Affymetrix Human Gene 2.0 ST GeneChip array (Life Technologies). Array probes were normalized and summarized to transcript-level signals by the RMA algorithm using the Affymetrix Expression Console software v1.1. Gene Set Enrichment Analysis (GSEA) was performed as described  using ge2 First Strand Kit (Cat# 330401). The cDNA was used on the real-time RT2 Profiler PCR Array (Cat# PAHS-090Z) in combination with RT2 SYBR Green qPCR Mastermix (Cat# 330529). The CT values were then uploaded on to the data analysis web portal at http://www.qiagen.com/geneglobe. Samples were assigned to either control or test groups. The data was normalized based on a manual selection from full panel of reference genes. The data analysis web portal calculated fold change/regulation using ΔΔCT method, in which ΔCT is calculated between gene of interest and an average of reference genes , followed by ΔΔCT calculations (ΔCT (Test Group)-ΔCT (Control Group)). Fold Change was then calculated using 2^ (-ΔΔCT) formula.Briefly, cells were seeded in 6-well plate, mock infected or HAstV-1 (MOI 10) infected and collected at the indicated time point in TRIzol reagent (Thermo Fisher Scientific). Then, RNA was isolated according to the manufacturer’s instructions. RNA quality was determined and was reverse transcribed using Qiagen’s RTOCLN (cat# QT00081844), CDH1 (cat# QT00080143), SERPINE1 (cat# QT00062496), SNAI1 (cat# QT00010010), TGFB1 (cat# QT00000728), TWIST1 (cat# QT00011956), ZEB1 (cat# QT00020972), ZEB2 (cat# QT00008554), and VIM1 (cat# QT00095795). The resulting Ct values were normalized to GAPDH (cat# QT00079247). The log transformed ΔΔCt values are reported as fold changes over untreated.Caco-2 cells were infected with HAstV-1 or mock infected and RNA extracted at indicated timepoints using TRIzol (AMbion) according to manufacturer’s specifications. Then qRT-PCR was performed using the QuantiTect SYBR green kit (Qiagen) primer assays for Following immunofluorescent staining and imaging, basal or apical localization of sodium-potassium ATPase was determined by measuring mean fluorescent intensity above and below cell midline using ImageJ 1.50i software. Results were expressed as a ratio of basal fluorescent intensity to apical fluorescent intensity.4). These cells were inoculated with supernatants (100 μl) taken from HAstV-1 (MOI 10) or mock-infected Caco-2 cells at various times post-infection and incubated at 37°C for 16–20 hours. The inoculum was removed, and the cells washed twice with PBS. Cell lysates were prepared and assayed for luciferase activity using the Luciferase Assay System (Promega) and imaged on the Cytation 5 Cell Imaging Multi-Mode Reader (BioTek).TGF-β activity was measured using a luciferase reporter cell line, as previously described . Briefly4 cells were seeded into transwells , and once confluent, transferred into serum free media for at least 1 hour. The cells were treated with 10μM U0126 (Promega) or 20μM SB431542 (Tocris) 1 hour prior to infection. Then the cells were infected with HAstV-1 (MOI 5), TGF-β treated (20 ng/ml), or mock infected, according to experiment, in serum free media. Following the virus adsorption period of 1 hour, the inoculum was removed and fresh media containing 10μM U0126 or 20μM SB431542 was replaced. DMSO was used as the vehicle control in both experimental setups.Briefly, 5 × 10Supernatants were titered via fluorescent focus assay as previously described . Viral RCytotoxicity was measured using the LDH Assay Kit (abcam) per the manufacturer’s protocol. Briefly, at 8- and 24-hours post-infection, 100μl of supernatant was combined with 100μl of LDH Reaction Mixture and incubated for 30 min in the dark at room temperature. The absorbance of the samples at 490nm was read on a microtiter plate reader. The percent of cytotoxicity was calculated as a percent of the positive control (cells treated with 1% Triton X-100) normalized to mock-infected wells.Data were analyzed by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test , ordinary one-way ANOVA followed by a test for trend , two-tailed student t-test , two-way ANOVA followed by Sidak’s multiple comparisons test , ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test , and ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (E-cad expression and RT-PCR with SB431542) using GraphPad Prism version 9. Asterisks show statistical significance as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.S1 FigGene set enrichment analysis was performed on HAstV-infected (MOI of 10) and uninfected Caco-2 intestinal epithelial cells. Shown are top upregulated hallmark pathways’ normalized enrichment scores with false discovery rate estimated by Benjamin-Hochberg method cut-offs of q<0.001 (red), q<0.05 (blue), and q>0.05 or non-significant (gray).(TIF)Click here for additional data file.S2 Fig2 Profiler PCR Array Human Epithelial to Mesenchymal Transition (EMT). RNA samples were collected from HAstV-infected or mock-infected cells at 8 and 24 hpi. Gene expression values are colored corresponding to the up (red) or downregulation (blue) relative to mock-infected cells.Heatmap showing fold regulation of EMT associated genes from Qiagen’s RT(TIF)Click here for additional data file.S3 FigCell toxicity was measured by lactate dehydrogenase (LDH) assay at 8- and 24-hours post-infection of HAstV-1, VA1, T1L, and T3SA (MOI 10). Error bars indicate standard deviations of two independent experiments performed in triplicate.(TIF)Click here for additional data file.S4 Fig(A) Caco-2 monolayers on grown coverslips, infected with HAstV-1 (lab-adapted), SJ054.225 (HAstV-1 isolate), HAstV-8 (lab-adapted), SJ60.212 (HAstV-8 isolate), HAstV-2 (lab-adapted), SJ177.110 (HAstV-2 isolate) or mock infected. Cells were fixed at 24 hpi and stained for E-cadherin (red) and DAPI (blue). (B) Expression of E-cadherin was quantified by immunoblot of HAstV-1 (lab adapted), SJ054.225 (HAstV-1 isolate), HAstV-8 (lab adapted), SJ60.212 (HAstV-8 isolate), HAstV-2 (lab adapted), SJ177.110 (HAstV-2 isolate) infected (MOI of 10) or mock infected Caco-2 cell lysates. Bands were then quantified by densitometry and normalized to β-actin then compared to mock-infection. Error bars indicate standard deviations of two independent experiments performed in triplicate, and asterisks show statistical significance as measured by ordinary one-way ANOVA followed by Dunnett’s multiple comparisons test as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Na/K ATPase (red) and ezrin (green) localization in Caco-2 cells infected with lab adapted and clinical isolate HAstVs is disrupted compared to mock infected cells. All images are representative of two independent experiments.(TIF)Click here for additional data file.S5 FigSERPINE1, SNAI1, and CDH1 mRNA levels were measured in mock and HAstV-1 infected (MOI 5) Caco-2 and HT29 cells at 24 hpi. Error bars indicate standard deviations from two independent experiments performed in duplicate, and asterisks show statistical significance as measured by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test as follows: *, P < 0.05; ****, P < 0.0001.(A) HT29 and Caco-2 monolayers infected with HAstV-1 were fixed at 24 hpi and stained for E-cadherin (red), HAstV capsid (green) and DAPI (blue). (B) Expression of E-cadherin was quantified by immunoblot of mock- and HAstV-1 infected (MOI 10) Caco-2 and HT29 cell lysates. Bands were then quantified by densitometry and normalized to β-actin then compared to mock-infection. Error bars indicate standard deviations of one experiment performed in duplicate. (C) (TIF)Click here for additional data file.S6 Fig(A) Western blot of E-cadherin at 1-, 2-, 3-, and 4-days post inoculation with 20 ng/ml active TGF-β compared to mock. Bands were quantified by densitometry and normalized to β-actin then compared to mock-infection. (B) Caco-2 inoculated with 20ng/ml active TGF-β or mock treated (as indicated). Cells were fixed at 1-, 2-, or 3-days post-inoculation in 100% ice-cold methanol and then stained for ezrin (green), Na/K ATPase (red), and DAPI (blue).(TIF)Click here for additional data file.


Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer. Natural killer (NK) cells are innate lymphocytes that kill virus-infected, stressed, or transformed cells ,2. NK ceNK cells expressing a single type of KIR are highly cytotoxic against HLA class I-mismatched allogeneic acute myeloid leukemia blasts ,13. MorePrevious studies have indicated that the presence of the non-classic major histocompatibility complex class I molecule HLA-E (cognate ligand for NKG2C and NKG2A) is necessary for the expansion of adaptive NK cells ,17. Liu Our group has examined the frequency of occurrence of FcεRIγ-deficient NK cells (termed g-NK cells), which appear in HCMV-positive donors and most often express the activating receptor NKG2C  in cord We have studied and developed effective protocols for the ex vivo expansion of NK cells from peripheral blood mononuclear cells (PBMCs) using both conventional K562 cells and genetically modified K562 cells expressing cofactor OX40L and cytokines mbIL-18 and -21 ,23,24,25Based on these results, we reasoned that genetically modified K562 cells expressing HLA-E would perhaps function as effective feeder cells for the improvement of the expansion rate of adaptive NKG2C+ NK cells. To validate this in the present study, we compared conventional K562- and K562-HLA-E-based culture methods and investigated the phenotypic and functional effects exerted by K562 cells expressing HLA-E on the expansion of adaptive NKG2C+ NK cells.HLA-E expression in K562-HLA-E cells was confirmed at both the transcriptional and translational levels. Real-time quantitative polymerase chain reaction (Rq-PCR) analysis performed using the total RNA isolated from K562 and K562-HLA-E cell lysates showed a significantly higher (18.7-fold) level of HLA-E mRNA expression in K562-HLA-E cells than in K562 control cells A. This wTo study the effect exerted by K562-HLA-E feeder cells on NK cell expansion rate, we compared the purity and fold expansion of the NK cells cultured on K562 and K562-HLA-E cells. Similar purity was observed in the eNK cells after 7 weeks, irrespective of whether they were cultured with K562 or K562-HLA-E cells A. HoweveTo test whether K562-HLA-E feeder cells were responsible for promoting adaptive NK cell expansion, NKG2C+ cells obtained from 16 healthy donors were cultured on irradiated K562 and K562-HLA-E feeder cells, and the KIR expression was compared. The clinical characteristics of the donors are presented in Comparisons of the phenotypic characteristics of NK cells expanded with K562 and K562-HLA-E cells were made on days 14 and 49 of culture. The eNK cells co-cultured with K562-HLA-E cells exhibited “adaptive” receptor features such as reduced CD16 (day 14) and NKG2A (days 14 and 49) expression, increased CD57 (day 14) expression, and slightly reduced NKp30 and NKp46 (day 14 and day 49) expression. Interestingly, eNK cells showed slightly higher NKG2D expression when expanded with K562-HLA-E cells than with K562 cells .Along with a high frequency of NKG2C expression, a deficiency of FcεRIγ expression is a characteristic feature of adaptive NK cells. Therefore, we investigated whether the K562-HLA-E-based eNK cells preferentially boosted the NKG2C+ FcεRIγ- NK cell population. Before expansion, the percentage of FcεRIγ- NK cells was significantly correlated with the percentage of NKG2C+ NK cells A, althouTo investigate the difference in the functionality of eNK cells when K562 and K562-HLA-E cells were used, we measured CD107a degranulation and IFN-γ production using K562 and MCF7 as target cells. Similar levels of CD107a degranulation and IFN-γ production were observed in eNK cells stimulated with K562 or MCF7 target cells irrespective of whether they were generated with K562-HLA-E or K562 cells A,B.We further examined cytotoxic effector functions using a (carboxyfluorescein succinimidyl ester) CFSE-based killing assay against K562 target cells and Raji cells coated with rituximab because ADCC is generally considered an essential function of adaptive NK cells ,20,27. HA trend of higher cytotoxicity was observed against HLA-mismatched MCF7 target cells (C2C2) in the eNK cells that used K562-HLA-E cells from C1C1 donors compared with those from C2C2 donors D, althouThe expansion rate of NK cells is affected by various factors such as the source of NK cells, cytokines, feeder cells, media, and sample status (fresh vs. cryopreserved) ,28,29,30The presence of HLA-E, which serves as a cognate ligand for NKG2C, is necessary for the expansion of adaptive NKG2C+ NK cells ,14,17,31To further characterize the expanded adaptive NK cells, we performed flow cytometric analysis of various NK cell receptors. Consistent with previous findings ,33,34, tTraditionally, adaptive NK cells have an FcεRIγ-deficient phenotype ,35,36. HDespite varied expression of several receptors, the expanded adaptive NK cells remained highly functional and showed similar cytotoxicity and CD107a degranulation levels against K562 cells, as did conventional NK cells. In contrast to previous studies ,16, K562Therefore, this expansion process can influence the adaptive NK cell phenotype and functional features. Further studies comparing isolated adaptive and expanded adaptive NK cells are needed to understand the potential mechanism underlying this alteration.Moreover, our data showed that expanded NKG2C+ NK cells could persist for up to 13 weeks when cultured with genetically modified K562 cells expressing HLA-E. Even after long-term culture, they remained functional and expressed the same patterns of surface markers, unlike adaptive NK cells after short-term culture C. SimilaOur study had several limitations. Although K562-HLA-E cells induced a higher NK cell expansion rate than in the previous study, a direct comparison with other feeder cells (including HLA-E-transfected 721.221 cells) in the same culture system should be verified by future studies. In addition, the combination of HLA-E, other co-factors, and cytokines  required to optimize the expansion rate of adaptive NK cells needs investigating. Another limitation of our study is that the frequency of the HLA-C2 ligand is much lower in the Korean population than in Caucasians (3.2%) , and theFurther studies with larger patient populations are required to confirm the influence of KIR ligand status on the generated specific adaptive NK cells (with single KIR expression) for the clinical targeting of cancer across HLA barriers in cell therapy.In conclusion, we successfully developed a remarkable ex vivo expansion method using K562-HLA-E feeder cells to mimic CMV triggers and selectively expand long-lived adaptive NKG2C+ NK cells expressing self-specific KIR. Furthermore, long-lived expanded adaptive NK cells demonstrated sustained cytolytic activity. Further studies are needed to uncover the relevant aspects of expanded adaptive NK biology in order to optimize their clinical applications.Peripheral blood samples from 16 healthy donors were collected in heparinized tubes. Informed consent was obtained prior to study participation and blood sample collection. This study was approved by the Institutional Review Board of Samsung Medical Center, Seoul, Korea (IRB No. SMC 2020).2 incubator.K562 (human myelogenous leukemia cell line) and Raji (human Burkitt’s lymphoma cell line) cells were obtained from the American Type Culture Collection . The cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) , penicillin (100 units/mL), and streptomycin (100 μg/mL)  at 37 °C in a humidified 5% CO5 cells/well and then transduced with the lentiviral supernatant for 24 h in the presence of polybrene  at a multiplicity of infection of 30. After 24 h of incubation, the virus-containing medium was removed and replaced with 2 mL of fresh culture medium. When cell confluency was over 90%, the cell culture was expanded to a T-25 flask. The transduction efficiency was evaluated daily using an inverted fluorescence microscope. Two weeks after transduction, GFP-positive cells were sorted using BD FACSAria TM III and maintained in RPMI 1640 with 10% FBS.The hybrid HLA-E sequence was synthesized after the signal domain of HLA-A2 was fused with the mature HLA-E sequence, as reported previously . As the Total RNA for mRNA Rq-PCR analysis was isolated using an RNeasy Mini Kit  according to the manufacturer’s instructions and quantified using an IMPLEN nanophotometer P330 . Isolated RNAs were converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). All PCRs were performed using a QuantiTect SYBR Green PCR Kit (Qiagen) and Rotor-Gene Q (Qiagen) in standard 20 µL reactions. A sense primer (5′-ACCCTCGTCCTGCTACTCTC-3′) targeting the signal sequence of HLA-A2 and an antisense primer (5′-CCACGTAGCCCACAGAGATG-3′) targeting the mature sequence of HLA-E were used to exclusively amplify the HLA-E gene. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using the sense primer 5′-ACATCGCTCAGACACCATG-3′ and antisense primer 5′-TGTAGTTGAGGTCAATGAAGGG-3′. All the samples were processed in triplicate to increase the reliability of the results. Each assay was performed using positive and negative controls. The threshold cycle (Ct) value for each gene was measured for each sample. The Ct value of GAPDH was used as an endogenous reference for normalization. The values obtained were normalized to those of the negative control and expressed as fold-changes.Recombinant human interleukin (IL)-2 and IL-15  were used to expand NK cells. Phycoerythrin (PE)-conjugated anti-human HLA-E (clone 3D12)  was used to measure HLA-E expression on generically modified K562-HLA-E during culture. Allophycocyanin (APC)-Cy7-conjugated anti-human CD3, PE-Cy7-conjugated anti-human CD56, pacific blue-conjugated anti-human CD16, fluorescein isothiocyanate (FITC)-conjugated anti-human CD57, pacific blue-conjugated anti-human NKp46, PE-conjugated anti-human NKp30, peridinin chlorophyll protein complex (PerCP)-conjugated anti-human NKG2D , FITC-conjugated anti-human CD69, APC-conjugated anti-human DNAM-1 , APC-conjugated anti-human NKG2A, PE-conjugated anti-human NKG2C, PerCP-conjugated anti-human KIR2DL1 , FITC-conjugated anti-human KIR2DL2/L3, and BV421-conjugated anti-human KIR3DL1 (BioLegend) were used to evaluate purity and surface expression of NK cell receptors. The PE-conjugated anti-human CD107a mAb was used as a surrogate marker for quantifying degranulation. BV421-conjugated anti-human interferon (IFN)-γ and FITC-conjugated anti-FcεRIγ subunit (FcɛRγ)  were used for intracellular staining.Human PBMCs were isolated from healthy adult donors using density-gradient centrifugation with Ficoll-Hypaque  and washed twice with phosphate-buffered saline . PBMCs were co-cultured with irradiated (100 Gy) K562 and K562-HLA-E cells in a 24-well plate with RPMI 1640 medium , streptomycin (100 µg/mL), and L-glutamine (4 mmol/L)) containing recombinant human IL-2 (10 U/mL). After day 7, the IL-2 concentration was increased from 10 to 100 U/mL, and soluble IL-15 (5 ng/mL) was added to the medium. The irradiated feeder cells were re-stimulated on days 7 and 14. The medium was replaced every 2–3 days. The expansion rate of NK cells is presented as the “expansion fold”, which was determined by dividing the absolute number of NK cells at time points of interest by the respective number on day 0.HLA-E expression on conventional K562 and K562-HLA-E cells and the expression of NK cell receptors on expanded NK (eNK) cells were examined by staining with an appropriate combination of fluorochrome-conjugated monoclonal antibodies  for 15 min in the dark. To detect signaling adaptors, the cells were fixed, permeabilized, and stained with FITC-conjugated anti-FcɛRγ. After washing with fluorescence-activated cell sorting (FACS) buffer, stained cells were acquired using a FACS Verse instrument .2 for 5 h. Cells were then harvested, washed with FACS, and stained with anti-human CD3 and CD56 monoclonal antibodies (mAbs) for 20 min on ice. After washing, fixation, and permeabilization, NK cells were further stained with PE-conjugated anti-human IFN-γ mAb on ice for 30 min, washed, and analyzed using FACS Verse and Kaluza 1.3 .To measure IFN-γ production, eNK cells were incubated in a 96-well U-bottom plate with K562 cells (effector-to-target ratio [E:T] = 1:1) in the presence of brefeldin A (BD Biosciences) at 37 °C and 5% CO5) were incubated with or without 2 × 105 K562 cells in a 96-well U-bottom plate in the presence of PE-conjugated anti-human CD107a (5 μL). Monensin and brefeldin A were added after 1 h, and the plate was incubated for an additional 4 h. NK cells were stained with anti-human CD3 and CD56 mAbs before acquisition.The eNK cells  and incubated with eNK cells at the same E:T ratios used for the cytotoxicity assay. The plates were centrifuged at 1500 rpm for 3 min and then incubated at 37 °C and 5% CO2 for 4 h. The mixed cells were transferred to FACS tubes after incubation and 1 μL of PI (1 mg/mL) (Invitrogen) was added to each tube. The cells were acquired on a FACS Verse and analyzed using the Kaluza software 1.3 .The cytotoxicity of eNK cells against target cells  was measured using a CFSE-based assay for 4 h, as previously described ,23. TargHLA-C1 and HLA-C2 levels were determined by PCR amplification with sequence-specific primers (PCR-SSP) and low resolution using the HLA-C SSP PCR kit .HCMV serology was determined using a BioMerieux VIDAS kit , and the cut-off value of seropositivity was ≥6 with CMVG and ≥0.90 with CMVM.p < 0.05.Statistical analysis was performed using GraphPad Prism 5 . The Mann–Whitney U-test (non-parametric) was used to analyze specific sample pairs for significant differences. All tests were two-tailed, and differences were considered statistically significant at


PTEN are observed in many cancers. PTEN is a dual-specificity phosphatase that harbors lipid and protein-phosphatase activities. The Caenorhabditis elegans PTEN ortholog is daf-18, which has pleiotropic effects on dauer formation, aging, starvation resistance, and development. Function of 3 daf-18 point-mutants, G174E, D137A, and C169S, had previously been investigated using high-copy transgenes in a daf-18 null background. These alleles were generated based on their mammalian counterparts and were treated as though they specifically disrupt lipid or protein-phosphatase activity, or both, respectively. Here, we investigated these alleles using genome editing of endogenous daf-18. We assayed 3 traits relevant to L1 starvation resistance, and we show that each point mutant is essentially as starvation-sensitive as a daf-18 null mutant. Furthermore, we show that G174E and D137A do not complement each other, suggesting overlapping effects on lipid and protein-phosphatase activity. We also show that each allele has strong effects on nucleocytoplasmic localization of DAF-16/FoxO and dauer formation, both of which are regulated by PI3K signaling, similar to a daf-18 null allele. In addition, each allele also disrupts M-cell quiescence during L1 starvation, though D137A has a weaker effect than the other alleles, including the null. Our results confirm that daf-18/PTEN is important for promoting starvation resistance and developmental arrest and that it is a potent regulator of PI3K signaling, and they highlight challenges of using genetic analysis to link specific DAF-18/PTEN enzymatic activities to particular phenotypes.Mutations in the well-known tumor suppressor  Tumor suppressor Phosphatase and Tensin homolog (PTEN) is frequently mutated in many human cancers , PTEN(Y138L), and PTEN(C124S) are most widely used. PTEN(G129E) has little activity against PIP3 but retains a large percentage of the protein-phosphatase activity (PTEN(Y138L) has barely any protein-phosphatase activity but retains most activity against PIP3 (PTEN(C124S) eliminates nearly all lipid and protein-phosphatase activity , and developmental arrest and survival of starved L1-stage larvae (L1 arrest), each of which is also regulated by PI3K signaling (PTEN transgenes can rescue daf-18 dauer formation defective (Daf-d) and shortened-lifespan phenotypes  is the C. elegans homolog of PTEN(G129E), and it is presumed to specifically disrupt lipid-phosphatase activity (daf-18(G174E) is unable to rescue the daf-18 Daf-d, shortened-lifespan, and L1 arrest-defective (Q-cell division) phenotypes, but it can weakly rescue the vulval-induction phenotype . Nonetheless, PTEN(D92A), which “traps” substrate but does not dephosphorylate it  (daf-18(D137A) was able to rescue the daf-18 L1 arrest-defective (Q-cell division) phenotype, but daf-18(G174E) was not (daf-18(D137A) selectively disrupts protein-phosphatase activity while leaving lipid-phosphatase activity intact. They also suggest that DAF-18 regulates Q-cell division during L1 arrest independent of its protein-phosphatase activity. daf-18(C169S) corresponds to PTEN(C124S) (daf-18(D137A), transgenic expression of daf-18(C169S) fails to rescue the daf-18 L1 arrest-defective (Q-cell division) phenotype  . By exteN(C124S)  and is tC. elegans embryos hatch without food, they remain developmentally arrested in the first larval stage (L1 arrest or L1 diapause) (daf-16 and daf-18 are starvation-sensitive and L1 arrest-defective (If iapause) . The traiapause) . AGE-1/Pdaf-18(G174E), daf-18(D137A), and daf-18(C169S) in the context of L1 arrest, with focus on starvation resistance. Our initial objective was to identify which DAF-18 enzymatic activities are required for starvation resistance, but we were also concerned about the specificity of each allele with respect to enzymatic activity. Rather than relying on transgenes, we used CRISPR-Cas9 genome editing to modify the endogenous daf-18 locus to produce daf-18(G174E), daf-18(D137A), and daf-18(C169S) mutants. We used 3 complementary assays for L1 starvation resistance: L1 starvation survival, growth during recovery from L1 starvation, and fecundity following L1 starvation  examining somatic gonad and germ-cell quiescence in dauer larvae. Collectively, these 2 studies suggest the need for caution in interpreting results of genetic analysis using these missense alleles.Here, we present phenotypic analysis of arvation . We alsoWild-type strain N2 is from the Sternberg Lab collection.ayIs6[hlh-8p::GFP + dpy-20(+)] X was obtained from the CGC.PD4666 daf18(ok480) IV was obtained from the Chin-Sang Lab at Queen’s University.IC166 daf-16(ot971[daf-16::GFP]) I was obtained from the Hobert Lab at Columbia University.OH16024 daf-18(syb1516) IV, PHX1615 daf-18(syb1615) IV, and PHX1618 daf-18(syb1618) IV were generated by SunyBiotech and have CRISPR-Cas9 engineered endogenous alleles C169S, D137A, and G174E, respectively.PHX1516 syb1615 backcrossed 4 times to SunyBiotech N2LRB434: syb1618 backcrossed 4 times to SunyBiotech N2LRB435: syb1516 backcrossed 4 times to SunyBiotech N2LRB436: daf-16(ot971[daf-16::GFP]) I; daf-18(ok480) IVLRB455: daf-16(ot971[daf-16::GFP]) I; daf-18(syb1618) IVLRB465: daf-16(ot971[daf-16::GFP]) I; daf-18(syb1615) IVLRB467: daf-16(ot971[daf-16::GFP]) I; daf-18(syb1516) IVLRB468: daf-18(syb1615) IV; ayIs6[hlh-8p::GFP + dpy-20(+)] XLRB472: daf-18(ok480) IV; ayIs6[hlh-8p::GFP + dpy-20(+)] XLRB477: daf-18(syb1516) IV; ayIs6[hlh-8p::GFP + dpy-20(+)] XLRB478: daf-18(syb1618) IV; ayIs6[hlh-8p::GFP + dpy-20(+)] XLRB479: Escherichia coli OP50 nematode growth medium (NGM) plates and were well-fed for at least 3 generations before being used in experiments. Worms were grown and starved at 20°C. Gravid adult worms were hypochlorite treated (“bleached”) to collect embryos, which were then resuspended, washed, and cultured in S-basal (including 0.1% EtOH). Cultures had ∼1 embryo/µl in 5 ml and were placed in 16 mm glass tubes on a tissue-culture roller drum at 20°C so they hatch and enter L1 arrest . Two days later, the number of live worms on the lawn was recorded . Proportion alive was determined as total alive divided by total plated.All strains assayed in this study were maintained on °C. Five hundred microliters of starvation culture was plated on 10 cm NGM plates seeded with E. coli OP50 to support larval development (recovery plates). For the control condition (no starvation), embryos were plated directly after hypochlorite treatment, before starvation cultures were placed on the roller drum. For the starved condition, 500 µl of the starvation culture was plated 4 days after hypochlorite treatment. Worms were washed off plates with S-basal and transferred to unseeded (no OP50) 10 cm NGM plates for imaging using ZEISS SteREO Discovery.V20. Because L1 larvae take ∼12 h to hatch following hypochlorite treatment, control and starved worms were imaged 60 and 48 h after plating, respectively. This approach enabled measurements of body length after ∼48 h of feeding for both populations. The image-analysis program WormSizer was used to determine worm length , 18 worms were singled out per genotype from the control recovery plate of the growth-rate assay onto 6 cm NGM plates seeded with OP50 the day after hypochlorite treatment. For the starved condition, a 50 µl aliquot of the starvation culture was plated on a 6 cm NGM plate with OP50 24 h after hypochlorite treatment (∼12-h starvation) for each genotype, and 18 worms were singled out onto 6 cm NGM plates with OP50 the next day. Starting the day after singling out, and every day after for 5, each worm was transferred to a new 6 cm NGM plate with OP50. The total brood size of each worm was determined from the number of progeny on each plate. Worms that died during the process, including due to internal hatching (“bagging”), were recorded, and their total brood sizes were included in the reported results since gonad abnormalities and bagging are consequences of L1 starvation , a 700 µl aliquot of the starvation culture was centrifuged in a 1.7 ml Eppendorf tube at 3,000 rpm for 30 s to pellet L1 larvae. 1.5 µl of worm pellet was pipetted into the center of a slide with a 4% Noble agar pad, and a glass cover slip was immediately placed on top. A timer was set for 3 min, and the slide was systematically scanned with each individual worm scored for nucleocytoplasmic localization specifically within intestinal cells at 1000x on a Zeiss Axio Imager A1. At least 50 larvae were scored per replicate. Nucleocytoplasmic localization of DAF-16::GFP was scored in intestinal cells and assigned to one of 4 categories: nuclear, more nuclear, more cytoplasmic, and cytoplasmic. “More nuclear” and “more cytoplasmic” are intermediate categories between nuclear and cytoplasmic, with localization being more nuclear or cytoplasmic, respectively. Scoring for each slide stopped after 3 min in order to avoid confounding effects of the worms being mounted on slides.E. coli HT115 colonies carrying pAD12 for empty vector (EV) or pAD48 for daf-2 RNAi grown on 12.5 µg/ml tetracycline and 100 µg/ml carbenicillin LB plates at 37°C were inoculated into LB starter cultures with the same concentration of tetracycline and half the concentration of carbenicillin. Starter cultures were incubated overnight at 37°C with shaking and then used to inoculate larger cultures of terrific broth (TB) with 25 µg/ml carbenicillin which were incubated overnight at 37°C with shaking. Cells from large cultures were spun down and resuspended at a final density of 250 mg/ml in S-complete with 15% glycerol. Aliquots were frozen and thawed only once and seeded onto 6 cm NGM plates containing 25 µg/ml carbenicillin and 1 mM IPTG. Lawns were allowed to grow overnight at room temperature before plating worms. For each genotype and each replicate, 7 L4 worms were picked onto EV or daf-2 RNAi plates and grown at 27°C. Twenty hours after plating, they were removed but their progeny were allowed to continue growing at 27°C. Forty-eight hours later, the total number of worms and the number of dauers were recorded for each plate and the proportion of dauers was calculated. Dauers were identified by visual inspection on plates , and worms that had bypassed dauer formation had vulvas and were typically adults.Single daf-18(G174E), daf-18(D137A), and daf-18(C169S) mutants (daf-18 (ok480). The ability to survive starvation is clearly an indication of starvation resistance, but L1 starvation also affects growth and reproductive success after recovery, and there are genotypes and conditions that affect growth and fecundity following starvation without appreciably affecting starvation survival  (presumed lipid-phosphatase defective) and daf-18(C169S) (presumed both phosphatase-activities defective) were expected to be starvation-sensitive. In contrast, it was unclear whether daf-18(D137A) (presumed protein-phosphatase defective) would affect starvation resistance.We used CRISPR-Cas9 to edit the laboratory reference N2 genome, producing  mutants . We backsurvival . We thersurvival . Becausedaf-18 are very sensitive to L1 starvation. All 3 mutants and the null mutant survived L1 starvation for a significantly shorter time than wild type , daf-18(D137A), and daf-18(C169S) are each very sensitive to L1 starvation (based on 3 different assays) comparable to a null allele.We found that all 3 missense alleles of ild type . Likewisild type . Furtherild type . This ladaf-18, so we assayed M-cell divisions in starved L1 larvae. We scored the number of M cells using an hlh-8p::GFP reporter (daf-18(G174E), daf-18(D137A), and daf-18(C169S), as well as daf-18(ok480) and wild type, after 3 days of starvation  (presumed lipid-phosphatase defective) and daf-18(C169S) (presumed both phosphatase-activities defective) to have M-cell divisions, but it was unclear whether daf-18(D137A) (presumed protein-phosphatase defective) would affect M-cell arrest.In addition to adapting to survive and recover from L1 starvation, wild-type worms remain in a developmentally arrested state while starved. There is a single M mesoblast cell at hatching, and it divides approximately halfway through the first larval stage in fed worms and goes on to produce 18 descendants by the L1 molt . Howeverarvation . M-cell daf-18 display M-cell divisions during L1 starvation (daf-18(D137A) (presumed protein-phosphatase defective) was clearly different than the other alleles, with significantly fewer animals with a divided M cell, though still significantly more than wild type (zero). These results show that each of the daf-18 missense mutations confers an L1 arrest-defective phenotype, but they also reveal a difference in penetrance among the alleles.We found that all 3 missense alleles and the null allele of arvation . HoweverD137A and G174E alleles disrupt protein and lipid-phosphatase activities, respectively. We decided to look at a direct target of AGE-1/PI3K signaling, DAF-16/FoxO, at the subcellular level. When insulin/IGF signaling is low, such as during starvation, DAF-16 localizes to the nucleus to regulate transcription, but when insulin/IGF signaling, and thus AGE-1/PI3K signaling, is activated, DAF-16 localizes to the cytoplasm where it is inactive , daf-18(D137A), daf-18(C169S), and daf-18(ok480). These results demonstrate that all 3 missense alleles as well as the null allele have a strong effect on DAF-16::GFP localization. It is a formal possibility that DAF-18 regulates DAF-16 localization independent of AGE-1/PI3K signaling through its protein-phosphatase activity, but we believe the simplest interpretation of these results is that the D137A allele (presumed protein-phosphatase defective) lacks specificity and also disrupts the lipid-phosphatase activity of DAF-18.We scored DAF-16 subcellular localization after 6 h of L1 starvation  and 4a. daf-2/IGFR mutants is suppressed by disruption of daf-18  developed into dauer larvae, and that over 30% of worms grown at high temperature with daf-2 RNAi (dauer-forming conditions) became dauers, as expected. In contrast, no daf-18(G174E), daf-18(D137A), or daf-18(C169S) worms became dauers in either control or dauer-forming conditions  also report that daf-18(D137A) (presumed protein-phosphatase defective) is defective at dauer development while focusing on somatic gonad development and germ cell proliferation in dauer larvae induced by daf-7/TGF-β mutation. As for the other phenotypes we examined, it is possible that DAF-18 regulates dauer formation through an unknown mechanism involving its protein-phosphatase activity, but these results along with those of Wittes and Greenwald  challenge the notion that D137A disrupts DAF-18 protein-phosphatase activity without appreciable effects on its lipid-phosphatase activity.Insulin/IGF and AGE/PI3K signaling is also a critical regulator of dauer formation , and thenditions . These rdaf-18(D137A) actually disrupts the lipid-phosphatase activity rather than being selective for the protein-phosphatase activity. We reasoned that by analyzing D137A/G174E trans-heterozygotes we could assess whether these 2 alleles have overlapping effects on DAF-18 function or if they are actually specific to each enzymatic activity. That is, if these alleles are truly specific, then they should complement each other and trans-heterozygotes should be wild-type-like. We produced daf-18(D137A/G174E) trans-heterozygotes (trans-heterozygotes) for both assays. We found that growth of daf-18(D137A/G174E) animals was more impaired after 4 days of starvation than wild-type (daf-18(D137A/G174E) animals produced fewer progeny than wild type after 12 h of starvation , daf-18(D137A), and daf-18(C169S) alleles, and we determined their effects on starvation resistance and developmental quiescence during C. elegans L1 arrest. We hoped to use these missense alleles to dissect the enzymatic activities of DAF-18/PTEN, delineating the function of its lipid and protein-phosphatase activities with respect to some of its mutant phenotypes. We found that all 3 alleles reduced starvation resistance, eliminated nuclear localization of DAF-16::GFP during starvation, and suppressed dauer formation. In each case, penetrance was comparable to a null allele . Each missense allele also caused an L1 arrest-defective phenotype (M-cell divisions), though penetrance varied, with D137A (presumed protein-phosphatase defective) displaying significantly lower penetrance. There is ample evidence that DAF-18 affects each of these phenotypes through its lipid-phosphatase activity, but there is no evidence that its protein-phosphatase activity is involved, though it is possible that DAF-18 dephosphorylates a protein in the AGE-1/PI3K pathway  and possibly G174E (presumed lipid-phosphatase defective) disrupt more than one of these 2 DAF-18 activities. Either allele could also affect DAF-18 expression levels, but we cannot distinguish between these possibilities with genetic analysis alone.We used genome editing to produce D137A/G174E trans-heterozygotes and also observed lack of complementation. They also showed that daf-18 is not haplo-insufficient. These results further suggest overlapping effects of each allele on DAF-18 function, and they also concluded that these alleles do not specifically disrupt one or the other enzymatic activity of DAF-18.Wittes and Greenwald  analyzed dauer development in PTEN alleles to the homologous daf-18 alleles should be made very cautiously.In vitro and in vivo phosphatase assays performed with PTEN greatly outnumber those performed with DAF-18. In vitro experiments actually suggest that PTEN(D92A) has disrupted lipid-phosphatase activity (daf-18(D137A) had significantly less effect on M-cell divisions during L1 starvation than the other alleles examined. Likewise, a high-copy daf-18(D137A) transgene functioned like wild-type daf-18 when used to rescue Q-cell quiescence during L1 starvation in the null daf-18(ok480) background  and daf-18(G174E) transgenes did not rescue quiescence. These results suggest that the effects of D137A and G174E on DAF-18 function do not completely overlap, as if there is a degree of specificity that can distinguish them in some phenotypic assays . The fact that daf-18(D137A) has less of a relative effect on M or Q-cell divisions than starvation resistance, DAF-16::GFP localization, or dauer development could reflect a difference in the relative sensitivity of the underlying biological process to the lipid and/or protein-phosphatase activities of DAF-18. However, our work together with that of Wittes and Greenwald  cautions against using missense alleles (D137A and G174E in particular) in an effort to causally connect specific DAF-18 enzymatic activities with specific phenotypes.In contrast to the other phenotypes assayed, https://github.com/jc271828/Chen2022 [accessed 2022 Apr 25].Data generated during the process of this study are available in this article. Strains are available upon request. Code for performing statistical tests with starvation survival data is available at


BNT162b2 induced increased expression of soluble proteins in adult blood , but demonstrated reduced proteins in elder blood , including 30–85% lower induction of TH1-polarizing cytokines and chemokines . Elder TH1 impairment was validated in mice in vivo and associated with impaired humoral and cellular immunogenicity. Our study demonstrates the utility of a human in vitro platform to model age-specific mRNA vaccine activity, highlights impaired TH1 immunogenicity in older adults, and provides rationale for developing enhanced mRNA vaccines with greater immunogenicity in vulnerable populations.mRNA vaccines have been key to addressing the SARS-CoV-2 pandemic but have impaired immunogenicity and durability in vulnerable older populations. We evaluated the mRNA vaccine BNT162b2 in human  The self-adjuvantation effects, particularly from ionizable lipids and mRNA8, can enhance immunogenicity. Self-adjuvantation has been associated with high efficacy in live-attenuated vaccines, arising from pathogen-associated molecular patterns activating and enhancing innate immune responses11. Investigating mRNA vaccine-induced immune activation may provide insight into human- and age-specific immunogenicity.Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines were developed and rapidly implemented to address the public health threat of coronavirus disease 2019 (COVID-19). The originally observed ~90–95% efficacy of BNT162b2 (Comirnaty) and mRNA-1273 (Spikevax) against wildtype SARS-CoV-2, reducing severe disease and/or hospitalization13. These effects can contribute to lower T follicular helper CD4+ T cell (TFH) activity and B cell class switching, and increased T regulatory cell (Treg) frequency restraining responses17. Likely reflecting such immunosenescence, SARS-CoV-2 mRNA vaccines have been less effective at preventing hospitalization and symptomatic infection with a 9.5% impairment in older populations  versus adults (18–65Y)2, and 20% less efficacy in >80Y compared to 60–69Y18. Vaccine effectiveness (VE) has also been impacted, with a meta-analysis identifying a 9.3% decrease of VE preventing infection in older populations compared to the general populace19, and another review identifying consistently lower VE in those ≥65 than <65, with up to 15.3% less VE in the elderly20. Generally, age-associated susceptibility has been attributed to increased disease severity and reduced vaccine-induced protection23. Vulnerability, linked with immunosenescence in older adults, has been observed both with respect to SARS-CoV-2 infection induced immunogenicity24 and reduced immunogenicity of mRNA vaccines29. These observations mirror impaired immunogenicity responses in elders across a range of vaccines targeting encephalitis viruses, tetanus toxin, Salmonella spp., Streptococcus pneumoniae, influenza, and varicella zoster virus17, impacting both humoral and cell-mediated immunity, culminating in diminished VE in the elderly32.Advancing age has been associated with impaired functionality including reduced neutralization, phagocytosis, chemotaxis, and co-stimulatory molecule expression27. Older populations  have had lower cell mediated immunity (CMI), with impaired CD4+ and CD8+ activation following BNT162b2 immunization, compared to adults33. Impaired CD4+ and CD8+ T cell responses were also observed in older adults post-SARS-CoV-2 infection34, suggesting consistent age-dependent immunological differences. Additional immunizations with mRNA vaccines encoding Wuhan1 or bivalent Wuhan1 with BA.4/BA.5 mRNA encoding spike protein have been applied to overcome elder immunosenescence37. The third immunization with Wuhan1 mRNA had amplified immunogenicity against Wuhan1, and the variants Delta (B.1.617.2), and Omicron (B.1.1.529) transiently35, but failed to induce durable immunity as >65Y individuals had more rapid waning of immunity compared to <65Y36. The emergency use authorization of a 4th immunization in elders37 and bivalent mRNA vaccination lack the formulation changes needed to increase elder responsivity. We hypothesized that elder participant samples stimulated with BNT162b2 would have innate immune impairments that could contribute to the observed impaired adaptive immune induction observed in the elderly. This has been broadly observed against other pathogens and immune stimuli38.Age-associated T-cell changes could contribute to reduced cellular and antibody (Ab) functionality following mRNA vaccination of aged groupsin vitro employing primary human leukocytes and autologous plasma is a powerful tool to characterize vaccines, providing species- and age-specificity, and enabling paired comparisons of multiple conditions from the same study participant40. We hypothesized that elder participants could have differential immune activation in response to BNT162b2 stimulation, skewing the immune response towards a distinct activation state compared to adults, particularly an impaired production of analytes polarizing towards CD4+ T helper cell (TH) 1 differentiation. TH1 polarized immune responses can trigger effective intracellular pathogen responses41, including CD8+ T cell-mediated immunity42, alongside B cell class switching44 and induction of TFH-like activity for effective B cell responses in the absence of TFH45, functions that have been impaired in mRNA immunized elder individuals. To gain further insight into age-dependent mRNA vaccine responses, we characterized BNT162b2 immune stimulation in supernatants from adult and elder human whole blood assay (WBA) in vitro using targeted high performance liquid chromatography, mass spectrometry (LC/MS) proteomics, a second proteomics platform of high sensitivity dual Ab-based proximity extension assay , and a multiplex quantifying cytokines and chemokines. We also verified TH1 responses of age dependent BNT162b2 induced immunogenicity in young and aged mice, in vivo. We identified impaired TH1 polarization in both humans and mice that may contribute to the lower mRNA vaccine-induced humoral and cellular immunogenicity observed in elders.Modeling human immune activation in vitro for immune activation following stimulation with the BNT162b2 lipid nanoparticle (LNP) encapsulated mRNA vaccine (Pfizer/BioNTech). Participants were grouped by age, with younger and middle-aged adults 18–50Y, and elderly, older adults ≥60Y  analysis49, assessing fold change (FC) over matching vehicle control for baseline-normalization. Adult participants had 20 upregulated and 4 downregulated proteins (cis-trans isomerase A (PPIA) and neutrophil defensin 3 (DEFA3). Elder sample protein upregulation included serum amyloid A-1 protein (SAA1) and the fibrinogens-γ and -β (FGG and FGB), while downregulated proteins included DEFA3, tropomyosin alpha-4 chain (TPM4), apolipoprotein F (APOF), apolipoprotein C-II (APOC2), CPN1, and peptidase inhibitor 16 (PI16). Additional supernatant evaluation was performed with 4 Target 96 kits  of PEA-based proteomics quantifying 368 proteins. PEA assessment identified upregulation of predominantly inflammatory markers in adult BNT162b2-stimulated samples compared to vehicle  ligand 2 (CCL2), CCL3, CCL4, CCL7, CCL8, CCL11, chemokine (C-X-C motif) ligand 8 (CXCL8), IL-1β, and IL-656, Heparinized human whole blood was stimulated with BNT162b2 in a whole blood assay (WBA) to measure both immune activation and age-associated differences by supernatant proteomics. LC/MS has been employed to identify disease severity-associated responses after SARS-CoV-2 infection in humansproteins , while eproteins . Heatmapproteins –D. BNT16in vitro BNT162b2 stimulation identified broadly dampened immune responses in elder participant samples across 2 proteomic platforms.BNT162b2-induced proteomic protein profiles in the WBA differed by age, with adults and elders expressing 21 and 13 unique proteins, respectively –B, 2A. OH1, TH2, TH17, or T regulatory (Treg) polarizing, chemokine, hematopoiesis-supporting, and trained immunity-associated. A linear modelling analysis, GEEGLM, evaluated if age interacted with each function. TH1 support was significantly impaired (p = 0.027) in elders compared to adults, by an average of 7.4% less in each analyte involved , CXCL8, tumor necrosis factor alpha (TNFα), and interferon gamma (IFNγ) in adult WBA samples . Other cinvolved –C. The oinvolved  indicatiIn vivo murine intramuscular BNT162b2 vaccination , IgG2a, and IgG1 Ab immunogenicity, with lower anti-receptor binding domain (RBD) Ab titers than adult mice  or TH2 (<1) polarization identified a transient impairment of TH1 on D28 post-prime in elder mice  such modeling demonstrates marked age-dependent differences; and (c) results in vitro can be verified in aged mice in vivo. While mRNA vaccines have been crucial in combatting the SARS-CoV-2 pandemic, much remains to be learned regarding mRNA vaccine-induced immune activation, and mechanisms contributing to age-associated decreased immunogenicity32. We hypothesized that elder participants would have differential immune activation post-BNT162b2 WBA stimulation skewing immune responses towards distinct functional states. As elders have higher rates of severe COVID-1960 and reduced vaccine immunogenicity61, understanding the mechanisms and contributing factors to weaker immunogenicity is an urgent unmet need that may inform future vaccine optimization, discovery and development. To our knowledge, we report the first combination of human in vitro WBA and murine in vivo systems modelling age-specific immune activation with the mRNA vaccine, BNT162b2.Herein, we have demonstrated for the first time that (a) human 65. Among other PRRs, SARS-CoV-2 can also activate MDA-5 signaling66, suggesting some similarity of sensory systems detecting mRNA vaccines and SARS-CoV-2 virions. Development of vaccine formulations that trigger similar innate immune activation as natural infection may enhance immunogenicity against microbial pathogens67. To assess similarities of BNT162b2 induced immunogenicity and SARS-CoV-2 infection, we evaluated BNT162b2-induced DEPs to proteins induced during infection, as described in literature. We employed two complementary proteomic approaches for the in vitro evaluation of BNT162b2-induced WBA responses. LC/MS proteomics identified DEPs of BNT162b2-stimulated WBA adult samples compared to vehicle control -2, TLR-3, TLR-4, TLR-7, TLR-8, retinoic acid-inducible gene I (RIG-I), and melanoma differentiation-associated protein 5 MDA-5) recognizing multiple vaccine components control . Adult ss, DEFA3 –D, 2A whA-5 recog results , F, obse results  support  results . The mar13, potentially impacting monocyte and antigen presenting cell (APC) chemotaxis to injection site and lymph nodes, respectively87. Network analysis of DEPs , can support CD4+ and CD8+ T cell differentiation89, such that downregulation in elders may reduce CMI.Analysis of the proteome in supernatants derived from BNT162b2-stimulated adult vs. elder WBAs via LC/MS –D and PE of DEPs –I furthein vitro WBA, BNT162b2 induced concentration-dependent production of IL-6, CXCL8, TNF-α, and IFNγ 92. Commonalities of immune activation across mRNA vaccine platforms may reflect similarities in innate immune responses reflecting inherent self-adjuvantation.A targeted multiplex assay quantifying cytokines and chemokines enabled analyses of functionally grouped analytes, yielding further insight. In the and IFNγ , each ofand CCL4  have beeH1 polarizing99 CXCL10, and IFNγ in elders  used in cancer RNA-LNP therapies101. This lipid differs from BNT162b2’s ALC-0315 ((4-hydroxybutyl)azanediyl)bisbis(2-hexyldecanoate))8, and BNT162b2 benefits from the reduced cytotoxicity from ionizable lipids8. This could impact self-adjuvanticity between formulations, and requires further investigation, as the head groups of cationic lipids can be toxic104.BNT162b2-induced WBA cytokine and chemokine induction was age-dependent with impaired production of IL-1RA and the Tn elders . ExpressH1 polarization has been observed following BNT162b2 immunization of the elderly29, and could reflect impaired production of polarizing cytokines, impaired naïve T cell frequency, reduced sensitivity to cytokines during differentiation, and/or impaired chemotaxis of APCs. We hypothesized functionally grouping analytes could identify age-dependent impairment or dysregulation of TH1, TH2, TH17, or Treg polarizing, chemokine, hematopoiesis-supporting, or trained immunity associated analytes. Interpreting individual cytokines from a multiplex is difficult for polyfunctional analytes and can cause overinterpretation of results. Pairing individual analysis with functionally grouped analytes can generalize changes to describe broader differences and assist with analyte redundancies105. The 41 multiplex-quantified analytes were categorized by function following targeted literature searches  immaturity marker in peripheral blood113. Impaired elder Ab isotypes IgG1 and IgG3 have been observed post-vaccination114, each markers of human TH1 polarization44. We present impaired human induction of TH1 polarizing IFNγ and CXCL10 - and age-specific responses, but may not completely reflect relevant vaccine responses in vivo. To investigate age-dependent BNT162b2-induced responses in vivo, we employed an age-specific murine model widely used to study mRNA vaccination119. Coupling human in vitro modeling with animal models represents a powerful approach to provide both species-specific and in vivo assessments. Increased age is associated with impaired human humoral immunity following BNT162b2 or mRNA-1273 vaccination120. Aged mice demonstrated impaired Ab induction at all immunization doses  additional boosters to extend protection127, albeit temporarily, due to rapidly waning immunity in elders36, and (c) use of TH1-polarizing adjuvants targeted towards elder populations22, that can enhance effective intracellular pathogen responses42, B cell class switching , and support TFH-independent B cell responses45, as successfully pursued with Alum:CpG, saponin or MF59 activation of elder immunity131.Impaired elder immune activation from BNT162b2 identifies an inherent difference within this population. Multiple approaches may amplify elder immunogenicity, including: (a) dose-escalationin vitro that is replete with age-specific cellular and soluble factors that could reflect or predict vaccine responses in vivo132, (b) use of three complementary proteomic approaches  to gain a comprehensive view of the impact of BNT162b2 on the WBA proteome, and (c) validation of findings using aged vs adult mice in vivo. The use of human in vitro assays is notable as such approaches enable species- and age-specific modeling with individuals serving as both control and test conditions permitting paired analyses of new and established/licensed vaccine formulations, thereby accelerating and de-risking vaccine discovery and development40.Our study features multiple strengths, including (a) use of a human WBA 136, but does not capture the gradient of immune impairments seen in >80Y135, or >100Y137, (b) age-dependent differences in vaccine uptake resulted in differential vaccination coverage between groups that could not be controlled for, (c) the in vitro WBA assay lacks fluid flow and interaction with other tissues  and may not completely reflect immune responses in vivo, (d) the WBA did not define the cellular origin of the TH1 polarizing cytokine and chemokines measured, and (e) immune proteins have redundancies105 that are progressively lost during immunosenescence138. We reduced the risk of over-interpreting the biologic significance of single analytes by evaluating impaired elder induction of both individual TH1 polarizing analytes, IFNγ and CXCL1098  grouping individuals into adult (18–50Y) and elder (≥60Y), which is commonCXCL1098 , and theCXCL1098 . We furtse model .in vitro, including IL-6, CXCL8, TNFα and IFNγ production  and oncology139. Identifying critically divergent functions between age groups is an essential step for optimizing the next generation of precision mRNA vaccines to overcome immunosenescence. Accordingly, translational research is needed to enhance elder immune responses including expanded adjuvantation efforts to enhance TH1 polarization, durable immunogenicity, and associated protection22.In summary, supernatants from adult and elder WBA  demonstroduction . Categorn elders , a potenrization . Our stuSupplement 1


Lactobacillus spp. are at reduced risk of acquiring sexually transmitted infections including HIV, but the biological mechanisms involved remain poorly defined. Here, we performed metaproteomics on vaginal swab samples from young South African women (n = 113) and transcriptomics analysis of cervicovaginal epithelial cell cultures to examine the ability of lactic acid, a metabolite produced by cervicovaginal lactobacilli, to modulate genital epithelial barrier function.Women with a cervicovaginal microbiota dominated by Lactobacillus-depleted microbiota, women dominated by vaginal lactobacilli exhibit higher abundance of bacterial lactate dehydrogenase, a key enzyme responsible for lactic acid production, which is independently associated with an increased abundance of epithelial barrier proteins. Physiological concentrations of lactic acid enhance epithelial cell culture barrier integrity and increase intercellular junctional molecule expression.Compared to women with These findings reveal a novel ability of vaginal lactic acid to enhance genital epithelial barrier integrity that may help prevent invasion by sexually transmitted pathogens.Video abstractThe online version contains supplementary material available at 10.1186/s40168-022-01337-5. Lactobacillus spp. is considered optimal . Figure S2. Multidimensional scaling (MDS) plot of genes differentially expressed by ectocervical epithelial cells treated with 0.3% L-lactic acid (L-LA) pH 3.9 or D-lactic acid (D-LA) pH 3.9 (yellow and blue respectively), HCl pH 3.9 (green) or untreated cells (red). Figure S3. Primary cervicovaginal epithelial cells were apically treated with 0.3% L-lactic acid pH , or low pH media alone  for 1 h and gene expression of tight junction factors claudin-1 (CLDN1) and claudin-4 (CLDN4) were assessed 4 h post-treatment by qRT-PCR.Additional file 2: Supplementary Table 1. Proteins with significantly different abundance in women with high as compared to low L-LDH and D-LDH abundance*. Supplementary Table 2. Barrier-related proteins with significantly different abundance in women with high as compared to low L-LDH abundance*. Supplementary Table 3. Genes differentially expressed by L-LA, D-LA and HCL treatment of Ect cells*. Supplementary Table 4. Gene ontology pathways significantly enriched by L-LA and D-LA treatment of Ect cells. Supplementary Table 5. Tight junction genes differentially expressed by L-LA, D-LA and HCL treatment of Ect cells*. Supplementary Table 6. Tight junction genes differentially expressed by treatment of VK2 cells with bacterial culture supernatants*.


Caenorhabditis elegans by engaging autophagy and the mitochondrial intrinsic apoptosis pathway. The anti-apoptotic protein CED-9 is the downstream effector that mediates CISD-1-dependent effects on proteostasis, neuronal integrity and lifespan. Moreover, intracellular iron abundance is critical for CISD-1 function, since mild iron supplementation is sufficient to decelerate ageing and partly ameliorate the disturbed mitochondrial bioenergetics and proteostasis of CISD-1 deficient animals. Our findings reveal that CISD-1 serves as a mechanistic link between autophagy and the apoptotic pathway in mitochondria to differentially modulate organismal proteostasis and ageing, and suggest novel approaches which could facilitate the treatment of Wolfram Syndrome or related diseases.Mitochondria preserve metabolic homeostasis and integrate stress signals, to trigger cytoprotective, or cell death pathways. Mitochondrial homeostasis and function decline with age. The mechanisms underlying the deterioration of mitochondrial homeostasis during ageing, or in age-associated pathologies, remain unclear. Here, we show that CISD-1, a mitochondrial iron-sulfur cluster binding protein, implicated in the pathogenesis of Wolfram neurodegenerative syndrome type 2, modulates longevity in the nematode  This is a neuronal RNAi sensitive strain which expresses the human alpha-synuclein (α-Syn) protein in dopaminergic neurons. We observed that nematodes subjected to cisd-1(RNAi) display accelerated dopaminergic neuronal loss compared to their control counterparts -associated segment of polyglutamine (Q40) repeats, fused to YFP [punc-54Q40::YFP] respectively, in their body wall muscles. We found that cellular proteostasis is impaired upon cisd-1 silencing, since the number of α-Syn::GFP and Q40::YFP aggregates are significantly elevated in cisd-1(RNAi)-treated animals, compared to the respective controls , and mitochondrial ROS, while mitochondrial mass remains unaffected  mutants (mev-1 encodes the succinate dehydrogenase cytochrome b560 subunit in complex II of ETC) shortens lifespan similarly to its effect on wt animals   and clk-1(e2519)  mutant animals upon cisd-1(RNAi) remains unaffected , a stress defense mechanism, required for ETC perturbation-mediated longevity  transcriptional reporter, under both normal and cox-5B(RNAi) conditions -treated animal lifespan. To further investigate whether CISD-1 is involved in other stress response mechanisms, we measured the expression of hsp-4 and hsp-16.2 genes by using the transcriptional reporters SJ4005 [phsp-4GFP] , while HSP-16.2 is the ortholog for human crystalline alpha B protein, which is transcriptionally induced when the Heat-Shock response (HSR) pathway is activated. Silencing of cisd-1 does not significantly alter UPRER or HSR under normal conditions. Interestingly, upon UPRER-inducing conditions (tunicamycin treatment) the cisd-1(RNAi) treated worms display increased hsp-4 activity compared to the controls. Given that CISD-1 deficient animals display impaired proteostasis  mutants shortens lifespan similarly to wt nematodes  does not further shorten the lifespan of gain-of-function ced-9(n1950) mutants  and ced-3(n717) mutants . Remarkably, in a similar manner, cisd-1(RNAi) does not lead to lifespan shortening in ced-13(sv32) mutants  loss-of-function mutants upon cisd-1 silencing. We found that, loss of CED-9 function (in ced-9(n2812) mutants) fully abrogates the detrimental consequences of cisd-1 downregulation on survival. Notably, CED-9/CISD-1 double-deficient animals are long-lived, compared to wt  and bec-1 (the nematode ATG6/ BECN1 ortholog) fully curtails lifespan extension triggered by loss of both CISD-1 and CED-9  or bec-1(RNAi) has an effect on nematode’s lifespan, we cannot exclude the possibility that CISD-1 and CED-9 act independently of the autophagy pathway. In the presence of CED-9 , downregulation of unc-51 further shortens the lifespan of cisd-1(RNAi)-treated animals -treated, wt animals  display significantly lower number of GFP::LGG-1 positive puncta, as expected  mutant animals  mutants, albeit to a lesser extent compared to that of double deficient animals. To examine whether autophagosomes accumulate due to a blockade in autophagic flux, we monitored the degradation of the autophagic substrate SQST-1, the nematode ortholog of p62/sequestosome , AM141: rmIs133 [punc-54Q40::YFP], TK22: mev-1(kn1) III, MQ887: isp-1(qm150) IV, CB4876: clk-1(e2519), MT4770: ced-9(n1950) III, MT7686: ced-9(n2812)/ qC1 [dpy-19(e1259) glp-1(q339)] III, RB1305: egl-1(ok1418) V, MT2547: ced-4(n1162) III, MT1522: ced-3(n717) IV, MD792: ced-13(sv32) X, DA2123: adIs2122 [plgg-1GFP::LGG-1 + rol-6(su1006)], MAH14: daf-2(e1370) III; adIs2122 [plgg-1GFP::LGG-1 + rol-6(su1006)], HZ589: him-5(e1490) V; bpIs151[psqst-1SQST-1::GFP + unc-76(+)], GA631: wuIs177 , SJ4103: zcls14 [pmyo-3GFP(mit)], SJ4143: zcIs17 [pges-1GFP(mit)], IR28: N2;Is[pmyo-3MYO-3::GFP], SJ4058: zcIs9 [phsp-60GFP + lin-15(+)], SJ4005: zcIs4 [phsp-4GFP], CL2070: dvIs70 [phsp-16.2GFP + rol-6(su1006)], CU1546: smIs34 [pced-1CED-1::GFP + rol-6(su1006)]. The initial null cisd-1(tm4993) mutant strain was obtained from the National BioResource Project (NBRP)  and UA49: baInl2 [punc-54α-Syn::GFP + rol-6(su1006)] were generously provided by Guy Cardwell. The following strains were generated in the current study: IR1571: N2;Ex; [rol-6(su1006)] transgenic animals, we used DNA microinjection in the syncytium region of C. elegans germlines. Wild-type (N2) young day-1 adult animals were injected with a mix containing the reporter plasmid [pCEOP2540CISD-1::GFP::unc-54-3’UTR] and the selection marker plasmid pRF4[rol-6(su1006)], at a final concentration of 25 ng/μl each.To generate the IR1571: N2;t = 1 (days of adulthood). Animals were transferred to freshly made RNAi plates every 2 days until the 12th day of adulthood and every 3 days until the end of the experiment. The animals were examined almost every day and scored as alive or dead (if they do not respond to touch). Nematodes with internally hatched eggs, protruding vulvas, or those which accidentally die during handling or due to dehydration in the edges of the plate, were scored as censored. Each lifespan experiment was repeated at least twice, with one representative experiment shown in the corresponding figures. Statistical analysis was performed using the Log-rank (Mantel–Cox) test of the Prism software package. The statistical analyses of each survival assay are summarized in Supplementary Table Lifespan assays were performed at 20 °C. Synchronous animal populations were generated by bleaching (hypochlorite treatment) a large amount of gravid adults of the desired strain grown on OP50-seeded NGM plates. Eggs were placed immediately on freshly made RNAi plates, seeded with IPTG-supplemented (2 mM) HT115 bacteria, which had already been transformed with either the empty RNAi vector pL4440 or the indicated RNAi construct. The progeny was grown through the L4 larval stage and then transferred to fresh plates in groups of 20–25 worms per plate for a total of 150–200 individuals per condition. The day after transferring L4 was used as Neuronal defects in mechanosensory neurons were assessed by using animals of the SK4005 strain, which had been growing on RNAi for two generations . For each condition 30–40 animals at the fourth day of their adulthood were selected for imaging. The mechanosensory neurons were classified into three categories depending on the axonal morphology: normal phenotype, intermediate phenotype (thin and beaded axons) and severe phenotype . For the detection of neuronal defects in dopaminergic neurons as a result of α-Syn aggregation, we used day-3 animals of the dopaminergic RNAi sensitive UA196 strain and determined the percentage of animals which had intact dopaminergic neurons. Each analysis was repeated at least three times. Independent experiments are represented as mean ± SEM in a single graph.For the analysis of α-Syn protein aggregation in body wall muscle cells, we used animals of the UA49 strain. 20–30 animals (second generation progeny grown on RNAi) at the fourth day of their adulthood were used for imaging. We imaged only the head region, to consistently measure the same body wall muscles in each worm. For the analysis of poly-glutamine aggregation in body wall muscle cells, we used animals of the AM141 strain. 20–30 day-1 adult animals (second generation progeny grown on RNAi), were used for imaging . Q40::YFP aggregates were measured in the whole animal body. In both cases, the animals were collected, anaesthetized with 20 mM tetramisole and mounted on agarose pads for microscopic observation. α-Syn::GFP puncta in the head region, as well as Q40::YFP puncta in the whole animal body, were quantified by using the “Analyze particles” tool of the ImageJ software . Each andaf-2(RNAi)- and unc-51(RNAi)-treated animals were used as positive and negative controls for increased autophagosome number, respectively. We also used the MAH14 strain as an additional control for induced autophagy and similarly measured the number of GFP::LGG-1 positive puncta in hypodermal seam cells. The sequestration of autophagic substrates as an indicator for autophagic flux, was monitored by using the SQST-1::GFP reporter strain (HZ589), as previously described , which were RNAi treated from hatching, were used for the determination of germline apoptosis. 25–30 animals per condition were mounted in microscope slides as previously described, and the number of CED-1 GFP positive engulfed cell corpses were scored by observing each gonad under a fluorescence microscope.Day-1 adult animals of the CU1546 strain [pThrashing behavior is typically assessed to identify differences in nematodes’ motility. Day-3 adult wt animals, which were RNAi treated from hatching, were individually placed in a 20 μl drop of M9 buffer on a microscope slide and observed under a stereoscope. The number of thrashes/body bends for each individual worm was counted every time the worm moved in the opposite direction of the previous body bend. The duration of each measurement was 15 s. For each condition, 20 individual animals were used and each one was measured at least three independent times.Worms were immobilized in a 20 mM tetramisole drop on microscopic slides, sealed with coverslips and analyzed with a Zeiss AxioImager Z2 epifluorescence microscope, or EVOS FV Auto 2 Imaging system (Thermofisher Scientific) or Zeiss LSM 710 multiphoton confocal microscope. Quantification of the mean pixel intensity or particle analyses were performed by using the ImageJ software .https://www.graphpad.com). Data are reported as the mean values ± standard deviation (SD), unless otherwise stated. For statistical analyses, p values were calculated by unpaired Student’s t-test and one-way ANOVA with Tukey’s multiple comparisons test. The significance was determined by the p-values: *(p < 0.05), **(p < 0.01), ***(p < 0.001), ****(p < 0.0001) and n.s. = not significant (p > 0.1).Statistical analyses were carried out using the Prism software package (version 8; GraphPad Software; Further information on research design is available in the Reporting Summary checklistSupplementary materialSupplementary materialSupplementary Figure 1Supplementary Figure 2Supplementary Figure 3Supplementary Figure 4Supplementary Figure 5Supplementary Figure 6Supplementary Figure 7