Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in  C. elegans daf-18 gene encodes the worm version of PTEN. In the C. elegans embryo, the two primordial germ cells (Z2 and Z3) are born, and they will eventually form the entire germ line. These two cells are quiescent in the embryo. They do not divide until after the L1 larva hatches and starts feeding. That is, if the L1 larva hatches in the absence of food, these cells will remain quiescent until the worm feeds. Previous studies showed that in worms that have lost daf-18 function, Z2 and Z3 inappropriately begin dividing in the L1 larva even in the absence of food. We found that daf-18 normally prevents this inappropriate division when daf-18 function is provided from the mother or from within the larva, and when it is provided from the germ cells or from the somatic cells. We also found that without daf-18, certain germline genes are inappropriately expressed. Finally, we found that one of the mechanisms known to regulate cell division in this context does not similarly regulate germline gene activity.PTEN is the second-most commonly lost tumor suppressor in human cancers. The  Depending on the cell type, arrested cells adopt a variety of altered metabolic states while they await an activation cue, usually a growth factor signal ) I; daf-18(ok480) IV, GC1537vbh-1(na110[GFP::vbh-1]) I; naSi2 II, GC1576vbh-1(na110[GFP::vbh-1]) I; naSi2 II; daf-18(ok480) IV, GC1456naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx261 (daf-18P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1460naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx264 (rab-3P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1464naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx268 (nlp-40P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1465naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx269 (dpy-7P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1485naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx275 (elt-2P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1484naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; naEx274 (ehn-3P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1479naSi22 (mex-5P::daf-18::nos-2 3’UTR::SL2::GFPo::PH::tbb-2 3’UTR + unc-119(+)) II; unc-119(ed3) III?; daf-18(ok480) IV, GC1483glh-1(sam24[glh-1::gfp::3xFLAG]) I; naSi22 (mex-5P::daf-18::nos-2 3’UTR::SL2::GFPo::PH::tbb-2 3’UTR) II; unc-119(ed3) III?; daf-18(ok480) IV, GC1627glh-1(sam24[glh-1::gfp::3xFLAG]) I; daf-18; naEx261 (daf-18P::daf-18::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR), GC1621ppw-1 (pk2505) I; naSi2 II, GC1390rrf-1 (pk1417) I; naSi2 II, GC1638naSi2 II; unc-119(ed3) III?; otIs45 (unc-119::GFP) V, GC1639naSi2 II; unc-119(ed3) III?; daf-18(ok480) IV; otIs45 (unc-119::GFP) V.Strains generated for this study are indicated below. All strains used and details on strain construction information are listed in Transgenic strains were generated by three different methods, as indicated below see :naSi2 II; daf-18(ok480) IV. All arrays used pJB134 (flp-17P::dsred) as a co-injection marker ), glh-1 (sam24 [glh-1::GFP]), naSi2 [mex-5P::mCherry], and otIs45 [unc-119P::GFP]) were generated by heat shock, and populations were expanded and maintained by crossing to hermaphrodites of the same strain. 2–4 crosses were performed per strain, in both the daf-18(+) and daf-18 mutant backgrounds, with 25–30 males and 8–10 hermaphrodites per cross. Hermaphrodites carried an alternate-color PGC marker . P0 animals mated for 24 hours on NGM plates with small lawns of OP50, and then mated hermaphrodites were treated with sodium hypochlorite on unseeded NGM plates to isolate clean embryos .Males carrying homozygous fluorescently-tagged genes daf-18 mutants . For “fed” wild type we included an additional control of starved counterparts (“starved”) to ensure that feeding-induced transgene expression is a function of feeding and not simply a function of time. We prepared and imaged the “fed” and “starved” worms in parallel as follows. After clean mixed-stage embryos (from the sodium hypochlorite treatment of mothers) were allowed to hatch 12–19 hours on unseeded NGM plates, resulting starved L1 larvae were then moved (by mouth pipetting) to OP50 bacterial lawns on NGM plates (for “fed”) or left on unseeded plates (for “starved” controls) for 5 hours before analysis.The resulting embryos were handled in two different ways. For “short” starvation, comparing wild type and nts Figs , L1 larvlet-363 RNAi efficacy was confirmed by suppression of daf-18 PGC divisions in starved L1 progeny, and 100% larval (L3) arrest in progeny from crosses that had continued feeding on RNAi plates  was confirmed only by 100% larval arrest in RNAi-fed progeny.For zygotic gene expression experiments utilizing RNAi, methods were the same as described above, except: crosses were performed on standard RNAi plates (IPTG/Amp) seeded with small lawns of RNAi bacteria (HT115 carrying plasmids for dsRNA production) ,92. Hermi plates . RNAi efImaging was performed with a Zeiss Z1 AxioImager equipped with an AxioCamMRm digital camera, ApoTome for optical sectioning, a plan apo 63X/1.4 oil DIC objective, and Zeiss filters for DAPI, CY3, and eGFP.daf-18 GFP transgenes in L1 larvae starved for 1–3 days was documented by taking epifluorescence image stacks of animals mounted on 2% agarose pads, paralyzed with 1mM levamisole. Images are shown as Z-projections (Tissue expression of jections .daf-18), exposure times were determined by wild-type stained animals and held constant for all samples and replicate experiments. For anti-pgl-1 co-staining, anti-GFP of an encoded germline::GFP, or DIC), in the image slice where it (based on surrounding P granules (GLH-1::GFP)) was most in focus. The nearest somatic cell/somatic gonad precursor (SGP) mean fluorescence. In addition, background within the worm but outside of nuclei, was measured in the same image. PGC and SGP fluorescence values were corrected for background fluorescence, and PGC/SGP ratio was calculated. When two PGC/somatic values could be calculated for the same animal (cases where both PGCs and surrounding somatic cells showed staining), they were measured separately with the cognate SGP and the ratios were averaged.Fluorescence image stacks were taken using Apotome for sectioning . For naSi2 [PGC::mCherry]). GFP, mCherry, and DIC channels were collected for each animal, within 1 hour of mounting. Exposure times were held constant for each individual experiment but varied based on zygotic transgene. When possible, both PGCs were imaged for each animal and values were averaged, resulting in a single fluorescence value for each animal. If one PGC was obscured , it was omitted, and only one PGC was imaged . This allowed identification of PGC nuclei by manual thresholding of each image in the red channel (PGC::mCherry), and automatic measurement of mean GFP channel fluorescence intensity of a 1μm-wide ring surrounding the nucleus.naSi2 (PGC::mCherry) expression, regions of interest were hand drawn around PGC nuclei and mean fluorescence measured.For zygotic unc-119P::GFP head fluorescence measurements, ellipsoid regions of interest were drawn over a single neuron in the head  and a single tail neuron near the rectum, and mean fluorescence was measured. The lack of any visible unc-119P::GFP in PGCs (even upon long exposure times) was noted in all animals that were imaged and measured for head fluorescence.For For all zygotic transgene expression analyses, background fluorescence was measured for each animal in a region within the worm but outside of fluorescent tissues, and was subtracted from zygotic fluorescence-of-interest mean values.RNA was isolated using TRIzol Reagent (Thermo-Fisher Scientific) following the manufacturer’s protocol with exceptions noted below. The procedure was scaled down in a linear fashion, using only 100 uL Trizol. 5 ug linear polyacrylamide (Sigma-Aldrich) was included as a neutral carrier for RNA precipitation. RNA was eluted in nuclease-free water and incubated at 55°C for approximately four minutes to resuspend the pellet. 140 ng of total RNA was used for each library preparation. NEBNext Ultra II RNA Library Prep kit (New England Biolabs) was used to perform poly-A selection and prepare libraries for sequencing. The final libraries were enriched with 11 cycles of PCR. Libraries were then sequenced using Illumina NovaSeq 6000 to obtain 50 bp paired-end reads.C. elegans genome was used to map sequencing reads (file name c_elegans.PRJNA13758.WS273.genomic.fa downloaded from WormBase). Bowtie version 1.2.2 [2 mean normalized CPM values for the genes included in differential expression analysis. PCA separates libraries based on strain 979 genes enriched in sorted, labeled embryonic primordial germ cells, Z2 and Z3, over all embryonic cells 306 genes 5x enriched in the L2 germline , data ac4980 genes expressed in embryonic primordial germ cells, Z2/Z3, for which all three “pseudotime” bins are above 0 TPM.daf-18 mutant compared to wild-type, the cumulative distribution of log2 fold changes for each group of genes in the GC1459 / GC1171 comparison was plotted along with the cumulative distribution of log2 fold changes of all 12,592 detected genes in the GC1459 / GC1171 comparison. Differences between the two cumulative distributions were assessed using the Kolmogorov-Smirnov test. In a complementary approach, the overlap between each gene group of interest and differentially expressed genes that were up- or down-regulated in the GC1459 / GC1171 comparison (FDR < 0.05) was assessed, and the hypergeometric test was used to determine significant overlap. Gene groups used in this analysis can be found in To determine whether gene groups of interest ,45,58,65S1 FigPGC::mCherry, naSi2) animals, and 49 daf-18 mutant animals .The number of L1 larvae hatched from embryos isolated at the 2–4 cell stage was monitored hourly. 4 replicate experiments , containing a total of 40 WT ((TIF)Click here for additional data file.S2 Figdaf-18(ok480) mutants, and all carry PGC::mCherry (naSi2), except for panel (B). Scale bars represent 10μm, white arrows indicate GFP expression in expected tissues-of-interest, pink arrows indicate PGCs, and white stars indicate a transgenic co-injection marker (flp-17P::dsRed) which was used to identify all extrachromosomal array-bearing animals. All daf-18(+) constructs (various promoters driving daf-18(+)::SL2::GFP::H2B) are carried as extrachromosomal arrays (with nuclear GFP for cell identification), except for germline::daf-18(+) (in panel (B)), which is a single copy insertion. Arrays carry the daf-18 3’UTR on the daf-18 rescuing portion, and the unc-54 3’UTR for the GFP reporter portion to allow broad somatic expression [rab-3P::daf-18(+)::daf-18 3’UTR::SL2::GFP::H2B::unc-54 3’UTR). (B) The germline:: daf-18(+) construct (mex-5P::SV40 NLS::daf-18 coding region::nos-2 3’UTR::SL2::GFPo::PH::tbb-2 3’UTR) has several features. First, the GFP is fused to a PH domain thereby targeting the GFP to the cell membrane. Second, there are two different 3’ UTRs, one following the daf-18 portion and another following the GFP. Specifically, the nos-2 3’UTR follows the daf-18 rescuing portion , which restricts expression to the PGCs . The tbb-2 3’UTR was used for the GFP portion, which allows somewhat broader expression as is visible in somatic tissues. Nevertheless, daf-18(+) expression should be limited to PGCs, as evidenced by the PGC::mCherry reporter naSi2 which expresses mCherry from the exact same mex-5 promoter and the same nos-2 3’ sequence. Last, an NLS is fused in frame with DAF-18; it is possible that it enhances DAF-18 nuclear activity. (H) Approximate embryonic transcript levels [(A-G) All animals are pression  Click here for additional data file.S3 Figdaf-18 dsRNA (feeding from P0 to F2) were assessed for number of PGCs after 2 days of starvation as L1s. All strains carry naSi2 (PGC::mCherry). n values display the total number of L1 larvae examined for each genotype across three biological replicates in which all three strains were subject to RNAi in parallel in each replicate. Two-sided Fisher’s exact tests were performed to compare within genotype and statistical significance is displayed .F3 progeny of animals fed HT115 bacteria expressing control (Ctrl), empty vector L4440, or (TIF)Click here for additional data file.S4 Figdaf-18(ok480) mutant L1s, starved 6–8 hours after hatching (synchronized within 2 hours of hatching and imaged 6 hours later). PGCs were identified with anti-PGL-1 antibody (OIC1D4). Each dot represents a single PGC/somatic cell pair . (B) Antibody ab8580 (H3K4me3) was used to stain WT and daf-18(ok480) mutant L1s hatched and starved overnight (~16h). PGCs were identified with encoded glh-1::GFP. Each dot represents one value per worm . Statistical significance determined by two-tailed T-test. **p<0.01, ****p<0.0001.. Mean staining fluorescence was measured for each PGC nucleus and its nearest somatic cell nucleus (from single image slices), and is displayed in the graph as a ratio. (A) Antibody ab32356 (H3K4me2) was used to stain wild type (WT) and (TIF)Click here for additional data file.S5 Figdaf-18(ok480) mutant 3-fold embryos. PGCs were identified with glh-1::GFP. Mean fluorescence was measured for each PGC and its nearest somatic cell (from single image slices), and is displayed in the graph as a ratio. Each dot is a single PGC/somatic cell pair. Statistical significance determined by two-tailed T-test. ***p<0.001. (B) and (C) Zygotic expression of GFP::VBH-1 was examined by epifluorescence  are marked with asterisks. Scale bars represent 10μm.(A) Antibody ab32356 (anti-H3K4me2) was used to stain wild type (WT) and ce as in  in embry(TIF)Click here for additional data file.S6 Figgermline::daf-18::SL2::GFP::PH) (top panels) or intestinal cells (anti-GFP in L1s carrying intestine::daf-18::SL2::GFP::H2B) (bottom panels). PGCs were identified by anti-GFP membrane marker (top) or DIC (bottom). Transgenic animals carrying daf-18(+) rescuing arrays were identified by anti-GFP. (B) “daf-18 control*” represents non-transgenic siblings of daf-18; intestine::daf-18(+) animals in the middle column (black dots), and a mix of transgenic and non-transgenic daf-18; neurons::daf-18(+) (blue dots). Since these are F3 progeny of P0 mothers that were selected (transgene-positive), possible maternal contributions of transgenes are unknown.(C) Magenta marks germ cell P granules  to identify PGCs. PGCs were also identified by DIC (top panels). Transgenic animals in daf-18; daf-18 genomic (+) were identified by anti-dsRed, which stains an encoded flp-17P::dsRed coinjection marker, expressed in a pair of head neurons. (D) “daf-18 control*” represents non-transgenic siblings of daf-18; daf-18 genomic(+) animals in the middle column. Since these are F3 progeny of P0 mothers that were selected (transgene-positive), possible maternal contributions of transgenes are unknown.Antibodies ab8580 (anti-H3K4me3)  and CMA303 (anti-H3K4me2)  were used to stain L1 larvae starved for up to 24 hours. Tricolor images (left panels) show DNA (DAPI/blue), histone mark immunofluorescence (green), and either a PGC marker (see below) or transgenic animal marker (magenta). Maximum intensity projections of image stacks are shown, except for (A) (top panels), which show a single slice. Dashed circle indicates one PGC in each image, and dashed smaller ellipse indicates its nearest somatic cell (likely its SGP). White arrowheads point to PGCs, and green arrowheads point to neighboring somatic cells. Scale bars represent 10μm. Mean staining fluorescence was quantified for each PGC nucleus and its nearest somatic cell nucleus (from single image slices), and is displayed in the correlated graph as a ratio. Each dot in the graph represents one animal . Dot colors represent data gathered on different days / in different experiments. Triangles represent animals shown in the images, and open circles represent animals with 3 or more PGCs. Statistical significance determined by one-way ANOVA. **p<0.01, ***p<0.001. (A) Magenta marks germ cell membranes (anti-GFP in L1 larvae carrying (TIF)Click here for additional data file.S7 FignaSi2 PGC::mCherry or sam24 glh-1::GFP) were allowed to hatch and starve up to 24h, with or without 10ug/mL α-amanitin, before assessing PGC numbers (see Embryos (from a bleach preparation) carrying a PGC marker (bers see  and dead(TIF)Click here for additional data file.S8 Figdaf-18 mutants than starved wild type (WT). Dashed ellipses show regions measured for mean fluorescence. As in daf-18 mutant PGCs than starved WT PGCs. L1s were imaged up to 19 hours after clean embryo preparation. Arrows point to zygotic PGC::mCherry expression in PGCs, and asterisks mark auto-fluorescent gut granules. Nuclear PGC::mCherry fluorescence was measured by hand-drawing regions of interest around PGC nuclei. One experimental replicate is graphed, and 2 others showed similar results. Statistical significance determined by one-way ANOVA with Tukey’s multiple comparisons test. (C) Zygotic unc-119P::GFP is not expressed in the PGCs of starved daf-18 mutants. However, it is expressed at a slightly higher level in neurons of starved daf-18 mutants. L1s were imaged up to 18 hours after clean embryo preparation. Dashed circles indicate approximate neuronal regions measured . Graph shows the pooled results of 2 experiments. Statistical significance determined by two-tailed T-test. *p<0.05.(A-C) Images show epifluorescence. Mean fluorescence intensity (AU = Arbitrary Units) values are plotted to the right of each set of images. Each dot represents one animal. Non-cross progeny were identified by the lack of somatic GFP ((A) and (C)) or lack of PGC::mCherry (B), which are all visible when present, even in starved animals. Mean +/- SEM. All scale bars represent 10μm. (A) Zygotic GFP::VBH-1 expression in the soma (head neurons) is not expressed more highly in starved (TIF)Click here for additional data file.S9 Figdaf-18(ok480)) starved L1s in RNA Sequencing experiment, highlighting low variance within each genotype and substantial variance between genotypes. (B) Correlation matrix of GC1171 (wild type) and GC1459 (daf-18(ok480)) showing that replicates of the same genotype were highly correlated.(A) Principal component analysis (PCA) of GC1171 (wild type) and GC1459 ((TIF)Click here for additional data file.S10 Fig(A) Venn diagram showing the overlap of all four germline gene sets analyzed ,45,58,65(TIF)Click here for additional data file.S11 FigIndividual Z-scores of these 40 genes (those detected in this dataset out of 42 identified in (TIF)Click here for additional data file.S12 Figdaf-18 , and tissues that may not express daf-18  [Cumulative distribution function (CDF) plots of transcript sets of interest (red line) versus the background set of all 12,592 transcripts detected (black line). p-values determined by Kolmogorov-Smirnov (KS) test are shown. Numbers of transcripts from published gene sets detected in our experiment are shown in graph titles (parentheses). (A-F) CDF plots for lists of transcripts from tissues of interest known to express l gland) ,45. Tiss(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Correction to: BMC Microbiol 21, 330 (2021)https://doi.org/10.1186/s12866-021-02389-7Following the publication of the original paper , the autE. coli isolate included in the current study were deposited at the National Center for Biotechnology Information (NCBI) with the following GenBank accession numbers. Data will be made available upon manuscript publication.The whole-genome sequencing datasets generated and analyzed for each Accession numberSCBcol-1 JAHTMT000000000SCBcol-2 JAHTMU000000000SCBcol-3 JAHUTW000000000SCBcol-4 JAHUTX000000000SCBcol-5 JAHUTY000000000SCBcol-6 JAHUTZ000000000SCB5 JAHUUA000000000SCB12 JMQO00000000SCB29 JAHUUB000000000SCB34 JMKH00000000SCB58 JAHUUC000000000RS218 JWZW00000000.1The original article has been corrected.


Retrospective observational studies have reported that statins improve clinical outcomes in patients previously treated with programmed cell death protein 1 (PD-1)-targeting monoclonal antibodies for malignant pleural mesothelioma (MPM) and advanced non-small cell lung cancer (NSCLC). In multiple mouse cancer models, de novo synthesis of mevalonate and cholesterol inhibitors was found to synergize with anti-PD-1 antibody therapy. In the present study, we investigated whether statins affect programmed death-ligand 1 (PD-L1) expression in cancer cells. Four statins, namely simvastatin, atorvastatin, lovastatin, and fluvastatin, decreased PD-L1 expression in melanoma and lung cancer cells. In addition, we found that AKT and β-catenin signaling involved PD-L1 suppression by statins. Our cellular and molecular studies provide inspiring evidence for extending the clinical evaluation of statins for use in combination with immune checkpoint inhibitor-based cancer therapy. An increasing number of studies have demonstrated that abnormal de novo synthesis of mevalonate and cholesterol are closely associated with tumor development, growth, and progression . CholestCD274 gene and a major co-inhibitory checkpoint molecule [CD274 through genomic amplification and translocation was identified in primary mediastinal large B-cell lymphoma (PMBCL), NSCLC, and gastric adenocarcinoma, consequently leading to immune escape [CD274 expression, respectively, via epigenetic regulation [CD274 promoter and activate CD274 gene expression [CD274 expression in malignant melanoma, NSCLC, and gallbladder cancer [T-cell-mediated immunity primarily regulates the elimination of pathogens and abnormally transformed tumor cells to maintain homeostasis in the body . Programmolecule . PD-L1 pmolecule . Multiplmolecule . Considemolecule . For insmolecule . The expmolecule . Upregule escape ,29. Furtgulation ,31. An ipression . Hyperacr cancer ,33,34. Ir cancer . TherefoLung , breast , colorectal (HCT116), hepatocellular (SK-HEP-1), and cervical (HeLa) cancer cells were obtained from the Korean Cell Line Bank . Melanoma cell lines (A375 and A2058) were obtained from the American Type Culture Collection . Cells were cultured in 10% fetal bovine serum (FBS) as well as penicillin/streptomycin-contained Dulbecco’s modified Eagle’s medium (DMEM) and Roswell Park Memorial Institute (RPMI) 1640 at both 20% O2 and 5% CO2. Cell culture medium, FBS, and antibiotics were purchased from HyClone Thermo Scientific . Simvastatin (S6196), Lovastatin (438185), Lonafarnib (SML1457), and GGTI-2133 (G5294) were purchased from Sigma Aldrich . Atorvastatin (S5715), Fluvastatin (S2061), and MK-2206 (S1078) were obtained from Selleckchem . All chemicals stock solution was dissolved in dimethyl sulfoxide (DMSO).To analyze protein expression, cells were washed by cold phosphate-buffered saline (PBS) and lysed by using 1% NP-40 lysis buffer containing 150 mM NaCl, 5 mM ethylenediaminetetraacetic acid (EDTA), 50 mM Tris-HCl (pH7.4), protease inhibitor, and phosphatase inhibitor cocktail. Protein contents were normalized using the Bradford protein assay. Total proteins (30 μg) were separated following the molecular weight through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then proteins were transferred onto polyvinylidene difluoride (PVDF) membrane . Transferred proteins on the PVDF membrane were reacted with primary antibodies for 12 h at 4 °C and secondary antibodies for 1 h at room temperature. Proteins incubated with primary and secondary antibodies were washed by using TBST  to remove non-specific reactions between target proteins and antibodies. Target proteins’ expression, such as for PD-L1, β-actin, phospho-ERK1/2, ERK1/2, phospho-AKT-S473, AKT, phospho-β-catenin-S552, and β-catenin, were analyzed by using the Enhanced Chemiluminescence (ECL) Prime kit . For western blotting, antibodies against anti-PD-L1 (CST-29122), anti-β-actin (sc-47778), anti-phospho-ERK1/2 (CST-4370), anti-ERK1/2 (sc-94), anti-phospho-AKT-S473 (CST-4060), anti-AKT (CST-4691), anti-phospho-β-catenin-S552 (CST-9566), and anti-β-catenin (CST-8480) were purchased from Cell Signaling Technology  and Santa Cruz Biotechnology .36B4 gene and relative mRNA expression was calculated versus human 36B4 expression as previously described [Total RNA was isolated with TRIzol  and 2 µg of total RNA was used for cDNA synthesis using a high capacity cDNA reverse transcription kit . Purity of isolated total RNA was measured by using the ratio of absorbance at 260 nm and 280 nm (A260/A280), and the ratio of A260/A280 higher than 1.8 was considered to be of acceptable purity. To analyze the gene expression, quantitative PCR was performed using the SYBR Green PCR Master Mix  and LightCycler96 real-time PCR . Ct values were normalized to the human CD274 promoter were 5′-ATGTAGCTCGGGATGGGAAGT-3′ (forward) and 5′-TGTGTGTGTGTGTATGGGTGTA-3′ (reverse) as described previously [A chromatin immunoprecipitation (ChIP) assay was performed by using the EZ-ChIP assay kit . A2058 and Calu-1 cells were incubated with 10 μM of simvastatin or DMSO in the absence or presence of 25-hydroxycholesterol (25-HC) for 16 h, and then cells were fixed in 1% formaldehyde-contained DMEM for 15 min. The fixed cells were lysed in ChIP-lysis buffer containing sodium dodecyl sulfate (SDS), protease inhibitor cocktail, and PMSF, and then cell lysates were sonicated by using an ultrasonic homogenizer  for four cycles of 5 min . Samples were incubated with normal rabbit IgG or anti-β-catenin antibody for 16 h at 4 °C and then the target chromatin complex was obtained by using protein A and G agarose bead  pre-blocked with salmon sperm DNA . Samples were washed with buffer containing 150 mM NaCl , 500 mM NaCl , and 250 mM LiCl. Immunoprecipitated DNA was isolated by using a phenol:chloroform:isoamyl alcohol (25:24:1) as previously described  and theneviously .CCND1, BIRC5, ENC1, MMP7, AXIN2, BCL2, and TP53, in pravastatin, simvastatin, lovastatin, or fluvastatin-treated lung  and pancreatic (PANC1 and MiaPaCa2) cancer cells were obtained from publicly available microarray datasets (GSE47458 and GSE149566). Microarray datasets were downloaded from the Gene Expression Omnibus  and analyzed to compare mRNA levels of β-catenin target genes between vehicle control and statins-treated lung and pancreatic cancer cells. Data are represented as means ± standard error (SE). Statistical analyses were conducted using Student’s t-test and one-way ANOVA post hoc test. Statins-treated samples were compared to the vehicle control.β-catenin target genes’ expression, such as for t-test for two experimental comparisons and one-way ANOVA post hoc test for multiple comparisons. A p value of < 0.05 was considered statistically significant.Illustrative figures were generated by using GraphPad Prism version 5 . Gene expression and chromatin immunoprecipitation (ChIP) experiments were performed in triplicates. Data are represented as means ± standard error (SE) and means ± standard deviations (SD). Statistical analyses were conducted using Student’s CD274) levels in various cancer cells. Here, we found that PD-L1 encoding CD274 mRNA levels were predominantly expressed in lung cancer , breast cancer (SKBR3 and MDA-MB-231), and melanoma (A375 and A2058) -neutralizing monoclonal antibodies ,19, the d A2058) A. PD-L1 d A2058) B. SimilaCD274 mRNA-encoding PD-L1 protein expression was measured in melanoma (A2058) and lung cancer  cells. Decreased CD274 mRNA levels were observed following statins treatment  and measured PD-L1 protein expression in lung cancer and melanoma cells. reatment G. These CD274 mRNA levels in simvastatin and lovastatin-treated lung cancer and melanoma cells were reversed by 25-hydroxycholesterol (25-HC) treatment  and geranylgeranyltransferases (GGTase) on PD-L1 expression were evaluated in lung cancer and melanoma cells A. In thireatment D. These It is clear that oncogenic-signaling, including phosphoinositide 3-kinases (PI3K) and mitogen-activated protein kinase (MAPK), is attenuated by statins in multiple types of cancer cells ,52,53,54CD274 expression in response to hypoxia, inflammatory cytokines, the epidermal growth factor (EGF), and Wnt-signaling [CD274 expression and immune evasion in glioblastoma [Multiple types of transcription factors, such as hypoxia-inducible factor 1 alpha (HIF1α), nuclear factor-κB (NF-κB), MYC, the signal transducer and activator of transcription 3 (STAT3), and β-catenin, are known to increase ignaling . Du et ablastoma . ConsideCD274 expression at the transcriptional level [AXIN-2 expression, which is a well-known β-catenin target gene, in statin-treated lung cancer and melanoma cells. Consistent with the suppression of CD274 by statins, decreased AXIN-2 mRNA was observed following statins treatment  and melanoma (A2058) cells  cells B. To strer cells C. Similaer cells D,E. ThesWhen viewed as a comprehensive study, our observations could provide an understanding of the molecular mechanisms by which statins have beneficial effects for improving the therapeutic efficacy of immune checkpoint inhibitors targeting PD-1 and its ligand PD-L1. Furthermore, extended preclinical and clinical evaluations based on these findings could help determine whether statins could improve the anti-cancer efficacy of immune checkpoint inhibitors and may be useful to expand the clinical practice guidelines for the treatment of lung cancer and melanoma therapy.


TNFα and IFN-γ may synergize to induce cytokine-driven lethal hyperinflammation and immune exhaustion in COVID-19 illness.≥ 50 compared to baseline and sustained for 48 hours. Secondary endpoints included 28-day mortality, dynamic cytokine profiles (Human Cytokine 48-Plex Discovery Assay), secondary infections, duration of supplemental oxygen support and hospitalization.To assess TNFα-antagonist therapy, 18 hospitalized adults with hypoxic respiratory failure and COVID-19 pneumonia received single-dose infliximab-abda therapy 5mg/kg intravenously between April and December 2020. The primary endpoint was time to increase in oxygen saturation to fraction of inspired oxygen ratio (SpO2/FiO2) by Consort diagramHospitalized patients with SARS-COV2 infection and pneumonia that were referred to the infliximab-abda study team for evaluation.≥ 65 years age with pre-existing lung disease or multiple comorbidities were attributed to secondary lung infections. Mean plasma IP-10 levels declined sharply from 9183 pg/ml to 483 pg/ml at Day 3 and 146 pg/ml at Day 14/discharge. Significant declines in IFN-γ, TNFα, IL-27, IL-6 (baseline above 10pg/ml), CRP and ferritin were specifically observed at Day 3 whereas other cytokines were unaffected. Among 13 lymphopenic patients, six (46%) had resolution of lymphopenia by day 3, and 11 by day 14. CXCR3-ligand (IP-10 and CXCL-9) declines were strongly correlated among patients with lymphopenia reversal .Patients were predominantly in critical respiratory failure , male , above 60 years , race-ethnic minorities , lymphopenic , steroid-treated , with a median ferritin of 1953ng/ml. Sixteen patients (89%) met the primary endpoint within a median of 4 days, 15/18 (83%) recovered from respiratory failure, and 14/18 (78%) were discharged in a median of 8 days and were alive at 28-day follow-up. Deaths among three patients Demographics and clinical characteristicsDemographics, comorbidities, clinical features, inflammatory markers, and outcomes of 18 patients with COVID-19 respiratory failure treated with infliximab-abda between April and December 2020.Changes in oxygen support status following infliximab-abda treatmentColored bars indicate the maximal level of oxygen support for each individual following treatment with infliximab-abda. The status of the patient at last follow-up  is indicated. ECMO: extracorporeal membrane oxygenationControl of inflammatory markers and cytokines following infliximab therapyValues from individuals are connected with solid lines, with deceased individuals indicated in red. Statistics: n=18, paired ratio t-test compared to baseline; *: P<0.05, **: P<0.01, ***: P<0.001, ****: P<0.0001, n.s.: not significant.Consistent with a central role of TNFα, the clinical and cytokine data indicate that infliximab-abda may rapidly abrogate pathological inflammatory signaling to facilitate clinical recovery in severe and critical COVID-19. Randomized studies are formally evaluating infliximab therapy in this context. Funding: National Center for Advancing Translational SciencesAll Authors: No reported disclosures


Aging is associated with accumulation of various tryptophan degradation products that may having either bone anabolic or catabolic effects. In epidemiologic studies, elevated levels of 3-hydroxyanthranilic acid (3-HAA) are associated with a higher bone mineral density (BMD). We have previously shown that the C57BL/6 mouse loses bone mass with age. Thus, we hypothesized that administering 3-HAA via a daily intraperitoneal (IP) injection would result in preserved or increased BMD. In an IACUC-approved protocol, we injected 26-month-old C57BL/6 mice with either a low dose (0.5 mg) or high dose (5 mg) of 3-HAA IP five days a week for eight weeks. At the end of this time mice were sacrificed and body composition and bone mineral density measured by DigiMus. BMD was significantly lower in the high dose 3-HAA group: 0.0570 + 0.004 vs 0.0473 + 0.006 vs 0.0432 + 0.0075 gm/cm2, . 3-HAA had no significant impact on body composition . Thus, 3-HAA did not prevent bone loss in older mice but instead significantly worsened bone loss. 3-HAA is known to have both pro- and anti- oxidant effects depending on the environment. These data would suggest that at the higher concentrations 3-HAA is acting predominantly as a pro-oxidant molecule accelerating age-related bone loss.


Majority of SARS-CoV-2 epitopes identified have <67% amino acid sequence identity with endemic coronaviruses and are unlikely to elicit high avidity cross-reactive T cell responses. Four SARS-CoV-2 Spike reactive epitopes, including a DPB1*04:01 restricted epitope, with ≥67% amino acid sequence identity to endemic coronavirus were identified. SARS-CoV-2 T cell lines for three of these epitopes elicited cross-reactive T cell responses to endemic cold viruses. An endemic coronavirus Spike T cell line showed cross-reactivity to the fourth SARS-CoV-2 epitope. Three of the Spike cross-reactive epitopes were subdominant epitopes, while the DPB1*04:01 restricted epitope was a dominant epitope. Frequency analyses showed Spike cross-reactive T cells as detected by tetramers were present at relatively low frequency in unexposed people and only contributed a small proportion of the overall Spike-specific CD4+ T cells in COVID-19 convalescent individuals. In total, these results suggested a very limited number of SARS-CoV-2 T cells as detected by tetramers are capable of recognizing ccCoV with relative high avidity and vice versa. The potentially supportive role of these high avidity cross-reactive T cells in protective immunity against SARS-CoV-2 needs further studies.Class II tetramer reagents for eleven common DR alleles and a DP allele prevalent in the world population were used to identify SARS-CoV-2 CD4+ T cell epitopes. A total of 112, 28 and 42 epitopes specific for Spike, Membrane and Nucleocapsid, respectively, with defined HLA-restriction were identified. Direct  ex vivo, frequencies of these epitope-specific T cells were estimated in convalescent and pre-pandemic samples. Based on these frequencies, epitopes were stratified into immunodominant and subdominant epitopes. Amino acid sequences of epitopes identified were compared with 4 common cold coronaviruses. Potential cross-reactive epitopes were defined as having ≥67% sequence identity between common cold viruses and SARS-CoV-2. Four potential Spike specific cross-reactive epitopes were identified and functional cross-reactivity was demonstrated. Of the four cross-reactive epitopes identified, three were subdominant epitopes eliciting relatively low frequencies in both unexposed and convalescent subjects. In contrast to the results from the activation induced marker assays, the current data suggests that only a limited number of high avidity SARS-CoV-2 T cells as detected by tetramers are cross-reactive.Previous studies with activation induced marker assays in monitoring antigen-specific CD4+ T cells have shown that common cold coronavirus T cells can cross-react with SARS-CoV-2 antigens and these cross-reactive T cells are present in up to 60% of the unexposed population. In this current study, sets of overlapping peptides for Spike, Membrane, and Nucleocapsid proteins were used to identify epitopes across 11 HLA-DR and 1 HLA-DP alleles in SARS-CoV-2 convalescent samples using peptide-loaded MHC-II tetramers. Using these tetramers  Since the first reported index case of Coronavirus disease in December 2019 (COVID-19) . Other dex vivo studies of phenotypes and frequencies of CD4+ T cell responses at the epitope level have been very limited. Most of these CD4+ epitopes identified so far do not display an experimentally verified HLA restriction element.Studies examining T cell responses toward SARS-CoV-2 have been extensive. Several early investigations in this area utilized activation induced marker (AIM) assays, in which cells were stimulated with SARS-CoV-2 pooled peptides overnight. T cells which upregulated activation markers were identified as SARS-CoV-2-specific cells –12. Othecoronavirinae subfamily [Though SARS-CoV-2 is a newly emerging virus, multiple investigators have reported the presence of SARS-CoV-2-specific T cells in 20%-60% of unexposed persons ,22,25–27ubfamily ,29, pre-ubfamily ,30,31. Subfamily ,30,32. Tubfamily ,34. Thisubfamily . Howeverubfamily  we reasoIn the current study, we utilized class II tetramer reagents for epitope identification and examined SARS-CoV-2-specific CD4+ T cells in both COVID-19 convalescent individuals (exposed) and pre-December 2019 SARS-CoV-2 naïve individuals (unexposed). This study included 11 prevalent HLA class II DR alleles and 1 DP allele, covering at least 60% of the world population . Amongstth International HLA and Immunogenetics Workshop [With PBMCs from COVID-19 convalescent donors , the tetWorkshop   in length, the netMHCIIpan 4.0 prediction tool was used to identify the 9 AA core that constituted the minimum T cell epitope region within the 20mers . The mul97-116, M105-124, and N121-140 also have ≥67% AA sequence identity   , the seca ccCoV) , while ta ccCoV) . These rEx vivo tetramer staining show DR0101 SARS-CoV-2 S961-980, DR0301 SARS-CoV-2 S985-1004 and DR1501 SARS-CoV-2 S1001-1020 reactive T cells could not be consistently detected in unexposed individuals, suggesting that these are not immunodominant epitopes. It is unlikely the SARS-CoV-2 S961-980 is a dominant DR0401 restricted epitope, as T cells specific for this peptide cannot be detected in the TGEM studies in DR0401 COVID-19 subjects. In contrast, DP0401 SARS-CoV-2 S809-828 reactive T cells were detected in 5 out of 6 DP0401 unexposed individuals. Almost 100% of DP0401 SARS-CoV-2 S809-828 reactive T cells detected in unexposed individuals were CD45RA-CXCR3+, implicating that these are memory T cells from previous ccCoV infection. In addition to the increase in frequency of these cells post-infection, the phenotypes of these DP0401 cross-reactive T cells pre- and post-infection were also distinct. The cross-reactive T cells gained expression of CCR4 post SARS-CoV-2 infection, implying these cells are being activated for further expansion and differentiation during the disease process.809-828 peptide. The prevalence of both DP0401 and DP0402 in the world population implies that a high percentage of the general population that had previous exposure to ccCoV should have these cross-reactive T cells. Interestingly, Low et. al., Dykema et al., and Woldemeskel et al [DP0401 is a prevalent allele and is estimated to be present in greater than 50% in both Europe and North America, and approximately in 30–40% of the world population, ,43. The el et al ,32,44 ha809-828 reactive T cells as detected by tetramers in unexposed individuals in our cohort were still relatively low. The degree of expansion of the cross-reactive DP0401 SARS-CoV-2 S809-828 T cells after SARS-CoV-2 infection also appeared to be less vigorous compared to the mono-reactive DP0401 SARS-CoV-2 S161-180 epitopes. Less vigorous expansion of the cross-reactive DR1501 SARS-CoV-2 S1001-1020 T cells compared to another mono-reactive DR1501 SARS-CoV-2 S689-708 was also observed. It is unclear whether these cross-reactive TCRs have lower avidity to the MHC-II/SARS-CoV-2 peptide complexes compared to those of mono-reactive TCRs. We estimated that the cross-reactive DP0401 SARS-CoV-2 S809-828 and DR1501 SARS-CoV-2 S1001-1020 T cells contribute to less than 10% of the total Spike responses in DR1501-DP0401 individuals with no recent ccCoV infection. Similarly, the DR0301 SARS-CoV-2 S985-1004 contributed less than 3% of the overall S-restricted responses in subjects with the DR0301 haplotype.Despite these findings, frequencies of the DP0401 CoV-2 SThe current observation of near absence or very low frequency of SARS-CoV-2 T cells in unexposed subjects as detected by tetramers was in contrast with most of the published data using the AIM assays in which cross-reactive T cells at relatively high frequencies were being detected in 20–60% of unexposed people. This difference in outcomes could be explained by the different assay being used.A study showed SARS-CoV-2 T cells in unexposed subjects were 10–100 fold lower avidity compared to SARS-CoV-2 T cells in COVID-19 subjects . The abiA limitation of this study is our focus on epitope specific T cells targeted toward the structural proteins of the virus. We do expect that the extent of cross-reactivity for T cells that targeted other regions of the virus should be very similar to the structural proteins as observed here, as degree of AA sequence homology between SARS-CoV-2 and ccCoV are fairly similar between the structural and non-structural proteins. Another limitation of our study is that tetramer reagents are incapable of detecting low avidity T cells. Though the use of CD154 upregulation assays with SARS-CoV-2 and ccCoV T cell in the current study also did not demonstrate a higher degree of cross-reactivity between SARS-CoV-2 and ccCoV compared to the tetramer approach. We acknowledge that ccCoV T cells specific for ccCoV epitope with AA sequence identity of <67% to SARS-CoV-2 could still potentially respond to SARS-CoV-2. Thus, sequence mismatches cannot completely rule out low avidity cross-reactivity. The current study did not investigate the prevalence of ccCoV T cells that had low avidity for SARS-CoV-2 in unexposed subjects. The relative absence of high avidity T cell cross-reactivity between ccCoV and SARS-CoV-2 as shown here also raise the possibility that the pre-existing SARS-CoV-2 T cells reported in other studies can be due to cross-reactivity between SARS-CoV-2 and other microbes. As it is known that T cell cross-reactivity can occur with minimum AA sequence homology –47.In summary, we show that cross-reactive CD4+ T cells with high avidity for both SARS-CoV-2 and ccCoV as detected by tetramers are restricted to a very limited number of SARS-CoV-2 epitopes with AA sequence identity ≥67% between these viruses. Of the four Spike cross-reactive epitopes examined in more detail in the current study, only the dominant DP0401-restricted epitope was capable of eliciting a consistent T cell response in both unexposed and exposed persons. Though low avidity cross-reactive T cells may be prevalent, high avidity cross-reactivity that involves dominant SARS-CoV-2 epitopes is likely limited and will be restricted to persons with specific HLA alleles. As the structural proteins of SARS-CoV-2 are highly immunogenic and have multiple mono-reactive dominant epitopes, the contribution of high avidity cross-reactive CD4+ T cells to the overall SARS-CoV-2 specific T cell responses for individuals with no recent ccCoV infection may be minimal. The extent of protection that can be offered by these low and high avidity cross-reactive T cells warrants further studies.The study was approved by Benaroya Research Institute and University of Washington Institutional Review Boards and all blood samples were obtained with written informed consent from the participants.A total of 34 COVID-19-convalsecent subjects were recruited between April 2020 and April 2021. Subjects were recruited through Virginia Mason Hospital and University of Washington. Attributes of these subjects are described in HLA typing was performed with OLERUP SSP typing kit according to the manufacturer’s instruction or by sequencing at Scisco Genetics, Inc. .The following class II monomers and multimers were produced for this study: DRA1/DRB1*01:01 (DR0101), DRA1/DRB1*03:01 (DR0301), DRA1/DRB1*04:01 (DR0401), DRA1/DRB1*04:04 (DR0404), DRA1/DRB1*07:01 (DR0701), DRA1/DRB1*11:01 (DR1101), DRA1/DRB1*11:04 (DR1104), DRA1/DRB1*15:01 DR1501), DRA1/DRB3*01:01 (DRB3), DRA1/DRB4*01:01 (DRB4), DRA1/DRB5*01:01 (DRB5), and DPA1*0103/DPB1*04:01 (DP0401). Production of these molecules has been previously described 1, DRA1/D. MonomerPeptide libraries for SARS-CoV-2 Spike , Membrane , and Nucleocapsid  consisted of 20 amino acid (20-mers) long peptides with a twelve amino acid overlap. The S peptides consisted of 158 peptides. The M peptides consisted of 27 peptides. The N peptides consisted of 51 peptides. Peptide libraries for Spike of HKU-1 (Accession: YP_173238.1) were also 20-mers with a 12 amino acid overlap. Peptide libraries for Spike protein of NL63 (Accession: Q6Q1S2), 229E (Accession: NP_073551), and OC43 (Accession: NP_937950) cold viruses were 17-mers and were obtained from BEI Resources.The tetramer-guided epitope mapping (TGEM) procedure was done as previously described ,39. Brieex vivo enrichment procedure was done as previously described [The combinatorial escribed . BrieflyEach sample was collected to completion on a BD LSR Fortessa flow cytometer. A schematic of the gating strategy is shown in 5 cells per well in TCM and 1 μg/mL anti-CD40 blocking antibody was added. Cells were incubated with 2μg/mL peptides or an equal volume of DMSO. After incubation, cells were resuspended, washed, and stained with CD3 FITC, CD4 PerCP-Cy5.5, CD69 BV650, and CD154 PE for 20 minutes at room temperature. Cell lines were analyzed on a BD LSR II flow cytometer with 50,000 events collected.After TGEM, cells positive for a peptide that shared ≥67% sequence identity with ccCoV were harvested and stained with 0.5mg/mL of tetramer and incubated for 45 minutes at 37°C. Cells were stained with CD4 FITC and sorted at 20–40 cells per well in a 96-well round-bottom plate using a BD FACS Aria I. Cells were stimulated with 2μg/mL PHA and 10IU/mL IL-2 in the presence of irradiated feeder cells and expanded for 12–14 days in TCM. Cell lines were validated with corresponding tetramer and CD4 FITC. Cell lines were expanded until ≥700,000 cells were obtained with 10IU/mL IL-2 added every 2 days. Cells were rested for 5 days without the presence of IL-2 then resuspended and harvested. Cells were plated in a 96-well round-bottom plate at 1x104 cells per well in a 96-well round-bottom plate, co-cultured with 1x105 irradiated HLA-matched feeder cells and peptides from ccCoV and SARS-CoV-2 at concentrations of 2, 0.5 and 0.01μg/mL or DMSO (as negative control) for 72 hours. Cells were pulsed with 1μCi 3H-Thymidine for an additional 24 hours. Cells were harvested on Harvester 96 Mach II M. Uptake of 3H-Thymidine was measured on a Perkin Elmer MicroBeta2 scintillation counter to assess proliferation. Stimulation index (SI) was calculated by taking the average CCPM (corrected counts per minute) of peptide stimulation divided by the average CCPM of DMSO.T cells were plated at 5x10GraphPad Prism 9 was used for data analysis.S1 FigA. Bold FCS plots indicate positive peptides. Positive peptides are S25-44, S33-52, S929-948, and S937-956.(A) Pool mapping of a representative DR0401 SARS-CoV-2-exposed individual with pools of overlapping peptides for the Spike protein. Bold FCS plots indicate positive pools. Numbers indicate percent of CD4+ Tetramer+ T cells. (B) Example of fine mapping of pools 1 and 24. Bolded FCS plots in (TIF)Click here for additional data file.S2 Fig(A) 1. PBMCs were incubated with the first pool of tetramers conjugated to PE, PE-CF594, PE-Cy7, and BV421 for 100 minutes at room temperature following a 10 minute incubation with dasatinib at 37°C. 2. PBMCs were incubated with 40ul of anti-PE magnetic beads for 20 minutes at room temperature. 3. Tetramer-positive PBMCs were enriched on magnetic column. 4a. Tetramer-positive PBMCs were eluted from the column. 4b. Flow through of tetramer-negative PBMC was collected and incubated with second pool of tetramers and the process was repeated. 5. PBMCs were stained with antibody panel for 20 minutes at room temperature and then analyzed with flow cytometry. (B) Gating strategy to identify DRB1*04:01 HA306/HAp68 from an unexposed individual. Size gating was applied to select for singlet lymphocytes followed by a dump gate  to exclude macrophages, B-cells, and dead cells. Live CD14-CD19- cells were gated for CD4+ cells. Live CD4+ cells were gated on the four tetramer fluorochromes  and Boolean gating was applied to select for double-positive tetramer CD4+ cells. These double-positive cells were gated for memory cells on CD45RA-. Surface phenotypic markers were gated on CD45RA- memory cells.(TIF)Click here for additional data file.S3 Fig801-820/S857-876; closed circle S985-1004/S1073-1092/S1105-1124; closed triangle S345-364/S353-372. (B) DR1501 dominant (D) and subdominant (S) epitopes. Open circle S57-76/S97-116; open triangle S689-708/S745-764; closed circle S321-340/S425-444. (C) DRB3 dominant (D) and subdominant (S) epitopes. Open circle S25-44/S33-52; open triangle S209-228/S393-412; closed triangle S1249-1268. (D) DP0401 dominant (D) and subdominant (S) epitopes. Open circle S161-180; open triangle S809-828; closed circle S129-148/S337-356.(A) DR0301 dominant (D) and subdominant (S) epitopes. Open circle S(TIF)Click here for additional data file.S4 Fig1093-1109 cell line. (B) DP0401 NL63 S867-883 cell line. (C) DP0401 HKU1 S905-924 cell line. (D) Proliferation assay on HKU1 S905-924 cell line generated from pre-pandemic DP0401 PBMC. SI: Stimulation index.PBMC of unexposed individuals were stimulated with ccCoV peptides and cultured for 14 days. Cold virus specific T cell lines from unexposed individuals were established by sorting of ccCoV tetramer positive T cells followed by expansion of sorted cells. CD154 upregulation assays were carried out, numbers indicate the percentage of CD154+CD69+ cells. (A) DR1501 OC43 S(TIF)Click here for additional data file.S5 Fig689-708/S745-764 in PBMC of unexposed (left) and exposed (middle) individuals. A summary of all individuals’ S689-708/S745-764 reactive T cell frequencies in DR1501 individuals is shown (left). Student’s unpaired t-test; ** p≤0.01.Representative FCS plot of ex vivo staining of T cells of dominant epitope S(TIF)Click here for additional data file.S6 FigThe above shows the Spike protein mutations in four prevalent SARS-CoV-2 strains, B.1.1.7, B.1.351, P.1, and B.1.617.2. Tan indicates the signal peptide, pink indicates S1, green RBD, and blue S2.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 TableRed designated 100% AA identity to SARS-CoV-1. Green designated AA sequence with 1–3 AA mismatch to SARS CoV-1.(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


Anti-SARS-CoV-2 monoclonal antibodies afford prompt immunity, have demonstrated reduction in severe COVID-19 in high risk ambulatory patients, and are available through Emergency Use Authorization. Challenges exist, however, to widespread utilization. This operations study 11/23/20-4/30/21 identified patients meeting monoclonal AB EUA criteria by test results or referral. Outreach to harder-hit neighborhoods included connecting with primary care teams and testing sites. Infusion centers with staff trained in infection control, rapid response and drug preparation were utilized. The primary study outcome was treatment of qualifying patients. Secondary outcomes included infusion complications, hospitalization/death, and symptom resolution. Investigational review board approval was obtained.367 patients were treated: mean age of 63, 201(55%) male, 276(75%) white, 54(15%) black. All patients had a first positive direct SARS-CoV-2 test within 10 days, 232(63%) had > 1 high-risk qualification, 32(9%) were vaccinated for SARS-CoV-2. Of patients with available zipcodes, 135(38%) had a Community Need Index >3.5 and 157(45%) a Social Vulnerability Index >0.5. 190(52%) received bamlanivimab, 93(25%) casirivimab/imdevimab, 84(23%) bamlanivimab/etesevimab. Four patients experienced infusion reaction, 1 with anaphylaxis. 172(73%) of 236 patients were symptom free at day 5. 20 patients (5%) were hospitalized for COVID-19 within 30 days with a median time from symptom onset to infusion of 7 days, 11(55%) were admitted within 24 hours, 1 died. Patient CharacteristicsCOVID-19 courseCommunity Need Index and Social Vulnerability Index by ZipcodeOur study demonstrates that treatment with anti-SARS-CoV-2 monoclonal antibodies is feasible in a high resource setting. There were no related SARS-CoV-2 exposures and therapy was well tolerated. Trials of anti-SARS-CoV-2 monoclonal antibodies have reported lower rates of hospitalizations in treated patients than we found. This may reflect the expanded time frame for EUA therapy as compared to clinical trials, differences in real world patients or viral variants. Given potential benefit in unvaccinated patients or those at risk for poor vaccine response, the equitable utilization of anti-SARS-CoV-2 monoclonal antibody therapy in early COVID-19 should remain a focus for researchers and clinicians. All Authors: No reported disclosures


Caenorhabditis elegans, we identified the RabGAP tbc-11 as an important factor for the microRNA pathway. We show that TBC-11 acts mainly through the small GTPase RAB-6 and that its regulation is required for microRNA function. The absence of functional TBC-11 increases the pool of microRNA-unloaded Argonaute ALG-1 that is likely associated to endomembranes. Furthermore, in this condition, this pool of Argonaute accumulates in a perinuclear region and forms a high molecular weight complex. Altogether, our data suggest that the alteration of TBC-11 generates a fraction of ALG-1 that cannot bind to target mRNAs, leading to defective gene repression. Our results establish the importance of intracellular trafficking for microRNA function and demonstrate the involvement of a small GTPase and its GAP in proper Argonaute localization in vivo.Once loaded onto Argonaute proteins, microRNAs form a silencing complex called miRISC that targets mostly the 3’UTR of mRNAs to silence their translation. How microRNAs are transported to and from their target mRNA remains poorly characterized. While some reports linked intracellular trafficking to microRNA activity, it is still unclear how these pathways coordinate for proper microRNA-mediated gene silencing and turnover. Through a forward genetic screen using  MicroRNAs play an essential contribution among the many mechanisms used by cells to maintain a proper gene expression program. These endogenous small RNAs regulate gene expression by binding to target mRNAs and blocking their translation into functional proteins. To achieve this, microRNAs rely on Argonaute and other proteins that form a complex called the miRISC. While research on microRNAs has brought a lot of interest over the last years, it is still unclear what modulators affect the activity of the miRISC. Our study identified proteins implicated in intracellular trafficking as important factors for miRISC function. We showed that the intracellular location of the Argonaute protein needs to be tightly regulated to assure proper microRNA-mediated mRNA repression in animals. F2 animals were screened for GFP derepression. 19 candidates were isolated and named tard-1 to tard-19 . Candidate tard-10(qbc24) was mapped by crossing with a Hawaiian CB4856 strain in which λN::mCherry::alg-1 and gfp::box-B constructs were inserted. Recombinants were sequenced and mutation was mapped as described in  Nonidet P40, 1 mM DTT, 1 mM cycloheximide) before flash freezing. Thawed pellets were washed 3 times with lysis buffer and homogenized in 1 volume of lysis buffer supplemented with 0.4U/uL RNase using a Dounce homogenizer. For EDTA controls, a final concentration of 10mM of EDTA was added to the extracts following extraction. Concentration of RNA was measured with a spectrophotometer. 10 OD260 units were loaded onto a 15% to 55% sucrose gradient and further fractionated and analyzed as described in [2 and cycloheximide in sucrose gradients were replaced by EDTA. Recovered fractions were precipitated with 2 volumes of ethanol 100% and 10% of each fraction was loaded on an 8% SDS-polyacrylamide gel for western blot analysis.Synchronized young adult animals were collected and washed in M9 supplemented with 1mM cycloheximide then washed once with lysis buffer , Repbase 15.10 [Sequencing reads were mapped to the genome and cDNA using custom PERL (5.10.1) scripts and Bowtie 0.12.7 . Databasse 15.10 , and miRse 15.10 . The Gense 15.10  was usedtbc-11 to the sum of tbc-11 + wild-type reads.The samples were normalized to the total small RNAs including miRNAs, 22G-RNAs and 21U-RNAs. We used the average of triplicates for each strain to calculate the ratio of each small RNA reads of Data for main text Figs S1 Fig(A) GFP reporter repressed by λN::mCherry tagged ALG-1. GFP was fused to alg-1 endogenous promoter  and cog-1 3’ UTR in which the lsy-6 binding sites were replaced by 6 box B sequences (cog-1Δlsy-6 bs). (B) DIC and fluorescent microscopy of WT and tbc-11(qbc24) pharynx in young adults. GFP is derepressed in tbc-11(qbc24) animals. ALG-1 expression (represented by mCherry) is not affected in these animals. Images were taken using the same settings and exposition time for each animal. Scale bar: 50μm. (C) Percentage of alae breaks of wild-type (WT), tbc-11(qbc24) homozygous, and tbc-11(qbc24) heterozygous (tbc-11(qbc24)/+) young adult animals. tbc-11(qbc24) heterozygous animals were observed as F1 of a genetic cross between tbc-11(qbc24) and wild-type animals. P value were obtained by one-way ANOVA  (D) Number of seam cells in tbc-11(qbc24) and (ok2576) alleles. Strains were crossed with a strain expressing GFP in the seam cells (scm::GFP) in order to score them. Animals were observed as young adults. Wild-type (WT) animals have an invariable number of 16 seam cells. P value were obtained by Fisher’s exact test  (E) Left: DIC and fluorescent microscopy of col-10::gfp::lin-41 3’UTR reporter in hypodermal cells of L4 staged animals. Animals were fed with bacteria expressing RNAi against tbc-11 or control RNAi (no targeting gene) for 48 hours. Scale bar: 50μm. Right: Quantification of GFP fluorescence intensity in four hypodermal cells per animal. Images were taken using the same settings and exposition time for each animal. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test. . (F) Left: DIC and fluorescent microscopy of miR-228 mutated reporter in intestine cells of L2 staged animals. Scale bar: 50μm. Right: Quantification of GFP fluorescence intensity in four intestine cells per animal. Images were taken using the same settings and exposition time for each animal. The number of animals scored (n) is indicated. P value were obtained by two tailed t-test. ns: non-significant.(TIF)Click here for additional data file.S2 Figtbc-11(ok2576) were scored under DIC Nomarski microscopy. Animals were fed with bacteria expressing RNAi against rab-2 (upper panel), rab-14 (lower panel) or control RNAi (no targeting gene) for 48 hours and observed as young adults. 50 animals were observed for each condition. Each circle represents the mean of one independent RNAi experiment. P value were obtained by one-way ANOVA .Alae breaks of (TIF)Click here for additional data file.S3 Fig(A) Western blot of ALG-1 and the GW182 protein AIN-1 in wild-type (WT), tbc-11(qbc24) and tbc-11(ok2576) young adult animals. Extracts were prepared with standard conditions (0.5% triton). Actin is used as a loading control. (B) Western blot of the GW182 protein AIN-1 detected in wild-type (WT), tbc-11(qbc24) and tbc-11(ok2576) young adult worms extracts prepared with low (0.5% triton) or high (1.5% triton) detergent concentration. High detergent concentration allows better extraction of membrane associated proteins. Actin is used as a loading control. (C) Western blot of ALG-1 protein in wild-type (WT), tbc-11 (qbc24) and tbc-11(ok2576). Proteins were fully solubilized by boiling animals in Laemmli buffer for 10 minutes. Actin is used as a loading control. (D) Small RNA sequencing of tbc-11(qbc24) (left) and tbc-11(ok2576) (right) young adult animals compared to wild-type animals. The dotted line represents a two-fold change. The number of miRNA analyzed is indicated (n). p<0.22 for let-7 in tbc-11(qbc24), p<0.50 for miR-228 in tbc-11(qbc24), p<0.13 for let-7 in tbc-11(ok2576), p<0.25 for miR-228 in tbc-11(ok2576). P value for individual miRNAs were obtained by two tailed unpaired t test. The samples were normalized to the total small RNAs including miRNAs, 22G-RNAs and 21U-RNAs. (E) Representative western blot of immunoprecipitations of ALG-1 quantified in (TIF)Click here for additional data file.S4 FigA) and tbc-11(qbc24) (B) animals. Animals are expressing a plasmid containing the sequence of alg-1p::mCherry::MANS as a marker of the Golgi. Merge image represents the overlap of GFP (ALG-1), mCherry (MANS) and DIC images. The nucleus of the seam cell is indicated by an arrow. Zoomed in images of GFP::ALG-1 merged with mCherry::MANS in a seam cell are shown in insets. Images were taken using the same settings and exposition time for each animal. Scale bar: 10 μm.DIC and fluorescent microscopy of intracellular localization of endogenously tagged GFP::ALG-1 in seam cells of wild-type Click here for additional data file.S5 FigA) Top: Polysome profiles of wild-type (WT) and tbc-11(ok2576) young adult animals performed with extracts prepared with high detergent concentration (1.5% triton). Bottom: Detection of ALG-1 in different fractions by Western blot. Polysome profiles show no differences in overall translation. Low and high exposure of the same membrane are shown. (B) Polysome profiles for wild-type (WT) and tbc-11(qbc24) extracts presented in (C) Polysome profiles for wild-type (WT) and tbc-11(qbc24) extracts treated with EDTA presented in ((TIF)Click here for additional data file.S6 FigIn absence of TBC-11, RAB-6 is constitutively active and shuttles miRNA-bound and unbound ALG-1 to the perinuclear region.(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Background: Inflammatory immunity theory has raised considerable concern in the pathogenesis of atherosclerosis. Proviral integration site of murine 2 (Pim-2) kinases functions in apoptosis pathways and the anti-inflammatory response. Here, we investigated whether Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.-/- mice a high-fat diet. THP-1-derived macrophages were subjected to ox-LDL  conditions in vitro to mimic the in vivo conditions.Methods: An atherosclerosis animal model was established by feeding ApoE in vitro and in vivo, whereas knocked down Pim-2 (Pim-2 KD) markedly promoted atherosclerotic inflammation associated with upregulation of the mTORC1 signaling pathway. The plaque areas and lesions in the whole aorta and aortic root sections were alleviated in ApoE -/- mice with Pim-2 OE, but aggravated by Pim-2 KD. Additionally, an mTOR agonist (MHY1485) counteracted the anti-inflammatory effect of Pim-2 in ox-LDL-treated THP-1-derived macrophages after Pim-2 OE, whereas rapamycin rescued atherosclerotic inflammation in ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. Furthermore, si-mTOR and si-Raptor alleviated the atherosclerotic proinflammatory effect in ox-LDL-treated THP-1-derived macrophages in a the background of Pim-2 KD.Result: The protein expression of Pim-2 was upregulated in ox-LDL-treated THP-1-derived macrophages and an atherosclerotic mouse model. Additionally, ox-LDL upregulated the protein expression of p-mTOR, p-S6K1 and p-4EBP1, intracellular lipid droplets, free cholesterol and cholesterylester and the mRNA expression of inflammatory cytokines, including IL-6, MCP-1, TLR-4 and TNF-α, in THP-1-derived macrophages. Functionally, overexpressed Pim-2 (Pim-2 OE) attenuated atherosclerotic inflammation associated with the mTORC1 signaling pathway Conclusions:These results indicated that Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway. The mortality of cardiovascular and cerebrovascular disease is increasing yearly and atherosclerosis is the main cause of this ischemic disease . Inflamm-/- mice fed a western-type diet transplanted with bone marrow from macrophage-Raptor knockout mice, and the macrophage contents and expression of inflammatory factors in atherosclerotic plaques were significantly reduced. Ma et al. [-/- mice and vascular smooth muscle cells, which depended on the increased activity of mTORC1.Mammalian target of rapamycin (mTOR) is a conserved serine/threonine protein kinase that exists in the cytoplasm in a stable state . When aca et al.  found thProviral integration sites of murine (Pim) kinases are a distinct class of serine/threonine-specific kinases comprising Pim-1, Pim-2 and Pim-3. Studies have shown that Pim kinase plays an important role in the cell cycle and apoptosis by interacting with apoptotic genes or phosphorylating different substrates , 15. Pre-/- mice fed a high-fat diet.Considering that the exact role of Pim-2 in atherosclerosis remains unknown, we investigated whether Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway in ox-LDL-treated THP-1-derived macrophages and ApoE To determine whether ox-LDL affects the expression of Pim-2 and mTOR signaling pathway, THP-1-derived macrophages were sequentially treated with 50 μg/ml of ox-LDL for 12 h, 24 h, 36 h, and 48 h, respectively. Next, total protein was extracted and processed for western blot analysis. The protein levels of Pim-2 increased gradually and reached a peak at 36 h in response to ox-LDL stimulation , 1B. SimIntracellular lipid droplets were stained with oil red O working solution. Lipid droplets were significantly increased in ox-LDL-treated THP-1-derived macrophages , 1E. ComCompared with the control group, the protein expression levels of p-mTOR(Ser2448), p-S6K1(Thr389), and p-4EBP1(Thr37/46) were significantly elevated in ox-LDL-treated THP-1-derived macrophages, but markedly decreased after overexpression of Pim-2 (Pim-2 OE) –2C. As eCompared with the ox-LDL-treated NC group, the protein expression levels of the mTORC1 signaling pathway and concentrations of intracellular lipid droplets were significantly elevated in ox-LDL-treated THP-1-derived macrophages after the knockdown of Pim-2 (Pim-2 KD) . Further-/- control group, but markedly decreased in the OE group and obviously increased in the KD group compared with the NC group  group, the plaque areas and lesions in the whole aorta and aortic root sections as well as Mac-2 expression by immunohistochemistry were significantly increased in ApoE NC group –5C. FurtCompared with the ox-LDL-treated NC group, the protein expression levels of the mTORC1 signaling pathway and mRNA expression of inflammatory cytokines were significantly lowered in ox-LDL-treated THP-1-derived macrophages after Pim-2 OE, but MHY1485 markedly counteracted this downtrend  counteracted the anti-inflammatory effect of Pim-2 in ox-LDL-treated THP-1-derived macrophages after Pim-2 OE, whereas rapamycin rescued atherosclerotic inflammation in ox-LDL-treated THP-1-derived macrophages after Pim-2 KD. Furthermore, si-mTOR and si-Raptor alleviated the atherosclerotic proinflammatory effect in ox-LDL-treated THP-1-derived macrophages in a background of Pim-2 KD. These results indicated that Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway.Inflammatory immunity theory has raised considerable concern in the pathogenesis of atherosclerosis –6. We foPrevious reports  have sugInflammation is an important pathological mechanism of atherosclerosis , 30. TheThe mTOR signaling pathway contains two different protein multicomplexes , of whicThis study has some limitations. Our current data cannot exclude the role of Pim-2 in other cell types, such as vessel endothelial cells and smooth muscle cells in atherosclerosis progression. Additionally, we did not observe the location distribution of Pim-2 in the aorta, information that might be useful for further preclinical and clinical studies. Future studies should be performed using cell-specific Pim-2-knockout mice.In conclusion, these results indicated that Pim-2 kinase inhibits atherosclerotic inflammation by suppressing the mTORC1 pathway. Our findings offer novel insight that Pim-2 may serve as a therapeutic target in atherosclerosis.2+) oxidized human low density lipoprotein (ox-LDL) was purchased from the Peking Union-Biololgy . THP-1-derived macrophages were sequentially treated with 50ug/ml ox-LDL for 12h, 24h, 36h, and 48h, respectively.THP-1 monocytes were obtained from the Cell Bank of Shanghai Chinese Academy of Sciences . THP-1 cells were cultured in Roswell Park Memorial Institute medium  supplemented with 10% FBS  and 50 pM β-mercaptoethanol  in a humidified atmosphere of 5% CO2 at 37° C and grown to 70–80% confluence. Cells at passages three to eight were used in the experiments. THP-1 monocytes are differentiated into macrophages by 48 h incubation with 150 nM phorbol 12-myristate 13-acetate  in RPMI medium. After 48 h treatment with PMA, THP-1 cells became adherent. The PMA-containing medium was removed and the differentiated cells were cultured in fresh cell culture medium for 48 h to increase their cytoplasmic volume. Copper , adherent THP-1-derived macrophages were detached with 5 mM EDTA and placed in FACS tubes. The cell suspension was centrifuged for 5 min at 200 g, and the pellet was resuspended in PBS containing 2% FBS. The pellet was next resuspended in human truStain FcX  diluted 20 times in PBS. The suspension was then incubated at room temperature for 10 min. Next, the cells were centrifuged for 5 min at 200 g, and primary PE (phycoerythrin) mouse anti-human CD11b antibody  was used to stain the differentiated cells in PBS in the dark at RT for 30 min. The stained cells were then washed twice with PBS and analyzed on a BD AccuriC6 Plus flow cytometer. Cells were also incubated with PE mouse IgG1, κ isotype control  corresponding to each primary antibody. Unstained samples were prepared for cell size and granularity assessments. The data were collected and analyzed using FACSCalibur . The full-length cDNA sequence of human Pim-2 (GenBank no. NM_ 006875) and negative control were synthesized and subcloned into the lentiviral Ubi-MCS-3FLAG-CBh-gcGFP-IRES-puromycin vector (Addgene plasmid GV492). A shRNA sequence targeting human Pim-2 (5′- ccggGATGAACCCTACACTGACTTTctcgagAAAGTCAGTGTAGGGTTCATCtttttg-3′) corresponding to coding regions 571–1591 relative to the first nucleotide of the start codon of human Pim-2 (GenBank no. NM_ 006875) and a scrambled (5′-ccggTTCTCCGAACGTGTCACGTctcgagACGTGACACGTTCGGAGAAtttttg-3′) sequence were cloned into the lentiviral U6-MCS-Ubiquitin-Cherry-IRES-puromycin vector (Addgene plasmid GV298). Lentiviruses were generated by transiently transfecting the resulting lentiviral vectors along with packaging vectors pGC-LV, pHelper 1.0, and pHelper 2.0 into HEK 293T cells (from the human embryonic kidney cell line) using Lipofectamine 2000. The medium containing viruses was harvested 48 h following the start of transfection and concentrated using Millipore Centricon Plus-20 filters.For lentivirus infection, viral solutions (MOI = 50) were added to cell culture medium containing polybrene . After 8 hours of infection, fresh RPIM1640 medium was replaced. 48 hours after infection, THP-1 cells were selected using puromycin . The efficiency of lentiviral transfection was determined with fluorescence microscopy, PCR and western blot analysis , 3. The The concentrations of total cholesterol (TC), free cholesterol (FC) and cholesteryl ester (CE) were determined using the TC/FC Quantification Assay  according to the manufacturer's protocol. The fluorometric assay was performed using a microplate reader  at an absorption wavelength of 550 nm. The TC and FC results were presented as nmol/mg, and CE was determined by subtracting the value of FC from TC.The mTOR inhibitor rapamycin (A8167) and agonist MHY1485 (B5853) were purchased from APExBIO . THP-1-derived macrophages were treated with 100 nM rapamycin for 1 h or 20 μM MHY1485 for 30 min to inhibit or activate the mTOR pathway before exposure to 50 μg/ml of ox-LDL for 24 h.5 cell per well), this THP-1-derived macrophages were then transiently transfected with 100 nM siRNA target sequences or negative control siRNA using GP-transfect-Mate  according to the manufacturer’s protocol. The transfected cells were then exposed to 50 μg/ml of ox-LDL for 24 h for subsequent studies.Gene expression silencing was achieved using siRNA-specific target sequences. Human mTOR siRNA (Si-mTOR) and Raptor siRNA (Si-Raptor) were obtained from Gene Pharma . The sense and antisense strands of siRNA are presented in -/- mice (n=60) were obtained from the Department of Laboratory Animal Science of Peking University Health Science Center  and were housed conventionally at 22± 2.0° C and 50% ± 5% humidity under a 12-h light-dark cycle with free access to water and food. All the animal experiments were performed and analyzed by blinded experimenters. All the animal experiments were conducted in compliance with the National Institutes of Health (NIH) policies in the Guide for the Care and Use of Laboratory Animals and were approved by the Nanchang University Animal Care and Use Committee (SYXK(G) 2015-0001).Male 7-week-old C57BL/6 mice (n=12) and ApoE -/- mice were assigned to five groups randomly : (i) ApoE -/- control group that received a single tail vein injection of equivoluminal PBS at 12 weeks of age (n = 12); (ii) Overexpression-negative control group  that received a single tail vein injection of negative control virus corresponding to Pim2-overexpressing virus at 12 weeks of age; (iii) Pim2-overexpressing group  that received a single tail vein injection of Pim2-overexpressing virus at 12 weeks of age; (iv) Knockdown -negative control group  that received a single tail vein injection of negative control virus corresponding to Pim2-knockdown virus at 12 weeks of age; (v) Pim2-knockdown  that received a single tail vein injection of Pim2-knockdown virus at 12 weeks of age.After one week of adaptive feeding, all the mice were fed a high-fat diet  for 12 weeks to create an atherosclerotic model . Body weEight weeks after the tail vein injection, the mice were anesthetized using an i.p. injection of sodium pentobarbital (50 mg/kg), and the adequacy of anesthesia was confirmed by the absence of a reflex response to a foot squeeze. Afterward, blood samples were collected under anesthesia by cardiac puncture immediately before sacrifice. Next, the hearts were perfused and the aortas were harvested. Partial aortas were fixed in 4% paraformaldehyde for pathological analysis. The remaining aortas were freshly flash-frozen in liquid nitrogen for gene and protein expression analysis.12 viral genomes per ml (vg/ml). To produce vessel-specific rAAV, mouse Pim2 genes (GenBank no. NM_138606) was sub-cloned into the CMV-bGlobin-MCS-EGFP-3FLAG-WPRE-hGH-polyA vector (Addgene plasmid GV388). A shRNA sequence targeting mouse Pim-2 (5′- accggGGAAGGTGGGTGAAGGCAAttcaagagaTTGCCTTCACCCACCTTCCttttt-3′) corresponding to coding regions 440–458 relative to the first nucleotide of the start codon of human Pim-2 (GenBank no. NM_138606) and a scrambled (5′- accgg CGCTGAGTACTTCGAAATGTCttcaagaga GACATTTCGAAGTACTCAGCGttttt-3′) sequence were cloned into the U6-MCS-CAG-mCherry vector (Addgene plasmid GV480). rAAV were generated by transiently transfecting the resulting vectors along with packaging vectors pAAV-RC and Paav Helper into AAV 293T cells using CaCl2. The medium containing viruses was harvested 48 h following the start of transfection and concentrated using Millipore Centricon Plus-20 filters. Viral particles were purified by an iodixanol step-gradient ultracentrifugation method. The titers were ~2 × 1012 viral genomes per ml (vg/ml).The AAV2/8-CMV-NC recombinant, AAV2/8-CMV-Pim2 recombinant, AAV2/8-U6-NC recombinant and AAV2/8-U6-shPim-2 recombinant were constructed and ordered from Genechem , and the titers were ~2 × 1011 genome copies of AAV2/8-CMV-NC recombinant or AAV2/8-CMV-Pim2 recombinant at 12-week-old. In the AAV2/8-knockdown atherosclerosis model, each mouse from the KDNC or KD group received a single tail vein injection of 3 ×1011 genome copies of AAV2/8-U6-NC recombinant or AAV2/8-U6-shPim-2 recombinant at 12-week-old.In the AAV2/8-overexpressing atherosclerosis model, each mouse from the OENC or OE group received a single tail vein injection of 3 ×10The oil red O working solution was prepared by diluting the stock solution with distilled water (3:2). For staining, THP-1-derived macrophages were fixed in 10% paraformaldehyde. After being washed with PBS and dehydrated with 60% isopropanol, oil red O working solution was added to the culture dishes and incubated at 37° C for 10 min. Next, the cells were washed with 60% isopropanol until the intercellular space was clear using an optical microscope .At the end of the administration, the aortas were harvested, the adventitial fat was carefully removed, the whole aortas were opened longitudinally, and aortic root sections were cut to an 8-μm thickness. The aortas were stained with oil red O working solution as indicated above and then differentiated with 50% glycerol. The intracellular lipid droplets and lesion areas were quantified using Image-Pro Plus 6.0 .Immunohistochemistry was performed to detect the macrophage marker protein Mac-2. Briefly, the aortas were embedded in paraffin, and 8-μm slices were cut from the embedded blocks. Rehydrated sections were incubated with Mac-2 antibodies . The cell nuclei were stained with 4′,6-diamidino-2-phenylindole  and counterstained with hematoxylin . Semiquantitative analysis of the tissue staining was performed using Image-Pro Plus 6.0 .The concentrations of inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and C reactive protein (CRP) were quantified using the ELISA kits  according to the manuscript’s instructions. Briefly, serum samples of mice were added into 96-well plates coated with anti-TNF-α, anti-IL-6, and anti-CRP antibodies after blocking with 1% bovine serum albumin, followed by incubation with tetramethylbenzidine , and the reaction was stopped by 50μl of Stop Solution.–ΔΔCT using GAPDH as a reference gene.Total RNA was extracted from the aortas and cells using TRIzol reagent  according to the manufacturer's protocol. cDNA synthesis was performed using a TaqMan reverse transcription kit . Then, quantitative real-time PCR was performed using a SuperReal PreMix Plus (SYBR Green) Kit  on a 7500 Fast Real Time PCR system from Applied Biosystems . All RT-PCR analysis was performed in duplicate 20-μl reactions in 96-well plates. The resulting melting curves were visually inspected to ensure specificity of product detection. Primer sequences were listed in The aortas and THP-1-derived macrophages were homogenized and lysed using radioimmunoprecipitation (RIPA) buffer  supplemented with cocktail proteinase and phosphatase inhibitors . Protein concentration was measured with the BCA Protein Assay Kit . Equal amounts of protein were separated on SDS-PAGE electrophoresis and electrotransferred onto PVDF membranes . The membranes were blocked in TBST buffer and then further incubated primary antibodies  overnighAll data were presented as means ± standard deviation (SD) using the Graphpad Prism 8.0 software . Normality analysis was performed before any testing, via the Shapiro-Wilk test. Differences were evaluated using unpaired Student’s t test between two groups. One-way ANOVA was conducted followed by Bonferroni post hoc test for comparisons between >2 groups. The non-normal distributed data were analyzed using non-parametric testing . Differences between groups over time were evaluated using repeated measures analysis of variance. Statistical analysis was performed using SPSS23.0  software and P < 0.05 was considered statistically significant.All animal experiments were conducted in compliance with the National Institutes of Health (NIH) policies in the Guide for the Care and Use of Laboratory Animals and were approved by the Nanchang University Animal Care and Use Committee (SYXK(G) 2015-0001).The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.Supplementary FiguresSupplementary Tables


CD73 levels inversely correlate with overall survival. Using our pDC-MM coculture models, we found that blockade of CD73 with anti-CD73 Abs: decreases adenosine levels; activates MM patient pDCs; triggers cytotoxic T lymphocytes (CTL) activity against autologous patient MM cells. Combination of anti-CD73 Abs and an immune-stimulating agent TLR-7 agonist enhances autologous MM-specific CD8+ CTL activity. Taken together, our preclinical data suggest that the therapeutic targeting of CD73, alone or in combination with TLR-7 agonist, represents a promising novel strategy to restore host anti-MM immunity.Interaction of plasmacytoid dendritic cells (pDCs) with multiple myeloma (MM) cells, T- or NK-effector cells in the bone marrow (BM) microenvironment induces tumor cell growth, as well as inhibits innate and adaptive immune responses. Defining pDC-MM interaction-triggered immunosuppressive mechanism(s) will enable design of interventional therapies to augment anti-MM immunity. In the present study, we show that pDC-MM interactions induce metabolic enzyme Ecto-5' Nucleotidase/CD73 in both pDCs and MM cells. Gene expression database from MM patients showed that  Multiple Myeloma (MM) is characterized by clonal expansion of malignant plasma cells in the bone marrow (BM) . The Amevia catabolic activity of metabolizing enzymes ectonucleotidases. Specifically, CD39/ectonucleoside triphosphate diphosphohyrolase-1 converts ATP/ADP to adenosine monophosphate (AMP), followed by conversion of AMP to adenosine by CD73/ecto-5' nucleotidase/NT5E. Similarly, NAD+ to adenosine conversion occurs via sequential activity of CD38 (NAD glycohydrolase) and CD73. Prior studies showed that ATP-NAD+/ectonucleotidases/adenosine-signaling axis confer immune suppression in cancers, especially in hypoxic tumor microenvironment  Affymetrix Human Genome U133 Plus 2.0 Array platform (+ MM available on MMRF CoMMpass (version IA13) patients’ survival data platform .We also utilized RNAseq expression database on CD138+ MM cells were treated with anti-CD73 Ab (0.5 µg/ml) or isotype control Abs for 48 h; supernatants were subjected to Adenosine assay using human Adenosine assay kit , as per manufacturer’s protocol. Data presented shows change in adenosine conversion in supernatants from BM-MNCs or CD138+ MM cells treated with anti-CD73 Abs versus isotype control Abs. Adenosine measurement in coculture assays: MM BM pDCs were co-cultured with CD138+ MM cells in the presence of anti-CD73 Ab (0.5 µg/ml) or isotype control for 48 h; supernatants were subjected to Adenosine assay. Data presented shows change in adenosine conversion in supernatants from pDCs-MM co-cultures treated with anti-CD73 Abs versus isotype control Abs.MM patient total BM-MNCs or CD138hi MM cell populations. Cell viability was assessed by WST assay, as previously described [MM cells were co-cultured for 24 h with pDCs; cells were then stained with CD73-FITC and CD138 Abs, followed by multicolor flow analysis to quantify CD73escribed , 11.MM patient BM CD8 + T cells were co-cultured for 3 days with autologous pDCs at 1:10 (pDC:T) ratio, in the presence or absence of anti-CD73 Ab 0.5 µg/ml) or TLR-7 agonist (0.1 µM). After washing to remove drugs, cells were cultured for 24 h with autologous MM cells pre-stained with CellTrace Violet or CellTracker Violet , followed by 7-AAD staining and quantification of MM cell lysis by FACS, as previously described  µg/ml or.t-test, with the minimal level of significance at p-value < 0.05 (Graph Pad PRISM version 8).Statistical significance was obtained using Student’s n = 3; three different relapsed/refractory multiple myeloma patients) were first co-cultured with MM.1S cells (1pDC:5MM) for 48 h, followed by separation of MM.1S cells from pDCs using flow cytometry. Total RNA from MM.1S cells was subjected to RNA sequencing (RNAseq) analysis using Illumina NGS. As shown in Fig. p-value of <0.05 was considered significant (CI > 95). We used negative binomial distribution (DEseq2) and linear (Limma) statistical models for our RNAseq analysis. A total of 19,569 and 25,436 transcripts were analyzed through negative binomial (Deseq2) and linear (Limma) models, respectively; 9200 (~47.01%) and 9250 (~36.36%) genes, respectively, are differentially expressed with an adjusted p-value < 0.05. Since the adjusted p-values are <0.05, the alternations in these genes were considered significant irrespective of their log2FC (fold change) values  and biological difference (log ratios) of differentially expressed genes showed a large number of genes to be highly upregulated  or downregulated  in MM cells cultured with pDCs versus without pDCs  A volcano plot analysis to simultaneously assess the statistical significance DCs Fig. . TogetheGene Ontology and KEGG pathway analysis of RNAseq data provided evidence for the functional relevance of pDC-triggered genetic changes in MM cells . pDC-induced CD73 transcription in MM cells was confirmed using RT-PCR analysis . Although our data show changes in other purinergic signaling-associated genes, we focused on delineating the role of CD73 in the present study since: (1) CD73 is a common downstream ectonucleotidase mediating adenosine generation via both ATP- and NAD+-signaling axis triggered by CD39 and CD38, respectively; (2) adenosine is elevated in the MM BM milieu, which may contribute to immune deficiency characteristic of MM; (3) CD73 is expressed on exosomes [via regulating adenosine homeostasis, and represents a potential immunotherapeutic target in cancer; (5) Studies in solid tumor models have linked CD73 to tumorigenesis and drug-resistance [Importantly, pDCs increase exosomes , which isistance , 30; consistance ; and finsistance .CD73 expression has been linked with poor prognosis in patients with breast, lung, pancreas, gastric, or colon cancer [NT5E/CD73 [NT5E/CD73 expression can be regulated by ETS family of transcription factors. Our analysis further suggests the gene expression of several ETS family proteins is altered in MM after coculture with pDCs. . Interestingly, CD73 levels were also increased in pDCs after their coculture with MM cells . As a control, we have also evaluated the levels of adenosine in pDC alone in the presence or absence of anti-CD73 Ab  in pDC culture supernatant. We also performed additional studies with MM patient-derived total bone marrow mononuclear cells (MM BMSCs) treated with anti-CD73 Ab at various concentrations. Our results show that anti-CD73 Ab triggers a dose-dependent decrease in adenosine levels in MM BMNCs  Fig. . Convers Ab Fig. . The aden = 3, p < 0.05).A recent study showed that targeting CD73 enhances dendritic cell-mediated anti-tumor activity . Since Mn = 7, 1 pDC: 10 T cells), in the presence or absence of anti-CD73 Abs (0.5 µg/ml). After washing to remove the anti-CD73 Ab, cells were cultured for 24 h with autologous MM cells pre-stained with CellTracker or CellTrace Violet (10 T: 1 MM), followed by 7-AAD staining and quantification of MM cell lysis by FACS. Targeting CD73 induced significant MM-specific CD8+ CTL activity, evidenced by decreased viable MM cells . Furthermore, blockade of CD73 also decreased viable MM cells in patient BM-MNCs , suggesting that targeting metabolic pathway enzyme CD73 may impact the MM BM microenvironment and thereby evoke an anti-MM immune response. Importantly, our findings show that blockade of CD73 activates pDCs and restores pDC-induced T cell-mediated cytolytic activity against MM cells. It is noteworthy to mention here that anti-CD73 Ab does not have any direct cytotoxic effect on MM cells in the range of the concentrations used for the CTL assays  were co-cultured for 3 days with autologous pDCs (1 pDC:10 T cells) in the presence of isotype control Ab, anti-CD73 Ab, TLR-7 agonist, or anti-CD73 Abs plusTLR-7 agonist, and then evaluated in CTL assays against autologous MM cells (10 T: 1 MM cell). The combination of anti-CD73 Ab and TLR-7 agonist triggered more robust autologous MM-specific CD8 + CTL activity than either agent alone .The immunosuppressive BM microenvironment contributes to the induction of tolerogenic pDCs in MM , 5–16. Wone Fig. . These c+ CTL activity; and (6) the combination of anti-CD73 Ab and TLR-7 agonist induces more potent MM-specific CD8+ CTL activity against autologous tumor cells than either agent alone. Our preclinical data, therefore, provide evidence for an immunomodulatory role of CD73 enzyme in MM, and suggests that therapeutic targeting of CD73-mediated metabolic pathways  pDC-MM interactions induce transcription of adenosine-signaling pathway enzyme ecto-5' nucleotidase CD73 in MM cells (Fig. Supplemental-final-with FiguresSupplementary-file-1-NGS-RAW-data.csvSupplementary-file-2-volcano-plot.pdfAuthors-agreement-on-Change-of-authorshipAuthors-agreement-on-Change-of-authorship: Doc file


PBRM1, ARID2, and BRD7 expression by the previously unrecognized E2F1-mediated activation of their respective promoters. The functional significance of the MUC1-C→PBAF pathway is supported by demonstrating involvement of MUC1-C in associating with nuclear PBAF and driving the NRF2 antioxidant gene transcriptome in PC cells. Mechanistically, MUC1-C forms a complex with NRF2 and PBRM1 on the NRF2 target SLC7A11 gene that encodes the xCT cystine-glutamate antiporter, increases chromatin accessibility and induces SLC7A11/xCT expression. We also show that MUC1-C and PBRM1 are necessary for induction of other NRF2 target genes, including G6PD and PGD that regulate the pentose phosphate pathway. Our results further demonstrate that MUC1-C integrates activation of PBRM1 with the regulation of antioxidant genes, ROS levels, pluripotency factor expression and the cancer stem cell (CSC) state. These findings reveal a role for MUC1-C in regulating PBAF, redox balance and lineage plasticity of PC CSC progression. Our findings also uncover involvement of MUC1-C in integrating the PBAF and BAF pathways in cancer.The polybromo-associated PBAF (SWI/SNF) chromatin remodeling complex, which includes PBRM1, ARID2, and BRD7, regulates cell differentiation and genomic integrity. MUC1-C is an oncogenic protein that drives lineage plasticity in prostate cancer (PC) progression. The present work demonstrates that MUC1-C induces  The BAF and polybromo-associated BAF (PBAF) chromatin remodeling complexes of the SWI/SNF family are essential for mammalian gene transcription and development . The PBAThe oncogenic MUC1-C protein promotes lineage plasticity in the progression of castrate resistant prostate cancer (CRPC) to neuroendocrine prostate cancer (NEPC) by driving NE dedifferentiation, self-renewal capacity, and tumorigenicity . MUC1-C There is no known association between MUC1-C and the PBAF complex. Accordingly, studies to investigate their potential interactions were performed in MUC1-C-driven cell models of CRPC and NEPC progression , 18. We MUC1-C promotes lineage plasticity in the progression of CRPC cells . The PBAPBRM1 promoter (pPBRM1)  and BAF  subunits , 17, 18.M1) Fig.  and ChIPM1) Fig. . In concARID2 and BRD7 promoters  at positions +11552 to +11560 in intron 1  gene, which like G6PD regulates the PPP [PGD intron-3 region , converts NADP+ to NADPH in maintaining redox balance . G6PD isn-2 Fig. . ChIP-PCion Fig. . Moreoveion Fig. , E2F1  and found that silencing MUC1-C  NRF2 and PBRM1 occupancy, and (ii) chromatin accessibility. In addition and like PBRM1, silencing MUC1-C suppressed constitutive SLC7A11/xCT expression. Increases in ROS induce SLC7A11/xCT expression and the present results demonstrate that MUC1-C and PBRM1 are also necessary for this oxidative stress response. In further support of the MUC1-C→PBAF/PBRM1 antioxidant pathway, MUC1-C and PBRM1 were necessary for the expression of additional redox homeostasis effectors, such as G6PD and PGD, and for induction of GSTP1, HMOX1, and other enzymes that maintain redox balance. In concert with these findings, silencing MUC1-C and PBRM1 was associated with decreases in NADPH and GSH and abrogation of the capacity to control ROS levels in the presence of oxidative and genotoxic stress.Consistent with the association between replicative and oxidative stress , CSCs pre stress , in suppe stress , 6. MUC1e stress , 34. Like stress , NRF2 dre stress ; howeverynthesis . We founBRG1 gene is frequently mutated or silenced in multiple types of cancers, indicating a role as a tumor suppressor [The ppressor . In addippressor , 38. Of ppressor –41. BRG1ppressor . The finppressor . We founppressor , 43. In ppressor .Human DU-145 (ATCC) cells were cultured in RPMI1640 medium  containing 10% heat-inactivated FBS . Human LNCaP-AI cells were grown in phenol red-free RPMI1640 medium  containing 10% charcoal-stripped FBS  . Human NMUC1shRNA (MISSION shRNA TRCN0000122938), E2F1shRNA (MISSION shRNA TRCN0000010328), E2F1shRNA#2 (MISSION shRNA TRCN0000039658), PBRM1shRNA (MISSION shRNA TRCN0000235890), PBRM1shRNA#2 (MISSION shRNA TRCN0000015994), BRG1shRNA (MISSION shRNA TRCN0000231102), ARID1AshRNA (MISSION shRNA TRCN0000059092), a control scrambled shRNA (CshRNA)(Millipore Sigma), and vectors encoding MUC1-C or the MUC1-C(AQA) mutant were inserted into pLKO-puro or pLKO-tet-puro . Guide RNA (CATCGTCAGGTTATATCGAG) targeting MUC1-C was cloned into the lentiCRISPRv2 vector (Addgene #52961). The viral vectors were produced in 293T cells [Total cellular RNA was isolated using Trizol reagent (Thermo Fisher Scientific). cDNAs were synthesized and amplified as described . PrimersTotal lysates prepared from subconfluent cells were immunoblotted with anti-MUC1-C , anti-PBRM1 , anti-ARID2 , Danvers, MA, USA), anti-BRD7 , anti-β-actin , anti-E2F1 , anti-SLC7A11/xCT , anti-G6PD , anti-PGD , anti-BRG1 , anti-ARID1A , anti-OCT4 , anti-SOX2 , anti-KLF4 , anti-MYC , anti-NANOG , anti-NOTCH1 , anti-BMI1 , anti-CD44 , anti-CD133  and anti-GAPDH ., anti-E2F1 , anti-NRF2 , anti-PBRM1  or a control non-immune IgG (Santa Cruz Biotechnology). The DNA-antibody precipitates were reverse cross-linked at 65oC for 18 h. DNAs were purified using gel extraction columns  and analyzed by qPCR using the Power SYBR Green PCR Master Mix and the ABI Prism 7300 sequence detector (Applied Biosystems). Data are reported as relative fold enrichment 12. Primers used for ChIP qPCR are listed in Supplementary Table Formaldehyde cross-linked and sheered soluble chromatin was precipitated with pre-cleared magnetic dynabeads (ThermoFisher Scientific) and 2 μg/ml of anti-MUC1-C Assay for Transposase-Accessible Chromatin with high-throughput sequency (ATAC-seq) was performed on purified nuclei from 50,000 cells using Tn5 transposase as described . Sequenc3) were seeded per well in six-well ultra-low attachment culture plates (Corning Life Sciences) in DMEM/F12 50/50 medium (Corning Life Sciences) with 20 ng/ml EGF (Millipore Sigma), 20 ng/ml bFGF (Millipore Sigma) and 1% B27 supplement. Cells were treated with vehicle or DOX for 10–14 days. Tumorspheres were counted under an inverted microscope in triplicate wells.Cells , NADP/NADPH , GSH  and GSH/GSSG  levels were performed according to the manufacturer’s instructions validated with internal controls. Luminescence intensity was detected using an Infinite 200 PRO .Measurements of ROS, NADP/NADPH, GSH, GSH/GSSG levels. Assays for measurements of ROS  was considered statistically significant.Each experiment was performed at least three times. Data are expressed as the mean ± SD. The unpaired Mann–Whitney Supplemental Material