The immune checkpoint molecule PD-L1 (CD274) is a crucial regulator of the tumor immune response. Its expression has been reported in the therapeutic context in Head and Neck Squamous Cell Carcinoma (HNSCC), but it remains unclear how therapeutically approved molecules regulate PD-L1 expression in HNSCC cells.Three HNSCC cell lines  were used to analyze PD-L1 expression by immunoblotting, immunofluorescence and QPCR. Freely-available single cell RNAseq data from HNSCC were also used.5-Fluorouracil (5-FU) increased the expression of PD-L1 with high efficacy in HNSCC cells. Single cell RNAseq data suggested the specificity of the regulation of PD-L1 in this context. The effect of 5-FU on PD-L1 expression was related to its genotoxic effect and was prevented by extracellular application of thymidine or using a chemical inhibitor of the DNA damage Response kinases ATM/ATR. We found that the effect of 5-FU was additive or synergistic with IFN-γ, the canonical inducer of PD-L1 in epithelial cells. QPCR analysis confirmed this finding and identified JAK-dependent transcriptional activation of PD-L1/CD274 as the underlying mechanism. The induction of PD-L1 by 5-FU was partially prevented by Epidermal Growth Factor Receptor (EGFR) inhibition with cetuximab.Our study highlights the specific DNA Damage Response- and JAK- dependent induction of PD-L1 by 5-FU in HNSCC cells. This induction is regulated by the cytokine context and is potentially therapeutically actionable. CD274) and its receptor PD1, are approved for the treatment of recurrent/metastatic (R/M) HNSCC [Head and Neck Squamous Cell Carcinoma (HNSCC) are a heterogeneous group of tumors that require multimodal treatment with surgery and adjuvant radio(chemo)therapy ,2. DespiM) HNSCC ,4. PD1 tM) HNSCC ,4. PD-L1M) HNSCC ,7.i.e. those directly presenting with R/M HNSCC or those with high risk tumors . Platinum salts (cisplatin or carboplatin), taxanes (docetaxel or paclitaxel), and 5-fluorouracil (5-FU) are used in this setting de novo pyrimidine synthesis. This effect of 5-FU results in an inhibition of DNA synthesis and a block in cell cycle progression Chemotherapy is currently indicated for patients with advanced HNSCC, T cell infiltrate were detected in vitro studies found that cisplatin induced PD-L1 expression in HNSCC cells [An immune response directed against cancer cells is emerging as a mechanism that contributes to the efficacy of therapeutic protocols used against solid tumors ,11. InteCC cells ,14. It iCell culture. The cell lines BICR6, PE/CA-PJ34 and PE/CA/PJ-41 are described in detail in the supplementary Materials and Methods section. Cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum, 2 mM glutamine, and penicillin / streptomycin.Reagents and chemicals. All chemical reagents were purchased from Sigma, unless stated otherwise. Afatinib and VE821 were purchased from Selleckchem. The JAK inhibitor1 (JAKi) was purchased from Calbiochem . The Human Phospho-RTK Array Kit was purchased from R&D Systems . The antibodies used in this study are listed in the Suppl. Materials and Methods.Single cell gene expression analysis. Single cell RNAseq data (5902 cells sequenced from 18 HPV-negative HNSCC) were retrieved from Puram et al. (2017) Gene ontology (GO) analysis. We used the PANTHER classification system (http://pantherdb.org/) to perform a statistical overrepresentation test. The enrichment of Gene Ontology (GO) terms in our gene set was compared to the whole Homo Sapiens genome , with a False Discovery Rate (FDR) correction QPCR. Total RNA was extracted and reverse-transcribed using High Capacity cDNA Reverse Transcription kit and random hexamers (Applied Biosystems). Amplification was performed with the TaqMan Universal PCR master Mix on an ABI 7900HT Sequence Detection System (Applied Biosystems). Primers and probe sets for PD-L1 and Glyceraldehyde-Phosphate Dehydrogenase (GAPDH) are described in the Suppl. Material and Methods.Immunoblotting. Complete cell extracts were transferred to nitrocellulose membranes using standard procedures as previously described https://imagej.nih.gov/ij/download.html).Immunofluorescence. Immunofluorescence labeling of PD-L1 was performed on paraformaldehyde-fixed cells, according to standard procedures Statistical analyses. Analyses were done with R version 4.0.3 (https://www.r-project.org) using packages “Hmisc” and “venneuler”. Student's t-test and ANOVA were used as indicated (GraphPad Prism). The Spearman test was used for gene correlation analyses. p<0.05 was used as the threshold of significance. False discovery rate (FDR) correction was applied as indicated using the Bonferroni method.n = 8 chemotherapeutic agents . Cetuximab was applied at a concentration of 50 µg/mL and was found to block EGFR phosphorylation without significant inhibitory effect on the growth of HNSCC cells in vitro (data not shown). We then analyzed the protein expression of PD-L1 as well as PD-L2, CD80, CD86, and MHC class I molecules by immunoblotting  that were exposed to c agents . All cheblotting . We obseblotting . In two r = 0.05). We identified the genes whose expression was significantly correlated with PD-L1 or PD-L2 mRNA in cancer cells from single cell RNAseq data , and compared them with the genes that correlated with PD-L1 mRNA in tumor infiltrating immune cells (Suppl. Table 4). A number of genes were significantly co-expressed with PD-L1, but not with PD-L2 in HNSCC cells (Suppl. Table 2 and 3). These non-overlapping gene contexts suggest the existence of specific regulation of PD-L1 mRNA in HNSCC cells. A statistical overrepresentation test of Gene Ontology terms was performed on the panel of genes found to be correlated with PD-L1 in HNSCC cells, and suggested a link between PD-L1 expression and xenobiotic/chemotherapeutic metabolism  A. We fouInterferon γ (IFN-γ) is the canonical inducer of PD-L1 in cancer cells CD274 in BICR6 and PE/CA-PJ41 cells, by performing immunoblot and QPCR analysis  EGFR In the present study, we examined the effects of various chemotherapeutic agents on HNSCC cells and observed that 5-FU robustly increased PD-L1 expression. This induction was observed in three independent genomic contexts, suggesting its potential broad relevance. A clear interaction  was observed between 5-FU and IFN-γ. PD-L1 was homogeneously distributed on the surface of the whole population of HNSCC cells, suggesting that it was not accounted for by a minor subpopulation of cancer cells and that PD-L1 is functional in this context. The effect of 5-FU on PD-L1 expression appeared to be related to its genotoxic effect and was prevented by the extracellular application of thymidine or by the DNA damage Response inhibitor VE-821, directed against the kinases ATM/ATR. Finally, we found that EGFR inhibition partially prevented PD-L1 induction by 5-FU., although the corresponding study did not examine the effects of xenobiotics/chemotherapeutics Recent studies point to the regulation of PD-L1 by oncogenic, inflammatory and hypoxic signaling in cancer cells We examined the possibility of targeting the induction of PD-L1 using already approved anti-cancer drugs. We observed that EGFR signaling is induced by 5-FU and that its inhibition by cetuximab prevents PD-L1 upregulation. Previous studies found that oncogenic signaling downstream of the growth factor receptors (EGFR and HGFR) positively regulates PD-L1 expression in HNSCC cells ,27,28. Hin vivo experimental part with an immunocompetent animal model. However, based on previous studies that examined the role of PD-L1 in cancer cells in human HNSCC samples [Activation of an adaptive immune response directed against cancer cells is emerging as a mechanism that contributes to the efficacy of chemotherapeutic protocols ,11. The  samples ,7, we poC.L. Conceptualization, Investigation, Writing: Review & Editing; M.L. Writing: Review & Editing; F.R. Investigation, Writing: Review & Editing; C.L.: Investigation; C.G.: Investigation; B.C.: Supervision, Funding acquisition, Writing: Review & Editing; A.G.: Conceptualization, Funding acquisition, Project administration, Writing: Original Draft; Z.S.: Conceptualization, Project administration, Writing: Original Draft.The authors declare that they have no conflict of interest to disclose for this work.


Platelet adhesion and activation through the interaction of von Willebrand factor (VWF) with platelet glycoprotein (GP) Ibα are the early key events in hemostasis and thrombosis especially under high blood shear stress. P-selectin translocation from α granule to the cell surface is a typical platelet function phenotype, which makes the platelet-induced inflammatory response of flowing leukocytes possible and can be induced by either chemical agonists  or high blood shear stress, but regulations of VWF mutation and blood shear stress on VWF-induced P-selectin translocation remain unclear. With flow cytometry, parallel plate flow chamber, and immunofluorescence staining techniques, we examined the P-selectin translocation of platelets on immobilized wild-type (WT) VWF-A1 domain and its two mutants, the gain-of-function (GOF) mutant R1308L and the loss-of-function (LOF) mutant G1324S, respectively. The results showed that the VWF-A1-induced platelet P-selectin translocation was triggered, accelerated, and enhanced by fluid shear stress and could be correlated with shear stress accumulation , and the PI3K/Akt axis was involved in the platelet P-selectin translocation. The force-triggered P-selectin translocation occurred quickly on partial platelet surface first and then extended gradually to the whole platelet surface as SSA increased. The P-selectin translocation process would be promoted by the GOF mutation (R1308L) but slowed down by the LOF mutation (G1324S). These findings demonstrated a force-enhanced regulation mechanism for the VWF-induced platelet P-selectin translocation through the PI3K/Akt pathway and provided a novel insight into the mechano-chemical regulation mechanism for the key events, such as platelet activation and functional phenotype change in hemostasis and thrombosis. Platelet activation is essential for thrombosis and hemostasis . P-selec2 is applied to platelets for 10 min  under various fluid shear stresses using parallel plate flow chamber (PPFC) and immunofluorescence staining. The present results showed that platelet P-selectin translocation was triggered, accelerated, and enhanced by shear stress but correlated with shear stress accumulation (SSA) instead of shear stress and mechanical stimulus time. Platelet P-selectin translocation was enhanced through GOF mutant R1308L but reduced by LOF mutant G1324S. The molecules PI3K and Akt were involved in the VWF-A1-induced P-selectin translocation. This work might provide a novel insight into the mechano-chemical regulation mechanism for the key early events in hemostasis and thrombosis.TM . All the other reagents were of analytical grade or the best grade available.Apyrase, phorbol 12-myristate 13-acetate (PMA), Akt1/2 kinase inhibitor, and wortmannin (PI3 kinase inhibitor) were gained from Sigma-Aldrich . FITC-conjugated anti-P-selectin antibody was purchased from AbD Serotec . The rabbit anti-human P-selectin polyclonal antibody was from Sino Biological, Inc. , and the Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody was obtained from Thermo Fisher Scientificg for 10 min at room temperature. The platelet-rich plasma collected was supplemented with 5 U/ml apyrase to block platelet aggregation and centrifuged again at 1,000 × g for 10 min. The supernatant was removed, and the pellet was resuspended at a final concentration of 3 × 107/ml in phosphate-buffered saline (PBS) containing 5% platelet-poor plasma.Washed platelets were prepared as reported elsewhere . BrieflyEscherichia coli as a fusion protein containing the His-tag in the N-terminus and purified as previously described . After being washed with 50 mM sodium phosphate, 500 mM NaCl, and 20 mM imidazole at pH 7.4, the bound protein was eluted with the same buffer containing 150 mM imidazole. Mutant (R1308L and G1324S) plasmids were constructed by site-directed mutagenesis with Quick-Change II XL Site-Directed Mutagenesis kit  using the WTA1 plasmid as a template. The recombinant mutants were prepared as described for the WTA1 protein. The purified proteins were verified by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot .Recombinant VWF-A1 (amino acids Q1238–P1458) polypeptide was expressed in escribed . Brieflyg for 5 min to remove excessive agonists. The platelets were resuspended and fixed in 2% paraformaldehyde for 30 min. After washing with PBS, the fixed platelets were blocked with 2% BSA at room temperature for 1 h. The PBS, PMA, WTA1, R1308L, or G1324S-treated platelets were incubated with a FITC-conjugated anti-P-selectin antibody in dark condition for 1 h. After washing with PBS, the samples were analyzed using flow cytometry . For each condition, three replicative experiments were performed.To confirm the role of shear stress in platelet P-selectin translocation induced by VWF-A1, the washed platelets were incubated in five distinct solutions with (i) PBS which served as a blank control, (ii) 1 μM of PMA as a positive control, (iii) 200 μg/ml of WTA1, (iv) 200 μg/ml of the mutant R1308L, and (v) 200 μg/ml of the mutant G1324S for 10 min at room temperature and then centrifuged at 1,000 × 7/ml) were perfused into the flow chamber. The washed platelets were incubated in petri dishes coated with WTA1, R1308L, or G1324S for 10 min and adhered to the petri dishes. At the end of the incubation, the platelets were stimulated by perfusing PBS at various fluid shear stresses for a range of time . Prior to incubation with a rabbit anti-human P-selectin polyclonal antibody at 5 μg/ml in PBS containing 2% BSA, the stimulated platelets were fixed in 2% paraformaldehyde for 30 min at room temperature and subjected to saturation with PBS containing 2% BSA. An Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody at 4 μg/ml was used as a secondary antibody. To explore the signal pathway involved in the platelet P-selectin translocation induced by VWF-A1 in flows, washed platelets were pretreated with 1 μM wortmannin  experimental system , the plartmannin  or 10 μMrtmannin  for 10 mFP  and AP (the mean of P-selectin coverage area per P-selectin-positive platelet) as well as FIP , were chosen herein. With the immunofluorescence experimental data, the characteristic parameters FP, AP, and FIP were calculated byTo quantify P-selectin translocation on activated platelet, three parameters, such as AP, FIj, and FIj).Data are expressed as means ± standard error of the mean (SEM) from at least three independent experiments. Statistical significance was assayed by one-way (or two-way) ANOVA for multiple comparisons with a Bonferroni 2 . The platelet adhesion was specific for VWF-A1 because of the high adhesion levels for platelet on VWF-A1  in comparison with the low non-specific adhesion of platelet on substrates coated with the blank group treated with 2% BSA  at a fluid shear stress of 1 dyn/cmh 2% BSA . The adhh 2% BSA  as shownh 2% BSA .2 for 8 min . The rabbit anti-human P-selectin antibody was used to identify P-selectin-positive members of the immobilized platelets. We observed that P-selectin-positive platelets were rare for the platelets without mechanical preloads but became abundant for the platelets preloaded with fluid shear stresses of 10 dyn/cmor 8 min . Force lor 8 min . PMA couor 8 min  but coulor 8 min . Besidesor 8 min  as demonor 8 min . These d2 for 8 min . The data showed that there was no significant difference between the vehicle (DMSO) and controls , we herein examined the effects of mechanical stimulus and mutation on VWF-A1-mediated P-selectin translocation on the immobilized platelets. Three parameters, such as the P-selectin-positive platelet fraction FP and the mean of P-selectin coverage area per positive platelets AP as well as the mean normalized P-selectin-related fluorescence intensity per positive platelets FIP , were exploited to weigh the platelet P-selectin translocation. The results were shown in the plots of FP, AP, and FIP against fluid shear stress τw with various mechanical stimulus time T of 0, 1, 2, 4, and 8 min for platelet on the immobilized VWF-A1  (τw = 0) should be rare and weak due to the low P-selectin-positive fraction FP and small P-selectin coverage area AP , and increasing stimulus time T would cause an upswing of both FP ∼ τw and AP ∼ τw curves obviously, especially for τw < 5 dyn/cm2  reduced either FP and AP under diverse preloading, and GOF mutant (R1308L) did inversely, suggesting that VWF affinity to GPIbα regulated the force-dependent VWF-induced translocation of platelet P-selectin -coated substrates at fluid shear stresses of 0, 2.5, 5.0, and 10 dyn/cm G1324S) . For pla area AP . Both FP dyn/cm2 . It was nditions , suggestselectin .τw and mechanical stimulus time T on the VWF-A1-induced platelet P-selectin translocation were shown not only in FP ∼ τw and AP ∼ τw curves for each given T   .FP, AP, and FIP against SSA  made both FP∼SSA and AP∼SSA curves upswing, and the LOF mutation (G1324S) did inversely, while these two different mutations had almost no effects on FIP  but slowed down by the LOF mutation (G1324S). These results were consistent with previous work for SSA-prompted platelet activation using a modified prothrombinase assay (The plots of inst SSA  demonstrectively –3. All omparable , althougs on FIP . These rse assay .As the first step for hemostasis, VWF recruits the circulating platelets to the vascular injured sites . The intPrevious studies demonstrated a force-dependent platelet P-selectin translocation through VWF-GPIbα signaling in flows , but for2 (–1 was less than that on WTA1 (It was demonstrated that a mutation in the VWF-A1 domain might enhance or reduce platelet adhesion through changing VWF affinity to GPIbα, but less knowledge was relevant to the effects of VWF-A1 mutation on P-selectin translocation of the immobile platelets under shear stress. Similar to the previous work which found that the VWF cleaved by MMP-13 promoted the binding affinity to platelet and improved the platelet P-selectin translocation in comparison with intact VWF , our res2 , suggest2 , and LOF2 . In cont on WTA1 . Perhaps on WTA1 , but thi on WTA1 .In conclusion, our data suggested that VWF-induced platelet P-selectin translocation would be triggered, accelerated, and enhanced by fluid shear stress but correlated with shear stress accumulation. This SSA-dependent P-selectin translocation of platelets would be enhanced by GOF mutant R1308L but reduced through LOF mutant G1324S. The present work provided a novel insight into the mechano-chemical regulation on adhesive molecule-mediated platelet P-selectin translocation and its relevant biological processes, such as tumor cell hematogenous metastasis, hemostasis, and inflammatory responses under flows.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by the Guangzhou First People’s Hospital. The patients/participants provided their written informed consent to participate in this study.JW and YF designed this research. JFa supervised this study overall. XS, SL, PY, JFe, JL, MAC, and J-FD partly performed research and analyzed data. JFa, YF, and JW wrote this manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Bifidobacterium strains on human peripheral blood mononuclear cells and collagen-induced arthritis (CIA) mice were assessed. Significant differences in gut microbiota composition were observed in patients with different RF levels. The relative abundance of Bifidobacterium and Collinsella was lower in RF-high than in RF-low and RF-negative RA patients, while the relative abundance of Clostridium of Ruminococcaceae family was higher in RF-high than in RF-low and RF-negative patients. Among 10 differentially abundant Bifidobacterium, B. longum RAPO exhibited the strongest ability to inhibit IL-17 secretion. Oral administration of B. longum RAPO in CIA mice, obese CIA, and humanized avatar model significantly reduced RA incidence, arthritis score, inflammation, bone damage, cartilage damage, Th17 cells, and inflammatory cytokine secretion. Additionally, B. longum RAPO significantly inhibited Th17 cells and Th17-related genes—IL-17A, IRF4, RORC, IL-21, and IL-23R—in the PBMCs of rheumatoid arthritis patients. Our findings suggest that B. longum RAPO may alleviate RA by inhibiting the production of IL-17 and other proinflammatory mediators. The safety and efficacy of B. longum RAPO in patients with RA and other autoimmune disorders merit further investigation.The potential therapeutic effects of probiotic bacteria in rheumatoid arthritis (RA) remain controversial. Thus, this study aimed to discover potential therapeutic bacteria based on the relationship between the gut microbiome and rheumatoid factor (RF) in RA. Bacterial genomic DNA was extracted from the fecal samples of 93 RA patients and 16 healthy subjects. Microbiota profiling was conducted through 16S rRNA sequencing and bioinformatics analyses. The effects of  Rheumatoid factor (RF) is an autoantibody detected in approximately 80% of patients with the autoimmune disease rheumatoid arthritis (RA) , 2. In aPorphyromonas gingivalis, can trigger RA development -17 lymphocytes in the lamina propria, inducing autoantibody production and aggravating RA . Human siving RA . FurtherSKG mice . MeanwhiSKG mice .in vivo studies to determine whether a single strain of Bifidobacterium, which was found based on the microbiome analysis, could modulate RA symptoms in an RA mouse model.Notwithstanding the increasing evidence of the role of microbiome in RA, the relationship between the gut microbiome and RF is understudied. In this study, we conducted microbiome analyses using bioinformatics to investigate the association between intestinal microflora and RF levels. We also performed n = 16), RF-low (n = 24), and RF-high (n = 53) groups. Patients with RF ≤20 IU/ml were considered RF-negative, those with RF levels between 20 and 60 IU/ml were considered RF-low, and those with RF >60 IU/ml were considered RF-high. Subjects who had recently used antibiotics, probiotics, or prebiotics were excluded from the study. The demographic and clinical characteristics of the study subjects are summarized in In this study, we enrolled 93 RA patients and 16 healthy subjects Table 1.Individual human fecal samples were sent immediately with ice to the research site after collection in a plastic container and were stored at −70°C within 12 h of arrival. Isolation of the bacterial genomic DNA from the fecal samples was performed using a QIAamp DNA Stool Mini Kit  following the instructions of the manufacturer, followed by bead beating on a TissueLyser system (Qiagen). Quantification of the bacterial genomic DNA was carried out using a Qubit 3.0 Fluorometer . For sequencing, 16S rRNA gene amplification and index PCR were conducted following the Illumina 16S Metagenomic Sequencing Library preparation guide . For amplification of V3 and V4 regions, the 16S sequence was amplified using forward primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGCCTACGGGNGGCWGCAG-3′) and reverse primer (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGACTACHVGGGTATCTAATCC-3′). For indexing, Nextera XT Index 1 and 2 Primers from a Nextera XT Index kit (Illumina) were used. Each PCR product was purified using AMPure XP beads . DNA sequencing was conducted in using the paired-end method (300 bpx2) with an Illumina MiSeq instrument according to the Illumina protocol. Raw 16S rRNA sequences were bioinformatically analyzed using QIIME 2 version 2019.4 , as prevBifidobacterium, was used to separate Bifidobacterium from 100 mg of human fecal samples. After plating fecal samples diluted with phosphate-buffered saline (PBS) and culturing for 30 h at 37°C in an anaerobic jar under anaerobic condition, colonies were randomly taken from a plate from each sample and inoculated with cysteine De Man, Rogosa, and Sharpe (cMRS) broth and cultured for 24 h. The above process was performed in a glove box for blocking oxygen. Identification of bacteria strain was done with 16S rRNA sequencing service of Macrogen Inc. .Transgalactooligosaccharide (TOS) antibiotic mupirocin agar medium, a selective medium for via centrifugation  at 20°C and 7,000 rpm. After two washes with PBS, the bacterial samples were dried at 36°C and centrifuged at 2,000 rpm for 24 h using a Scanvac Speed Vacuum Concentrator . All bacteria used in this study were isolated from healthy subjects and RA patients. Donor characteristics are summarized in Bacterial samples (50µl) were inoculated into 5 L of cMRS liquid medium and incubated for 20 h at 37°C. After incubation, the bacteria were collected null (NSG) mice  were maintained under specific pathogen-free conditions in an animal facility with controlled humidity (55 ± 5%), light (12 h/12 h light/dark), and temperature (22 ± 1°C). The air in the facility passed through a HEPA filter system designed to exclude bacteria and viruses. Animals were fed mice chow  and water ad libitum. All experimental procedures were approved by the Institutional Animal Care and Use Committee at the School of Medicine and the Animal Research Ethics Committee of the Catholic University of Korea and were conducted in accordance with the Laboratory Animals Welfare Act according to the Guide for the Care and Use of Laboratory Animals. All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: CUMC 2019-0242-01). All procedures performed in this study followed the ethical guidelines for animal use.Seven-week-old male DBA/1J mice  and 7-week-old male NOD-scid IL2rγvia injection into the bottom of the tail. At 2 weeks following immunization, the mice were boosted with 100 µg of CII in incomplete Freund’s adjuvant (Chondrex Inc.). In addition, the obese CIA mouse group was fed a high-fat diet  at primary immunization. A HFD was maintained until the sacrifice.Collagen-induced arthritis (CIA) was established in male DBA/1J mice. Briefly, mice were inoculated with 100 µg of chicken type II collagen  liquified overnight in 0.1 N acetic acid (4 mg/ml) in complete Freund’s adjuvant. Immunizations were performed intradermally 5 peripheral blood mononuclear cells (PBMCs) of RA patients were injected into the tail vein. At 2 weeks after, orbital blood was collected to confirm engraftment by flow cytometry. Orbital blood was immunostained with CD4 T cells and CD8 T cells, and the cells were stained with Alexa Flour 700-conjugated anti-human CD4 antibody  and allophycocyanin (APC)-Cy7-conjugated anti-human CD8 (BD Biosciences). Thereafter, 1 week after confirmation of engraftment, the mice were sensitized with 50 µg of lipopolysaccharide . Each in vivo experiment was repeated a total of three times.To induce arthritis in humanized mice, 5 × 10Arthritis severity was determined using the mean arthritis index, which ranges from 0 to 4, as follows: 0, no evidence of erythema or swelling; 1, erythema and mild swelling confined to the midfoot  or ankle joint; 2, erythema and mild swelling extending from the ankle to the midfoot; 3, erythema and moderate swelling extending from the ankle to the metatarsal joint; and 4, erythema and severe swelling encompassing the ankle, foot, and digits. The severity of arthritis was determined as the sum of scores from all legs assessed by two independent observers blinded to the experimental groups.Mouse joint tissues were fixed in 4% paraformaldehyde , decalcified in a histological decalcifying solution , and embedded in paraffin wax for histological analyses. Sections (7 μm) were prepared and stained with hematoxylin , eosin , and safranin O (Sigma-Aldrich). Cartilage damage was marked as described previously .Joint tissues were incubated overnight at 4°C with primary antibodies against TNFα , IL-17 (Abcam), IL-6 (Abcam), and IL-1β . Subsequently, samples were incubated with a biotinylated streptavidin–peroxidase complex for 1 h, and the signals were developed using chromogen 3,3′-diaminobenzidine . The sections were examined under a photomicroscope . The number of positive cells in high-power digital images  was counted using Adobe Photoshop software . Stained cells were counted independently by three observers, and the mean values were evaluated.Serum was gathered 7 weeks after the first immunization for concentration of the IgA, IgM, IgG2a, and collagen-specific IgG2a levels, and the serum was stored at −70°C until further use. The levels of the IgA, IgM, IgG2a, and collagen-specific IgG2a antibodies in the serum were measured using ELISA .via intracellular staining with the following antibodies: fluorescein isothiocyanate-conjugated anti-IL-17, APC-conjugated anti- interferon-gamma (IFN-γ), and PerCP-conjugated anti-CD4. Before staining, cells were stimulated for 4 h with phorbol myristate and ionomycin in the presence of GolgiStop (BD Biosciences). To analyze regulatory T (Treg) populations, splenocytes were stained with PerCP-conjugated anti-CD4, APC-conjugated anti-CD25, and PE-conjugated anti-Foxp3 antibodies. T helper and Treg populations were determined using specific antibodies . After surface staining for 30 min, the cells were permeabilized with Cytofix/Cytoperm solution (BD Biosciences). Thereafter, the cells were intracellularly stained with fluorescent antibodies. Flow cytometry was performed with the aid of a cytoFLEX Flow Cytometer , and flow cytometry data were analyzed using FlowJo .Cytokine expression in mice was analyzed PBMCs were isolated from healthy donors. Briefly, blood from donors was mixed with PBS, and the mixture was cautiously layered onto 10 ml of Ficoll Plaque Plus solution . Blood samples were centrifuged for 30 min at 2,500 rpm and 20°C to separate blood contents. After centrifugation, the layer containing PBMCs was collected, and the cells were washed with PBS and cultured in RPMI medium containing 10% fetal bovine serum. PBMCs were incubated with plate-bound anti-CD3 (0.5 μg/ml) and then subjected to IL-17 analysis by ELISA for 3 days or subjected to RNA sequencing and real-time polymerase chain reaction analysis for 2 days.B. longum RAPO or vehicle treatment with anti-CD3 (0.5 μg/ml). IL-17 was analyzed by sandwich enzyme-linked immunosorbent assay . The absorbance at 450 nm was determined using an ELISA microplate reader (Molecular Devices).The supernatant was collected 3 days after Homo sapiens (hg38) using HISAT v2.1.0 (Homo sapiens (hg38) and the annotation data were downloaded from the UCSC table browser (http://genome.uscs.edu). Transcript assembly and abundance estimation used StringTie  and KEGG pathway (http://www.genome.jp/kegg/pathway.html).The relative abundances of gene were measured in read count using StringTie. We performed statistical analysis to find differentially expressed genes using the estimates of abundances for each gene in the samples. Genes with one more than zeroed read count values in the samples were excluded. To facilitate log2 transformation, 1 was added to each read count value of filtered genes. Filtered data were log2-transformed and subjected to TMM normalization. Statistical significance of the differential expression data was determined using exactTest using edgeR and fold change in which the null hypothesis was that no difference exists among groups. False discovery rate (FDR) was controlled by adjusting Total RNA was extracted using TRI reagent  and cDNA was synthesized with the Dyne First Strand cDNA Synthesis Kit  according to the protocol of the manufacturer. mRNA was quantified using the StepOnePlus™ Real-Time PCR systems  with SensiFAST SYBR Hi-ROX . mRNA levels were normalized to that of β-actin. The following primers were used: β-actin 5′-GGACTT CGAGCAAGAGATGG-3′ and 5′-TGTGTTGGGGTACAGG TCTTTG-3′; IL-17A 5′-CAACCGATCCACCTCACC TT-3′ and 5′-GGCACTTTGCCTCCCAGAT-3′; RORC 5′-AGTCGGAAGGCAAGATCAGA-3′ and 5′-CAAGAGAGGTTCTGG GCAAG-3′; IL-21 5′-TGTGAATGACTTGGACCCTGAA-3′ and 5′-AAACAG GAAATAGCTGACCACTCA-3′; IRF-4 5′-CCTGCAAGCTCTTTGACACA-3′ and 5′-GAGTCACCTGGAATCTTGGC-3′; and IL-23R 5′-AGGTACTGGCAGCCTTGGAGTT-3′ and 5′-CCCTGTAGAGATGGAAGCAACTG-3′.t-test or the Mann–Whitney U-test. Differences in the mean values of more than two groups were assessed using analysis of variance with a post-hoc test. p-values <0.05 (two-tailed) were considered to indicate statistical significance.Non-parametric statistical tests  were used to compare the alpha diversity of each group and the relative abundance of each taxon. Pairwise permutational analysis of variance (PERMANOVA) was used to compare beta diversity between groups. Spearman correlation analysis was used to analyze the relationship between the relative abundance of each taxon and RF levels. Data were analyzed using GraphPad Prism  and are presented as means ± standard deviations. Comparisons of numerical data between two groups were performed using Student’s p = 0.027, PERMANOVA; p = 0.010, PERMANOVA; p = 0.045, PERMANOVA; p = 0.003, PERMANOVA; To examine the relationship between RF levels and changes in intestinal microbial diversity, we performed gut microbiome profiling through 16S rRNA sequencing of fecal samples from 77 RF-positive patients, 16 RF-negative patients, and 16 healthy volunteers Table 1.R = −0.29, p = 00051; R = 0.22, p = 0.0322; Bifidobacterium were significantly more abundant in RF-negative than in RF-high RA patients  with B. pseudocatenulatum 20T2, B. pseudocatenulatum 20T1, B. pseudocatenulatum 18T6, B. pseudocatenulatum 5T4, B. longum 4L7, B. longum 4L6, B. adolescentis 8T3, B. adolescentis 8T1, B. bifidum 7T2, and B. longum RAPO. Among these strains, B. longum RAPO exhibited the most potent inhibitory effect on IL-17 secretion .To evaluate the effects of B. longum RAPO in obese CIA, obese CIA-induced RA mice were fed the HFD for 7 weeks. There was a significant decrease in the arthritis score and incidence in the B. longum RAPO-administered mice compared with the vehicle-treated obese CIA mice  population was reduced by the administration of B. longum RAPO; on the other hand, the populations of Th1 (IFN-γ+ in CD4 T cells) and Th2 (IL-4+ in CD4+ T cells) did not differ significantly between B. longum RAPO- and vehicle-treated mice with obese CIA .An obese arthritis model was reported according to a previous report . Obese CB. longum RAPO for 72 h. Flow cytometry showed that B. longum RAPO treatment significantly suppressed Th1 and Th17 cell proliferation .The PBMCs of RA patients were cultured under anti-CD3 0.5 μg/ml conditions with B. longum RAPO can have an effect on human avatar arthritis mice. The engraftment of the patient PBMC was confirmed by analyzing the expression of CD4 and CD8 T cells using human antibodies .Also, it was confirmed that Th17 cells were effectively inhibited like CIA and obese CIA  restored gut microbial dysbiosis and prevented bone destruction in a rodent model of RA  repository, accession number PRJNA759926. The RNA-sequencing data presented in the study is deposited in the GEO repository, accession number GSE183154.The studies involving human participants were reviewed and approved by the Institutional Review Board of Seoul St. Mary’s Hospital, The Catholic University of Korea . The patients/participants provided their written informed consent to participate in this study. All experimental procedures were evaluated and conducted in accordance with the protocols approved by the Animal Research Ethics Committee at the Catholic University of Korea (Permit Number: CUMC 2019-0242-01). All procedures performed in this study followed the ethical guidelines for animal use.All authors were involved in drafting the article or revising it critically for important content. M-LC, GJ, and S-HP had full access to all the data in the study and took responsibility for the integrity of the data and the accuracy of the data analysis. Conception and design of the study: YJ and JJ. Acquisition of data: YJ, JJ, S-YL, HN, JC, K-HC, SL, AL, and S-JP. Analysis and interpretation of data: J-WK, MP, and BK. Drafting the article: YJ and JJ. Revising the article critically: M-LC, GJ, and S-HP. All authors contributed to the article and approved the submitted version.This research was supported by the Bio & Medical Technology Development Program of the National Research Foundation (NRF) and funded by the Korean Government (MSIT) (No. NRF-2017M3A9F3041045).Authors YJ, S-JP, MP, BK and GJ was employed by company BIFIDO Co., Ltd.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


PP2A-B genes were identified from the soybean genome via homologous sequence alignment, which was distributed across 20 soybean chromosomes. Among soybean PP2A-B family genes, 26 GmPP2A-B members were found to be responsive to drought and salt stresses in soybean transcriptome data. Quantitative PCR (qPCR) analysis demonstrated that GmPP2A-B had the highest expression levels under salt and drought stresses. Functional analysis demonstrated that overexpression of GmPP2A-B in soybeans can improve plant tolerance to drought and salt stresses; however, the interference of GmPP2A-B in soybean increased the sensibility to drought and salt stresses. Further analysis demonstrated that overexpression of GmPP2A-B in soybean could enhance the expression levels of stress-responsive genes, particularly genes associated with ROS elimination. These results indicate that PP2A-BAbiotic stresses induce the accumulation of reactive oxygen species (ROS) and significantly affect plant growth. Protein phosphatase 2A (PP2A) plays an important role in controlling intracellular and extracellular ROS signals. However, the interaction between PP2A, ROS, and stress tolerance remains largely unclear. In this study, we found that the B Glycine max (L.) Merr.] is one of the main plant proteins and lipid resources and plays an important role in our national agricultural production; its planting area ranks first among the four oil crops. However, climate change continues to threaten soybean growth, which has resulted in serious losses in yield, especially abiotic stresses such as drought, soil salinization, and high temperatures , cold, and abscisic acid (ABA) at the transcriptional level, and overexpression of TaPP2AbB in transgenic Arabidopsis had more lateral roots under mannitol or NaCl treatment. These results indicated that TaPP2AbB involved in various stress responses and promoted lateral root growth under osmotic conditions , a well-conserved catalytic (C) subunit, and a regulatory (B) subunit . The B sructures . Motifs g domain , and furactivity . This in pathway , 2019. Tnditions . It was nditions . Our resArabidopsis, which mediated regulation of the cascade reaction of brassinolide (BR) signal from cell surface receptor kinase to gene regulation in the nucleus  of each PP2A-BWe did a protein BLAST search of the soybean genome in the Phytozome databaseGmPP2A-B genes were distributed across 20 soybean chromosomes; the distribution map of the 83 GmPP2A-B family genes on the chromosomes was made by Map Gene 2 Chromosome v2 (MG2C) software.3 Eighty-three GmPP2A-B and 43 AtPP2A-B were used for multiple sequence alignment analysis to construct a phylogenetic tree using the maximum likelihood (ML) method. The phylogenetic tree was constructed using MEGA-X with 1000 Bootstrap replicates.Through the Phytozome database, we found that the 83 identified 4 was used to determine the structures of the 83 GmPP2A-B genes ] (Cis-acting element diagrams were generated using GSDS 2.0. We used Soybase6 to identify the expression patterns of the 83 GmPP2A-B genes in different tissues (Gene Structure Display Server 2.0 (GSDS 2.0) software′′ genes . Motif asoftware . We seleent.be)] . Cis-act tissues , and the tissues .Actin (100500082) gene was used as the internal control (GmPP2A-B genes were determined using the 2–ΔΔCT method. All primers are listed in Soybean variety “Williams 82” was used for quantitative PCR (qPCR) analysis. Soybean seeds were sown into flowerpots and after 2 weeks of growth, soybean seedlings were irrigated with 200 mM NaCl and 250 mM mannitol, respectively. A total of 0.1 g of soybean leaf blade tissue was collected at 0, 2, 4, 6, 8, 10, and 12 h after the 200 mM NaCl induced salt stress and 250 mM mannitol induced drought stress treatments, and total RNA was extracted using an RNA extraction kit . Double stranded cDNA was obtained using a cDNA synthesis kit  . qPCR re control . The expGmPP2A-B was cloned into vector 16318h-GFP, containing a green fluorescent protein (GFP) reporter gene, and the recombinant vector was transformed into the Top10 strain of Escherichia coli. After sequencing, the GmPP2A-BArabidopsis protoplasts by the PEG4000-mediated method (The full length coding sequence (CDS) of d method . GFP sigGmPP2A-B was cloned into vector pCAMBIA1302 to construct recombinant vector pCAMBIA1302-GmPP2A-B. After sequencing, the recombinant vector was transformed into Agrobacterium tumefaciens strain GV3101 by the freeze-thaw method. Agrobacterium-mediated inflorescence impregnation was used to generate transgenic Arabidopsis plants. Arabidopsis plants (ecotype Col-0) were grown under normal conditions  until flowering. A. tumefaciens carrying the recombinant vector was cultivated in 50 mL of LB liquid culture medium with antibiotics (rifampicin and kanamycin). When the optical density (OD) of the culture reached 0.6–0.8, the culture was spun down and re-suspended in 1/2 MS liquid medium to generate the Agrobacterium impregnation solution to be used for Arabidopsis transformation, according to the methods described by The CDS of 0 generation of transgenic Arabidopsis plants were screened on 1/2 MS solid medium supplemented with hygromycin (30 mg/L). After screening for 14 days, positive transgenic Arabidopsis seedlings were transferred into soil for growth. The T3 generation of transgenic Arabidopsis plants were used for qPCR analysis to determine the expression level of GmPP2A-B. After identification, the T4 positive generation of transgenic Arabidopsis seedlings were used in experiments.The TGmPP2A-B transgenic Arabidopsis lines and wild-type (WT) plants were sown into flowerpots. Three weeks old of GmPP2A-B transgenic Arabidopsis and WT plants were used for stress tolerance analysis. For salt tolerance experiments, the 3 week old plants were irrigated with a 250 mM NaCl solution. After 5 days of salt treatment, leaf samples were collected for measurement of relative electrolyte leakage. After 7 days of salt treatment, phenotypes were observed and survival rates were calculated. In addition, we used different concentrations of NaCl (100 and 125 mM) for salt stimulation to observe the root system of GmPP2A-B transgenic and WT Arabidopsis seedlings. The seeds of GmPP2A-B transgenic and WT Arabidopsis plants were disinfected by sodium hypochlorite (0.9%), transferred to 1/2 MS solid medium for 3 days of low temperature treatment (4–8°C), then transferred to normal growth conditions. After 7 days of growth, the seedlings of GmPP2A-B transgenic and WT Arabidopsis plants were transferred onto 1/2 MS solid medium supplemented with different concentrations of NaCl (100 and 125 mM) for salt stress treatment. After 7 days of treatment, the fresh weight and root lengths were measured.The seeds of three homozygous GmPP2A-B for 10 days, after which leaf samples were taken for measurement of relative electrolyte leakage. After 14 days of drought treatment, phenotypes were observed and the soil water content of each flowerpot was measured; the resulting data is presented in GmPP2A-B transgenic and WT Arabidopsis plants were disinfected with sodium hypochlorite (0.9%), transferred to 1/2 MS solid medium for 3 days of low temperature treatment (4–8°C), then transferred to normal growth conditions. After 7 days of growth, the seedlings of GmPP2A-B transgenic and WT Arabidopsis plants were transferred to 1/2 MS solid medium supplemented with different concentrations of PEG6000 (4 and 6% PEG6000) to simulate drought stress. After 7 days of treatment, the fresh weight and root lengths were measured. Experiments were repeated three times. Each experimental repetition included 5 groups, and each group contained 10 each of GmPP2A-B-OE1, GmPP2A-B-OE2, GmPP2A-B-OE3, and WT (Col-0) plants. All plants of were cultured at 25°C under a 16:8 h light/dark photoperiod in a greenhouse.For drought experiments, watering was withheld from 3 week old GmPP2A-B was cloned into the pCAMBIA3301 vector by homologous recombination to create the OE construct. To create the GmPP2A-B RNA interference (RNAi) construct, 200 bp of the GmPP2A-B CDS containing a hairpin structure was selected as the inducer sequence, artificially synthesized (BGI China), and cloned into the pCAMBIA3301 vector. The overexpressing-, interfering-, and empty-vector (EV-control) were transformed into Agrobacterium rhizogenes strain K599. The resulting A. rhizogenes strains were cultured for 3 days in a 28°C incubator, then used to transform soybean plants using the methods described by A. rhizogenes carrying the respective recombinant vectors were injected into 7 day old soybean plants, 1–2 mm below the cotyledonary node. After injection, soybean seedlings were kept in darkness for 12 h of induction, then grown under normal conditions  for 3 weeks to encourage hairy root growth; after 3 weeks hairy roots grew up to about 5 cm. The part 1 cm below the inoculation site was removed, and soybean seedlings with hairy roots were transferred into new flowerpots to generate mature soybean plants with GmPP2A-B transgenic hairy roots.Seeds of soybean variety “Williams 82” were sown into flowerpots. The CDS of GmPP2A-B transgenic soybean plants with hairy roots were irrigated with a 250 mM NaCl solution. After 3 days of salt treatment, 0.1 g of hairy root samples were collected. The malondialdehyde (MDA) and proline (Pro) contents of samples were measured using the MDA and Pro Assay kits , and the catalase (CAT) and peroxidase (POD) activities of the samples were measured using the Micro Catalase (CAT) and Micro Peroxidase assay kits , respectively, following the manufacturers protocols. After 1 week of salt stress induction, phenotypes were observed and chlorophyll content was measured.In salt tolerance experiments, EV-control and GmPP2A-B transgenic soybean plants with hairy roots for 10 days of drought treatment, after which hairy root samples were collected and the MDA and Pro contents, and CAT and POD activities were measured as described. After 14 days of drought treatment, phenotypes were observed and chlorophyll and soil water contents were measured. Soil water content data is presented in GmPP2A-B-RNAi, EV-control, and GmPP2A-B-OE soybean plants. All soybean plants were cultured at 25°C under a 16:8 h light/dark photoperiod in a greenhouse.In drought tolerance experiments, water was withheld from EV-control and For drought tolerance experiments, soybean plants were grown in 15 cm diameter flowerpots containing 315.16 ± 0.1 g of soil (1:1 ratio of soil to vermiculite) and watered with 300 mL of water. To determine the soil water content of soil under normal growth conditions, soil samples were collected after 3 days of growth and the weight was recorded (W1), after which the samples were baked at 150°C until completely dry, and the weight was again recorded (W2); the soil water content was calculated using the following formula: (W1–W2) × 100/W1. After 14 days of no watering, soil samples were taken again to determine soil water content under drought conditions, as described for the samples taken under normal growth conditions.GmPP2A-B-RNAi, EV-control, and GmPP2A-B-OE soybean plants under salt and drought treatments, root tips and leaves of stress-induced plants were collected and soaked in Nitro Blue Tetrazolium (NBT) solution for 30 min and overnight, respectively. After 30 min of staining, the stress-induced root tips were observed under the microscope . Leaves were transferred to a 7:3 (v/v) acetone/glycerol decolorization solution and boiled (100°C) for 1 h then observed under the integrated microscope.To assess the degree of ROS accumulation of GmPP2A-B-OE, EV-control, and GmPP2A-B-RNAi), pulverized with a mortar and pestle, then incubated in 1 mL of 80% acetone solution for 12 h in darkness. The chlorophyll content of the samples was then measured using a Varioskan LUX enzyme-labeling measuring instrument  at wavelengths of 665 and 649 nm; the acetone solution alone was used as a blank control. Measurements were taken from three biological replicates.To measure the chlorophyll content in transgenic soybean leaves, 0.1 g of leaf tissue was sampled from each transgenic soybean line  (25°C). The hairy root samples were then heated at 100°C for 30 min, then allowed to cool to room temperature (25°C) to determine the final electric conductance (S2). The relative electrolyte leakage was evaluated as: REC = S1 × 100/S2.t-tests. Differences were considered significant when at p < 0.05. Results are expressed as the mean ± standard deviation (SD).SPSS 19.0 software  was used to compare the means of each treatment group using one-way ANOVA and GmPP2A-B to GmPP2A-B according to their chromosomal positions and Pfam databases  . Group X′′ genes .GmPP2A-B  staining analysis to observe the ROS levels of the GmPP2A-B-RNAi, EV-control, and GmPP2A-B-OE soybean plants under drought treatment. Our results demonstrated that the leaves and root tips of GmPP2A-B-OE plants had lower ROS levels with a lighter color of NBT staining than EV-control plants, indicating that GmPP2A-B overexpressed soybeans had stronger ROS-scavenging ability than EV-control plants under drought conditions. GmPP2A-B-RNAi plants presented the opposite phenotype, with higher ROS levels than EV-control plants. We further measured the chlorophyll content in the leaves and the Pro, MDA, POD, and CAT contents in the roots of GmPP2A-B-RNAi, EV-control, and GmPP2A-B-OE soybean plants under drought conditions. Our results demonstrated that Pro, chlorophyll, POD, and CAT contents in GmPP2A-B-OE soybean plants were higher than in EV-control plants, while the MDA content in GmPP2A-B-OE soybean plants was lower than in EV-control plants. However, GmPP2A-B-RNAi plants had lower Pro, chlorophyll, POD, and CAT contents and higher MDA contents than EV-control plants  family . PP2A, afamilies . Gene st2A-B′′58 , 3. MotiGroup VI . These rome data . Of thes pathway  and have pathway –9. Thesehomology , 8, 9, wHMG-CoA reductase (HMGR) gene is a key regulator in the MVA pathway, which is regulated by several endogenous signals and external stimuli , which gives rise to an imbalance in ROS homeostasis. Overexpression of CAT removes the excess H2O2, which can maintain a steady pattern of ROS to mediate plant growth regulation  peptide incorporation (GmPP2A-B-OE plants presented higher stress tolerance and lower ROS levels than EV-control plants, and the qPCR results demonstrated that the expression levels of GmLEA15, GmCAT1, GmPOD, and GmERF115 genes in GmPP2A-B-OE plants were significantly enhanced compared with those in EV-control plants, while the expression levels of these genes decreased in GmPP2A-B-RNAi plants. These results imply that GmPP2A-B gene maybe affect the expression of transcriptional regulation factor which was related with ROS signaling, such as GmERF115, to mediate regulation of critical gene transcription that was involved in ROS scavenging. Together, these results indicate that GmPP2A-B can enhance plant stress tolerance by regulating the ROS scavenging pathway, making it a candidate gene for plant tolerance to stress.Reactive oxygen species pathways in plants can be divided into ROS synthesis pathways and ROS scavenging pathways. However, the most important way to improve plant stress tolerance is to improve ROS scavenging capacity . Certainn plants . The defgulation . Harsh egulation . POD is us cells . These rus cells . When plus cells , LEA prous cells . Additiobidopsis . A recenbidopsis . These rbidopsis . A recenporation . This inThe original contributions presented in the study are included in the article/Z-SX coordinated the project, conceived and designed the experiments, and edited the manuscript. YX performed the experiments and wrote the first draft. X-HF, QW, Z-GY, and X-WS generated and analyzed the data. Y-ZM and JM reviewed and contributed valuable discussions. Y-ZM coordinated the project. All authors have read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


However, the pathogenic CD4+ T cell subset involved in gastritis and the potential regulators are still unclear. Here we identified an IL-21-producing gastric CD4+T cell subset, which exhibited tissue-resident CXCR5−BTLA−PD-1hi TFH-like phenotype in H. pylori-positive gastritis patients. Meanwhile, we identified glucocorticoid-induced tumor necrosis factor receptor (GITR) as an important regulator to facilitate IL-21 production by CD4+T cells and accelerate mucosal inflammation in gastritis patients with H. pylori infection. Moreover, GITR expression was increased in gastric CD4+T cells of gastritis patients compared to healthy controls, along with the upregulated expression of its ligand GITRL in mucosal macrophages (Mϕ) of gastritis patients. Further observations showed that the activation of GITR/GITRL signal promoted the IL-21 production of CD4+T cells via the STAT3 pathway. Besides this, IL-21 from CD4+T cells induced the proliferation of B cell and promoted the production of inflammatory cytokines IL-1β and IL-6 and chemokines MIP-3α and CCL-25 as well as matrix metalloproteinase (MMP)-3 and MMP-9 by human gastric epithelial cells, suggesting the facilitating effect of IL-21-producing CD4+T cells on mucosal inflammation and injuries. Taking these data together, we revealed that GITR/GITRL signal promoted the polarization of mucosal IL-21-producing CD4+T cells in H. pylori-positive gastritis, which may provide therapeutic strategies for the clinical treatment of H. pylori-induced gastritis.Gastric CD4 Helicobacter pylori (H. pylori)-induced gastritis usually results in mucosal injuries and extensive immune cell infiltration  cell is a special CD4ntiation . In secontiation . The unintiation . IL-21, ntiation . IL-21/Intiation –11. Furtntiation , synoviantiation , gastrointiation , and gutntiation , suggestH. pylori-infected patients  patients compared to ulcerative colitis (UC) patients and controls . The neupatients  and coulnfection . HoweverThe function of TFH cells is regulated simultaneously by the T cell antigen receptor binding strength  and the reg cells with high levels and at low levels on unstimulated macrophages, T cells, and B cells  is a member of tumor necrosis factor receptor superfamily (TNFRSF), which is constitutively expressed on T B cells . The trieg cells . GITR aleg cells –29. Santeg cells . Liao etocolitis . However colitis . BesidesH. pylori-infected gastritis remains unclear. Here we revealed the role of IL-21-producing TFH-like cells in H. pylori-positive gastritis. We identified a class of TFH-like CD4+ T cells with the capacity to secrete IL-21 in the gastric mucosa of H. pylori-positive gastritis patients and demonstrated that the GITR/GITRL axis promoted IL-21+CD4+ T cell polarization via the STAT3 signal pathway. Moreover, gastric CD4+T cell-producing IL-21 induced the proliferation of gastric B cells and the pro-inflammatory cytokines and chemokines as well as the secretion of MMPs by gastric epithelial cells. Collectively, our findings expand the understanding of IL-21+TFH-like CD4+ T cells in H. pylori-positive gastritis and investigate the underlying regulatory mechanism, which may provide potential interventions in the clinical treatment of H. pylori-induced gastritis.The role of GITR in H. pylori-positive gastritis, we firstly collected biopsy samples from H. pylori-positive gastritis patients and healthy controls . We then detected the expression of GITRL on mucosal Mϕф(CD11b+ HLA-DR+ CD68+). Consistently, the results showed that GITRL expression was upregulated in the mucosal Mϕфof H. pylori-positive gastritis patients  and CCL-25 in GES-1 cells compared to the supernatant of CD4+T cell from healthy controls  lymphoma . Informed written consents were obtained from all participants prior to the commencement of the study.This study was approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University . The biopsy specimens of H. pylori under a light microscopy. All 39 patients were diagnosed as cases of H. pylori-positive gastritis with pathological changes of the mucosa, whereas 20 healthy controls are H. pylori-negative and with normal mucosa .Mucosal mononuclear cells were isolated from the gastric mucosa biopsy of the patients as previously described . Briefly+T cells were isolated from mucosal mononuclear cells by positive selection with magnetic cell sorting system of BD Biosciences. The purity of sorted CD4+T cells was determined as >95% by flow cytometry, gated on CD3+CD4+T cells.CD4+ monocytes were sorted from human peripheral blood mononuclear cells by positive selection with human CD14 magnetic particles (BD). MDM was generated from monocytes as previously described  for 24 h. After the incubation, the Mϕ were irradiated, fully washed, and co-cultured with gastric CD4+T cells at a ratio of 1:5 in a 96-well flat-bottomed plate (2 × 104 Mϕ and 1 × 105 CD4+T cells per well). The percentage of IL-21-producing CD4+T cells was calculated by gating on live CD4+T cells.MDM were pre-incubated with +T cells, recombinant human GITR ligand , blocking antibody (Ab) of GITR , and agonist Ab of CD3  were added to the co-culture system. At 3 days later, the secretion of IL-21 by CD4+T cells was determined by flow cytometry.To evaluate the effect of GITR on the IL-21 production of CD4+T cells were restimulated with 1 μg/ml anti-CD3 Ab , 1 μg/ml anti-CD28 Ab , and 3 μg/ml brefeldinA  for 12 h. The intracellular fixation/permeabilization buffer set  was used in IL-21 staining. Flow cytometric detection was performed with FACS Canto II . Data was analyzed by the FlowJo software (Tree Star). The anti-human antibodies used in this study were purchased from Biolegend, BD Biosciences, or Miltenyi Biotec, namely: CD3 , CD4 , GITR , GITRL , IL-21 , BCL6 , STAT3 Phospho (Ser727) , BTLA , CXCR5 , and PD-1 .The procedure of cell staining and flow cytometry was previously described . For int® 488  and Alexa Fluor® 594  as well as DAPI  before the detection. Fluorescence images were captured under a ZEISS IMAGER A1 fluorescence microscope (CARL ZEISS).Paraffin-embedded samples were cut into 5-μm slices, and immunohistochemistry was conducted as previously described . In brieThe cells were collected, washed three times with cold phosphate-buffered saline , and lysed by RIPA lysis buffer  containing 1% (v/v) protease inhibitor cocktail , 1 mM phenylmethylsulfonyl fluoride , and 1 mM DTT . Equivalent amounts (20 μg) of cell lysates were separated by SDS-PAGE and then transferred to polyvinylidene difluoride membrane. The membranes were blocked in PBS-Tween20  containing 5% BSA for 1 h at room temperature (RT) and then incubated overnight with primary antibodies at 4°C. On the next day, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies at RT for 1 h, followed by visualization with an ECL kit  based on the recommendations of the manufacturer.CT value (actin–target gene).Gastric mucosa was collected and homogenized by bead-milling as previously described . The cel+T cells, gastric CD4+T cells were isolated and restimulated with 1 µg/ml CD3 Ab and 10 µg/ml recombinant GITRL protein for 3 days, followed by the collection of the culture supernatant for ELISA assay.The concentration of IL-21was detected by Human IL-21 ELISA kit  according to the manufacturer’s instructions. For IL-21 release of CD4t-tests. Data are shown as mean ± SD unless otherwise indicated. P-value less than 0.05 is considered significant.Statistical analysis was performed with GraphPad Prism 5.0 . Significance was analyzed by chi-square test, Spearman correlation analysis, one-way analysis of variance, or unpaired Student’s The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author.This study was approved by the Ethics Committee of Guangzhou Women and Children’s Medical Center, Guangzhou Medical University . The patients/participants provided their written informed consent to participate in this study.SM and YW initiated and designed the research. SM, YW, and SG wrote the manuscript with the help of other co-authors. SM, HY, and XL performed the experiments and analyzed and/or interpreted the results. YW, GZ, and SG were in charge of patient care and contributed to the discussion of the results. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Natural Science Foundation of China , the China Postdoctoral Science Foundation (2020T130131), the National Key Research and Development Plan (2019YFC0840602), the Guangdong Scientific and Technological Foundation (2020B1111170014 and 2019A1515110055), and the Shenzhen Scientific and Technological Foundation (KCXFZ202002011007083 and JCYJ20180228162321234).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Thymidine kinase 1 (TK1) is an intracellular protein associated with DNA synthesis, expressed during the G1 phase and remained elevated through the M phase, with a potential as a biomarker for cell proliferation. In this study, we explore the possible use of TK1 in Hodgkin lymphoma (HL).Serum concentrations of TK1 (S-TK1) were measured in 46 newly diagnosed HL patients using prospectively collected biobanked serum samples. The samples were analyzed using a novel antibody-based TK1 immunosorbent assay (ELISA).P = 0.003). A further increase in S-TK1 was observed during the treatment. The S-TK1 concentrations were higher in patients with advanced stage disease, low B-Hb, elevated P-LD and in those with B-symptoms. A high ESR correlated with low S-TK1.The concentrations of S-TK1 were elevated in HL patients compared with healthy controls (median 0.32 μg/L vs. 0.24 μg/L, The study results suggest that S-TK1, measured using a novel antibody-based assay, has the potential to be a biomarker in HL. However, while S-TK1 levels are elevated at baseline compared with healthy controls, a limited number of patients and comparatively short follow-up time render reliable conclusions difficult. Thymidine kinase (TK) is an intracellular protein associated with DNA synthesis with a potential as a biomarker for cell proliferation. Its use has been explored in several tumor diseases . TK exisTK1 is traditionally measured by its enzymatic activity and used clinically in non-Hodgkin lymphoma (NHL) but has been documented in few studies on Hodgkin lymphoma (HL) . HoweverIn HL tumors, the malignant Hodgkin and Reed–Sternberg (HRS) cells are limited in number, often making histological diagnosis difficult for pathologists, while the bulk of the tumor consists of normal reactive immune cells. The cell turnover in HL would thus be primarily attributed to apoptosis in healthy cells, with a controlled turnover of cell cycle-dependent proteins, including TK1. Our hypothesis is that TK1 is a more sensitive tumor cell turnover marker compared with lactate dehydrogenase (LD), commonly used as a biomarker in lymphoma. Thus, in this study, we investigated the feasibility of using the cell cycle-dependent TK1 as a possible biomarker and early treatment predictor in a cohort of HL patients.Serum TK1 (S-TK1) was measured in HL patients before and during treatment using the AroCell TK 210 ELISA, according to the manufacturer’s instructions . The technical specifications of the TK 210 ELISA assay, including comparison with the existing Abcam TK1 ELISA assay, have previously been published . The TK Fifty-eight patients with primary or recurrent HL were recruited as part of the Uppsala Umeå Comprehensive Cancer Consortium (U-CAN) project  between P-value of <0.05 was considered statistically significant. Multiple regression analysis was applied, with S-TK1 as the dependent variable and other numerical serum or plasma variables as independent variables. Statistical analysis was performed using the Medcalc Statistical Software version 19 .Non-parametric statistics were applied unless otherwise indicated. A P = 0.003), with a proposed cut-off of 0.45 g/L based on the upper 97.5 percentile limit of the healthy population  (P = 0.02) and high international prognostic score (IPS) (P = 0.02).High S-TK1 concentrations correlated with stage and were highest in those with stage IV disease  . In addiIn a multivariate analysis, high S-TK1 concentrations pre-treatment correlated with low blood hemoglobin (B-Hb) , high plP = 0.02, Wilcoxon’s paired test; 18F-FDG-PET/CT) defined as Deauville score ≥ 4 (During treatment, after two cycles of chemotherapy, the S-TK1 concentrations increased. The increment was observed in 9 of 11 patients for whom data were available (core ≥ 4 , HoweverCorrelation with overall or progression-free survival could not be determined due to the excellent prognosis of HL and limited follow-up time.To our knowledge, this research study presents the only analysis of TK1 protein concentrations in the serum of HL patients before and during treatment using a modern antibody-based ELISA assay. In our material of newly diagnosed HL patients, we have found elevated concentrations of S-TK1 compared with healthy controls, with a further increase during treatment. The S-TK1 concentrations were higher in patients with advanced stage, low B-Hb, elevated P-LD and B-symptoms. However, high S-TK1 correlated with low ESR. The correlations to stage, P-LD and B-symptoms as defined by the Ann Arbor staging criteria  an unex.The value of P-LD in NHL was early recognized  and is wThe results of this study reveal that the elevation of S-TK1 concentrations in HL is modest compared with healthy controls. Values are considerably higher in patients with leukemias, myelomas, myelodysplastic syndromes  and diffWhile current stratification of patients with HL follows the IPS , it oper18F-FDG-PET/CT has been found to be superior in predicting outcomes compared with IPS (18F-FDG-PET/CT in most patients shows a metabolic response after two cycles of chemotherapy, S-TK1 in our material increased during therapy. Given these dynamics of S-TK1, early assessment might be useful, ideally during the first cycle of chemotherapy.Interim with IPS  and measThe mechanism for S-TK1 increase during treatment has been established in cell culture experiments measuring TK1 release in cells treated with doxorubicin . ChemothAs previous studies have shown that the TK1 values vary over time in relation to the administration of chemotherapy, further studies with repeated analyses are needed to ascertain the best time to analyze TK1 in relation to the administration of chemotherapy. U-CAN samples are collected either midcycle or prior to cycle 3. TK1 concentrations are thus collected at their lowest point in relation to chemotherapy, which strengthens the argument for TK1 as a possible biomarker.In conclusion, our analysis suggests that relative change in TK1, before and after initiation of chemotherapy could be a potential biomarker in HL; however, further studies are needed to determine the role of TK1 in relation to chemotherapy administration.


Hijacking host ubiquitin pathways is essential for the replication of diverse viruses. However, the role of deubiquitinating enzymes (DUBs) in the interplay between viruses and the host is poorly characterized. Here, we demonstrate that specific DUBs are potent inhibitors of viral proteins from HIVs/simian immunodeficiency viruses (SIVs) that are involved in viral evasion of host restriction factors and viral replication. In particular, we discovered that T cell-functioning ubiquitin-specific protease 8 (USP8) is a potent and specific inhibitor of HIV-1 virion infectivity factor (Vif)-mediated apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3)G (A3G) degradation. Ectopic expression of USP8 inhibited Vif-induced A3G degradation and suppressed wild-type HIV-1 infectivity even in the presence of Vif. In addition, specific DUBs repressed Vpr-, Vpu-, and Vpx-triggered host restriction factor degradation. Our study has revealed a previously unrecognized interplay between the host’s DUBs and viral replication. Enhancing the antiviral activity of DUBs therefore represents an attractive strategy against HIVs/SIVs. Ubiquitin modification of proteins regulates their functions and is involved in virtually all aspects of cellular processes . UbiquitUbiquitin modification of proteins regulates their function , 4, 5 anThree other HIV/simian immunodeficiency virus (SIV) accessory proteins, Vpu, Vpr, and Vpx, are also essential components of viral replication and pathogenesis. Ubiquitin modification is vital for them on the process of target protein destruction. HIV-1 Vpu hijacks CUL1 and β-TrCP to form a CRL1 E3 ubiquitin ligase complex, triggering the degradation of BST-2  and PSGLIn this report, we demonstrate that DUBs are potent inhibitors of viral evasion of host restriction. We have identified various DUBs inhibiting functions of HIV Vif, Vpr, Vpu, and Vpx proteins. Collectively, our study reveals a previously unrecognized interplay between the host’s DUBs and viral replication. Enhancing the antiviral activity of DUBs therefore represents an attractive strategy against HIVs/SIVs.+ T cells are primary targets of HIV-1 infection. HIV-1 is potently inhibited by several human cytidine deaminases that are members of human APOBEC3 proteins. HIV-1 Vif neutralizes some APOBEC3’s antiviral functions by forming viral-specific CRL5 E3 ubiquitin ligase complexes  degradation . USP8 expression had no detectable effect on viral protein expression, as determined by the expression of intracellular HIV-1 Pr55Gag in HEK293T cells , which is overcome by HxB2 Vif , and A3F+ T cells by caspases. The C-terminal fragment of USP8, but not N-terminal fragment, possesses the enzymatic activity  assay to determine whether USP8 could interact with HIV-1 Vif. In the absence of Vif, USP8 was not detected in the co-IP sample . The intin vitro study  and then inserted between the SalI and BamHI sites of a C-terminal HA/Flag tag VR1012 vector. pET28a-USP8 C40 was constructed by PCR amplification from USP8-HA/Flag and then inserted into the pET28a-Plus vector with a 6xHis-tag at the N terminus. pET28a-USP8 C40 C786D was made from pET28a-USP8 C40 by site-directed mutagenesis. All the USP plasmids were purchased from Addgene. The infectious molecular clone pNL4-3 (WT HIV-1) , A3G-HA ; Addgene+ T lymphocytes were then purified from the PBMCs with anti-CD4-specific antibody-coated microbeads  according to the manufacturer’s instructions. CD4+ T lymphocytes were maintained in RPMI-1640 medium (HyClone) with 10% FBS and penicillin/streptomycin.HEK293T , HeLa , and TZM-bl  cells were maintained in Dulbecco’s modified Eagle’s medium  containing 10% heat-inactivated fetal bovine serum  and penicillin/streptomycin. H9 , Jurkat , and THP1  cells were purchased from the ATCC and maintained in Roswell Park Memorial Institute 1640 (RPMI-1640) medium (HyClone) with 10% FBS and penicillin/streptomycin. The peripheral blood mononuclear cells (PBMCs) were isolated through Ficoll gradient centrifugation, and the CD4DNA transfection was carried out using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. H9 and Jurkat cells were transfected using the Amaxa Cell Line Nucleofector Kit V  with the program G-014 or X-001 according to the manufacturer’s instructions.The antibodies used in this study are as follows: β-tubulin monoclonal antibody , Anti-CUL5 , anti-EloB , anti-CBFb , anti-Vif , anti-USP8 , anti-HA , anti-Myc , anti-V5 , anti-SAMHD1 , anti-HLTF , anti-BST2 , anti-A3G , anti-USP7 , anti-USP33 , anti-USP37 , anti-His . CAp24 mAb (1513) was purchased from the NIH AIDS Reagents Program. Secondary antibodies were alkaline phosphatase-conjugated anti-rabbit  and anti-mouse , HRP-conjugated anti-rabbit  and anti-mouse .For immunoblot analysis of cell-associated proteins, whole cell lysates were prepared as follows: Cells were collected in culture medium and centrifuged at 5,000 rpm for 5 min. Each supernatant was mixed with an appropriate volume of lysis buffer  and corresponding 4× loading buffer . Proteins were solubilized by heating for 30 min at 100°C, with occasional vortexing to shear cellular DNA. Cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). Proteins were transferred to NC membranes  and reacted with appropriate antibodies as described in the text, e.g., 1:500 rabbit polyclonal anti-USP8 to detect USP8. Membranes were then incubated with the corresponding secondary antibody, and proteins were visualized using a hypersensitive ECL chemiluminescence detection kit (Proteintech) according to the manufacturer’s protocol; the assembled HIV-1 in the culture supernatants was evaluated by immunoblotting in In ® Green PCR Master Mix (2x) . The primers used in this study are as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-F: GCAAATTCCATGGCACCGT; GAPDH-R: TCGCCCCACTTGATTTTGG; USP8-RT-F: CTGAAAGACTCTCTGAAAGCCT; USP8-RT-R: CCTTTCTCTTTGGTCTCACAT. Data were normalized to the housekeeping GAPDH gene, and the relative abundance of the transcripts was calculated using Ct models.Total RNA was extracted with TRIzol reagent  according to the manufacturer’s instructions. RNA reverse transcription used EasyScript First-Strand cDNA Synthesis SuperMix  according to the manufacturer’s instructions. The quantitative real-time polymerase chain reaction (qPCR) was carried out on an Mx3005P instrument  using Power SYBRTo generate knocking down USP8 cell lines, chemically synthesized short interfering RNA (siRNA) and a nonspecific control were purchased from RiboBio Co. Ltd. . The siUSP8 sequences are as follows: sense, #1: GCATAAAGGTGAAGTGGCA; #2: GAAAACAGGAAGAGAGGAT; #3: GCAAAGAGGGGCAAAGAAA.USP7/USP8/USP33/USP37-specific shRNAs with the following target sites were cloned in the lenti-retroviral vector pLKO.1-puro (Addgene). The shRNA sequences are as follows: USP7 shRNA: 5-CCGGCCTGGATTTGTGGTTACGTTACTCGAGTAACGTAACCACAAATCCAGGTTTTTG-3; USP8 shRNA: 5-CCGGTAAGAGTTATGTGCACAGTGCCCTCGAGGGCACTGTGCACATAACTCTTTTTTTG-3; USP33 shRNA 5-CCGGTCTCGACAGTGGCTTAATTAACTCGAGTTAATTAAGCCACTGTCGAGATTTTTG-3; USP37 shRNA 5-CCGGCCGGATTTGCAGAAGATGATACTCGAGTATCATCTTCTGCAAATCCGGTTTTTG-3. HEK293T cells were co-transfected with sh-USP7/USP8/USP33/USP37-pLKO.1 or pLKO.1 plus RRE, REV, and VSV-G expression vectors by using Lipofectamine 3000. At 48 h after transfection, supernatants containing packaged lentivirus were harvested and used to infect H9, Hela, or THP1 cells for 96 h as indicated. Puromycin  was then added into the culture to screen for stable cell lines.HEK293T cells were transfected with pNL4-3 (WT HIV-1) plasmid; 48 h later, the supernatant was collected and filtered through a 0.45-μm filter. Then, cells were infected with pNL4-3 virus in the presence of diethylaminoethyl-dextran hydrochloride  for 30 h. The culture medium was changed 30 h after infection and replaced with fresh medium and harvested at the indicated time. HIV-1 infectivity was assessed using TZM-BL indicator cells. LTR-luciferase was activated when TZM-BL cells were infected by HIV-1. TZM-BL cells were seeded in 24-well format plates (2 × 104 cells/well); 24 h later, the cells were infected with the equivalent of 0.5 ng of HIV-1 p24 antigen in the presence of 20 μg/ml DEAE. Cells were collected and lysed after 48 h. LTR-luciferase activity was measured with the Dual-Luciferase Reporter Assay System  according to the manufacturer’s protocol.in vitro deubiquitination assay, ubiquitinated A3G protein was incubated with USP8 or its mutant in the deubiquitination buffer  for 1 h at 37°C. The ubiquitinated A3G was analyzed by immunoblotting.Ubiquitinated A3G was isolated from HEK293T cells transfected with expression vectors of Ub-Flag, Vif-HA, and A3G-V5 and then purified from the cell extracts with anti-V5 antibody-conjugated protein G agarose beads. USP8 or its mutant was purified from HEK293T cells overexpressing USP8-HA or its mutant using anti-HA-Agarose antibody in IP buffer . For BL21(DE3) and purified by metal-affinity chromatography on chelation resin. For in vitro deubiquitination assay, ubiquitinated A3G protein was incubated with USP8 C40 or its mutant in the deubiquitination buffer (mentioned above) for the indicated time courses at 37°C. The ubiquitinated A3G was analyzed by immunoblotting.pET28a-USP8 C40 and pET28a-USP8 C40 C786D fusion protein were expressed in the strain of Data from the protein quantitative analysis and luciferase reporter assays are presented as means and standard derivations. Differences among groups were analyzed by ANOVA test  .The original contributions presented in the study are included in the article/Written informed consent was obtained from the individual(s) for the publication of any potentially identifiable images or data included in this article.XY and WG designed the experiments. WG, GL, YR, CZ, and JS performed the experiments. XY, SH, WG, and YR analyzed the data. HL, WWZ, BZ, WYZ, and YY provided technical support. XY, WG, and YR wrote the paper with help from all authors. All authors contributed to the article and approved the submitted version.This work was supported in part by funding from the National Natural Science Foundation of China , the Chinese Ministry of Science and Technology (2018ZX10731-101-001-014), and the National Natural Science Foundation of Zhejiang Province (LQ21C010001).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In response to our recent publication describing affinity-enhanced, long−half-life ACE2-based receptor traps for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) neutralization , Liu et 2+-binding ablated mutant CVD118 (ACE2(614)-Fc-H34V/N90Q/H374N/H378N) for hydrolysis of Ang II. Contrary to the Liu et al. , alongside controls including CVD208 (wild-type ACE2(740)-Fc), and a Znu et al. . For exau et al. A to yielu et al. B. We werive site C.6 against standard substrates, but was substantially restored by substrates containing a P2 His which can bind in the cavity created by H64A and participate in catalysis (What could account for the interesting differences in substrate-dependent activity for the H345L (inactive on both substrates) versus H345A (inactive on Dnp-APK(Mca) but active on Ang II)? It is possible the differences depend on the other mutations in CVD313. Alternatively, it is striking to note that Ang II contains a P2 His residue not found in Dnp-APK(Mca); it is possible that the P2 His cooperates with H345 to form part of the oxyanion binding D. Some yatalysis . By this


Methods: We used a novel MS mouse model that combines cuprizone and experimental autoimmune encephalomyelitis to reproducibly induce 2 brain inflammatory lesion types, differentiated by their T cell content. 18F-FAraG PET imaging, T2-weighted MRI, and T1-weighted contrast-enhanced MRI were performed before disease induction, during demyelination with high levels of innate immune cells, and after T cell infiltration. Fingolimod immunotherapy was used to evaluate the ability of PET and MRI to detect therapy response. Ex vivo immunofluorescence analyses for T cells, microglia/macrophages, myelin, and blood–brain barrier (BBB) integrity were performed to validate the in vivo findings. Results:18F-FAraG signal was significantly increased in the brain and spinal cord at the time point of T cell infiltration. 18F-FAraG signal from white matter  and gray matter  further correlated with T cell density. T2-weighted MRI detected white matter lesions independently of T cells. T1-weighted contrast-enhanced MRI indicated BBB disruption at the time point of T cell infiltration. Fingolimod treatment prevented motor deficits and decreased T cell and microglia/macrophage levels. In agreement, 18F-FAraG signal was decreased in the brain and spinal cord of fingolimod-treated mice; T1-weighted contrast-enhanced MRI revealed intact BBB, whereas T2-weighted MRI findings remained unchanged. Conclusion: The combination of MRI and 18F-FAraG PET enables detection of inflammatory demyelination and T cell infiltration in an MS mouse model, providing a new way to evaluate lesion heterogeneity during disease progression and after DMT. On clinical translation, these methods hold great potential for stratifying patients, monitoring MS progression, and determining therapy responses.Lymphocytes and innate immune cells are key drivers of multiple sclerosis (MS) and are the main target of MS disease-modifying therapies (DMT). Ex vivo analyses of MS lesions have revealed cellular heterogeneity and variable T cell levels, which may have important implications for patient stratification and choice of DMT. Although MRI has proven valuable to monitor DMT efficacy, its lack of specificity for cellular subtypes highlights the need for complementary methods to improve lesion characterization. Here, we evaluated the potential of 2′-deoxy-2′- Adaptive and innate immune cells play a critical role in the onset and progression of multiple sclerosis (MS) (18F-fluoro-9-β-d-arabinofuranosylguanine (18F-FAraG) is phosphorylated by cytoplasmic deoxycytidine and mitochondrial deoxyguanosine kinases  for MS  in the cuprizone-EAE model of MS. We also evaluated the ability of 18F-FAraG PET to detect response to fingolimod, a DMT that limits the infiltration of T cells into the CNS  a cuprizone-supplemented diet  for 3 wk, followed by 2 wk of normal chow. At the start of the sixth week, the mice were immunized with myelin oligodendrocyte glycoprotein 35–55 in complete Freund adjuvant. The mice received an intraperitoneal injection of pertussis toxin on the day of immunization and the following day (Hooke Laboratories). After immunization, the mice were evaluated daily for motor symptoms and were scored using an EAE scale as previously described (n = 4) immediately followed by ex vivo biodistribution studies to evaluate tracer distribution and accumulation. A second group of cuprizone-EAE mice (n = 10) underwent longitudinal PET and MRI sessions at the following time points: before disease induction (baseline), after 3 wk of cuprizone (W3), and at the end of the seventh week, which corresponds to 14 ± 1 d postimmunization (W7–14dpi). Euthanasia was performed after the last imaging session. A third group of cuprizone-EAE mice (n = 10) received a daily administration of a fingolimod solution by oral gavage  for 14 ± 1 d after immunization, starting on the day of immunization. PET and MRI were performed at W7–14dpi and immediately followed by euthanasia. A fourth group of mice that did not undergo any imaging procedures was used for histologic analyses . The experimental outline is shown in Supplemental Figure 1A .All animal research was approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco. The cuprizone-EAE model was induced by feeding 8-wk-old C57BL/6J female mice . For each imaging session, the mice were anesthetized using isoflurane (1.5%–2% in O2) and a 27-gauge catheter was placed in the tail vein for injection of the gadolinium contrast agent . The animals were placed in a water-heated cradle with the head secured to ensure similar positioning between experiments. Respiration was continuously monitored using PC-SAM software . T2-weighted MRI was performed using the following parameters: an echo time/repetition time of 11.8/3,000 ms, a rapid-imaging-with-refocused-echoes factor of 8, a slice thickness of 0.5 mm, a gap of 0.25 mm, 6 averages, a matrix of 256 × 256, and a field of view of 30 × 30 mm. T1-weighted MRI was acquired before and 2 min after injection of a gadobutrol bolus (1 mmol/kg of body weight) using an echo time/repetition time of 2.09/120 ms, a slice thickness of 0.8 mm, a gap of 0.2 mm, 10 averages, a matrix of 256 × 256, and a field of view of 20 × 20 mm.All in vivo MR experiments were conducted on a 14.1-T vertical MR system (Agilent Technologies) equipped with 100 G/cm gradients and a 18F-FAraG was synthesized as previously described (2), and 18F-FAraG  was injected into a catheterized tail vein. After 18F-FAraG injection, the mice were either imaged immediately for 60 min or were allowed to recover from anesthesia and be ambulatory for 60 min to permit tracer distribution, and whole-body static scans (a 15-min PET acquisition followed by a CT scan for anatomic reference and attenuation correction) were then performed under anesthesia using isoflurane (2% in O2) with warming and constant monitoring. PET images were reconstructed using the ordered-subsets expectation maximization algorithm provided by the manufacturer.All in vivo PET/CT scans were performed on a dedicated small-animal PET/CT scanner . n = 4) were euthanized and blood and brain were collected. The blood and brain tissue were weighed and radioactivity measured using a Hidex AMG γ-counter. The percentage injected dose per gram of tissue was calculated against a standard of known activity.Immediately after the dynamic PET acquisitions, control mice , as indicated in Supplemental Figure 1B. Normalized mean T2-weighted signal intensity for each region of interest was calculated as previously described . One-way ANOVA was used to assess the statistical significance of the MBP, Iba1, CD3, and fibrinogen immunofluorescence staining. Correlation analyses were performed using the Pearson correlation coefficient. 18F-FAraG PET acquisitions in control animals revealed a steady increase in 18F-FAraG signal in the brain over 60 min. The brain-to-blood ratio calculated from PET images (over the last 10 min) was 1.1 ± 0.2, and the brain-to-blood ratio calculated from subsequent ex vivo biodistribution studies was 0.73 ± 0.1 . These results indicate that 18F-FAraG accumulates in the brain and crosses the intact BBB in mice. Next, we evaluated the potential of 18F-FAraG PET to detect brain-infiltrating T cells in the cuprizone-EAE model. 18F-FAraG signal can be seen in the corpus callosum and hippocampal area at W7–14dpi. Quantitative analyses revealed that 18F-FAraG signal was significantly increased in the entire brain after immunization at W7–14dpi . A significant 18F-FAraG signal increase at W7–14dpi was also seen in the corpus callosum  and hippocampus . A higher 18F-FAraG signal was found in the somatosensory cortex (P = 0.0077 compared with baseline). Histologic analyses confirmed a significant increase in CD3 T cells after immunization at W7–14dpi, with the highest CD3 T cell density in the hippocampus (P < 0.0001 compared with both baseline and W3), followed by the corpus callosum  and cortex , in line with the PET imaging results .Dynamic  results . Also inP = 0.0418 and P = 0.05 compared with baseline and W3, respectively). The volume of T1-enhancing lesion varied greatly among animals, ranging from 13 to 117 mm3. T2-weighted MRI detected the presence of hyperintense lesions in the corpus callosum, but not in the hippocampus or cortex, after the cuprizone diet  and immunization .Next, we assessed the potential of MRI to detect brain alteration at W3 and W7–14dpi during inflammatory demyelination. A leaky BBB was observed after immunization (W7–14dpi) (P < 0.0001) and W7–14dpi (P < 0.0001) in the corpus callosum, along with demyelination (MBP) at W3 (P < 0.0001) and W7–14dpi (P = 0.0059) (P = 0.0353 compared with both baseline and W3). Similarly, an increase in innate immune cells (Iba1) was observed at W3 and W7–14dpi in the hippocampus (P = 0.0309 and P = 0.0002 compared with baseline) and at W7–14dpi in the cortex (P < 0.0001 compared with baseline) . Interestingly, demyelination (MBP) was present at W3 in the hippocampus (P = 0.0117 compared with baseline) and W7–14dpi in the cortex . Fibrinogen staining confirmed BBB breakdown in the hippocampus at W7–14dpi .Inflammatory demyelination at W3 and W7–14dpi was confirmed by immunofluorescence analysis. Innate immune cells (Iba1) were significantly increased at W3 ( 0.0059) . Fibrino18F-FAraG signal arising from the lumbar spinal cord can be observed at W7–14dpi. Quantitative analyses revealed a significant increase in 18F-FAraG signal in both the cervical/thoracic  and the lumbar  spinal cord at W7–14dpi. CD3 T cells were detected at W7–14dpi in the cervical/thoracic  and the lumbar  spinal cord . 18F-FAraG signal was also increased in the subiliac lymph nodes at W7–14dpi , in agreement with the elicited adaptive immune response after immunization . As shown in nal cord  and signnization .18F-FAraG could detect the effect of fingolimod treatment. Untreated animals showed weakness or paralysis of the limbs . In contrast, fingolimod-treated mice did not show any sign of EAE pathology , confirming the effect of fingolimod treatment.We evaluated whether 18F-FAraG signal was significantly lower in the entire brain (P = 0.0467), corpus callosum (P = 0.0011), hippocampus (P = 0.0169), and cortex (P = 0.0355) of fingolimod-treated animals , hippocampus (P < 0.0001), and cortex (P = 0.0077) after fingolimod treatment . animals . No T1-e18F-FAraG PET in the detection of T cell infiltration and accumulation within the CNS and lymphoid tissue in a MS model, and we demonstrated its ability to detect response to an immunomodulatory DMT.A way to noninvasively visualize immune cells within the CNS would tremendously help improve the diagnosis and monitoring of neurodegenerative disorders, particularly MS. Here, we showed the high potential of 18F-FAraG PET to detect various levels of infiltrating T cells. We showed that 18F-FAraG signal was strongly associated with T cell density in the brain and spinal cord. Prior in vitro studies have shown that 18F-FAraG uptake also occurs in macrophages, but the agent is not retained because of low levels of cytoplasmic deoxycytidine and deoxyguanosine kinases  within the same animal. Furthermore, in space, the differential load of T cells throughout the brain, with different levels in white and gray matter, enables evaluation of the capability of 18F-FAraG delivery to the brain . 18F-FAraG brain uptake in the 2 cohorts of control mice showed different values .In healthy conditions, the adult brain has a limited capacity for de novo synthesis of nucleosides and relies on uptake through nucleoside transporters present on the BBB, thus providing a means for 18F-FAraG signal coincided with the time point of BBB disruption measured by contrast-enhanced T1-weighted MRI and fibrinogen immunostaining—a timing that may facilitate 18F-FAraG accumulation in the brain. However, we showed in Supplemental Figure 9 that T1-enhancing lesion volumes did not correlate with 18F-FAraG signal and that 18F-FAraG signal was increased in non–T1-enhancing lesions containing high levels of CD3 T cells. These findings further support that 18F-FAraG provides additional information with regard to T cell accumulation. Discrepancies between imaging modalities might also be explained by differences in molecular weight and lipid solubility of the different agents used to evaluate BBB permeability  cytosine (18F-FAC) use in the EAE model . However, this method failed to detect fingolimod therapy response, as both demyelination and immune cell content influence the T2 tissue properties and are important components of lesions before and after DMT , and by the Dana Foundation’s David Mahoney Neuroimaging Program. Jelena Levi is employed by CellSight Technologies. CellSight Technologies is commercializing