At mid-term follow-up, a decrease in vaccine-induced anti-SARS-CoV-2 IgG antibodies was observed in all subgroups of patients receiving DMTs, although most patients receiving induction DMTs or natalizumab and non-treated patients remained protected. Cellular immunity was maintained above protective levels in all DMT subgroups except the fingolimod subgroup. Conclusions: SARS-CoV-2 vaccines induce robust and long-lasting humoral and cell-mediated specific immune responses in most patients with MS.Background: This study aimed to evaluate short- and long-term humoral and T-cell-specific immune responses to SARS-CoV-2 vaccines in patients with multiple sclerosis (MS) treated with different disease-modifying therapies (DMTs). Methods: Single-center observational longitudinal study including 102 patients with MS who consecutively received vaccination against SARS-CoV-2. Serum samples were collected at baseline and after receiving the second dose of the vaccine. Specific Th1 responses following in vitro stimulation with spike and nucleocapsid peptides were analyzed by quantifying levels of IFN-γ. Serum IgG-type antibodies against the spike region of SARS-CoV-2 were studied by chemiluminescent microparticle immunoassay. Results: Patients undergoing fingolimod and anti-CD20 therapies had a markedly lower humoral response than those treated with other DMTs and untreated patients. Robust antigen-specific T-cell responses were detected in all patients except those treated with fingolimod, who had lower IFN-γ levels than those treated with other DMTs (25.8 pg/mL vs. 868.7 pg/mL,  Over the past decade, the disease course of multiple sclerosis (MS) patients appears to have become milder. This may be partly due to the availability of effective disease-modifying therapies (DMTs) that better reduce inflammatory disease activity and delay disability progression . The varThe COronaVIrus Disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a rapidly evolving situation that continues to cause unprecedented disruption of normal life. As the COVID-19 pandemic spread worldwide, an increasing number of SARS-CoV-2 vaccines were developed. Preliminary data suggest that the humoral response to the SARS-CoV-2 vaccines is impaired in MS patients treated with fingolimod and anti-CD20 therapies ,7,8. HowWe aimed to evaluate the humoral and cellular immune responses to SARS-CoV-2 vaccines in patients with MS receiving different DMTs and to explore their protective role against COVID-19 during patient follow-up.We performed a single-center observational longitudinal study including 102 MS patients who consecutively received vaccination against SARS-CoV-2 at Hospital Universitario Ramón y Cajal in Madrid, Spain. Inclusion criteria were: diagnosis of MS according to McDonald 2017 criteria  and compAt baseline, before vaccination, demographic characteristics, time since first MS symptoms, MS phenotype, disability according to the Expanded Disability Status Scale (EDSS) score, current DMT, time since DMT initiation, last DMT administration (in patients undergoing pulsed therapy), and occurrence of COVID-19 before vaccination were recorded.Patients were subsequently evaluated every 3 months after vaccination to assess the occurrence of COVID-19.Serum samples (5 mL) were collected after vaccination , aliquoted, and stored at 80 °C until studied. Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized whole blood by Ficoll density gradient centrifugation (Abbott).6 cells/well resuspended in a volume of 200 µL of complete medium enriched with 10% human serum (Merck). Four culture wells were prepared for each patient: one well with complete medium (negative control), another well with 10 µL (10 µg/mL) of OKT3 as a positive control; the other two wells were stimulated with 4 µL (50 µg/mL) of spike (S) peptides of SARS-CoV-2  and with 4 µL (50 µg/mL) of nucleocapsid (N) peptides of SARS-CoV-2 . After 30 min, 6 µL (25 µg/mL) of CD28/CD49d co-stimulator was added to each well, and cells were incubated at 37 °C, 5% CO2, and 95% humidity for 24 h. Then, the cellular suspension was centrifuged, and supernatants were collected and stored at −80 °C until analysis.Fresh PBMCs were cultured in 96-well flat-bottom plates at 10Supernatant interferon-gamma (IFN-γ) levels were quantified in a SR-X instrument  using the single-molecule array IFN-γ Advantage Kit technique (Quanterix). Since no consensus has been established on the best cut-off value to consider a good cellular response to SARS-CoV-2, we considered a positive result when the concentration of IFN-γ was higher than 80 pg/mL, based on results obtained in our untreated patients.Serum IgG-type antibodies against the spike region of SARS-CoV-2 (S1 subunit) were studied by chemiluminescent immunoassay of micro-particles (CMIA) in the automated system ALINITY . Following the instructions for using the WHO International Standard and Reference Panel for anti-SARS-CoV-2 antibody ,14, antiTo evaluate the persistence of immune responses after two vaccine doses, serum samples obtained from each patient were classified into two groups: samples obtained between 0 and 90 days after the second vaccine dose (early sample) and samples obtained beyond 90 days after the second vaccine dose (late sample). The immune response was considered persistent when determinations were positive before and after 90 days or when a patient without an early sample presented a positive late sample.2 test. Continuous variables were reported as median  and were analyzed with Wilcoxon rank-sum test. Kruskal–Wallis test was used for between-group comparisons. Associations between demographic, clinical, and laboratory data and humoral and cellular immune responses were assessed using Spearman’s rank correlations. Two-tailed p-values < 0.05 were considered significant.Analyses were performed using the GraphPad Prism 6.0 software . Categorical variables were summarized using frequencies (percentages) and were analyzed with the χOne hundred and two patients were prospectively included in the study. Baseline demographic and clinical characteristics are shown in p = 0.0001 for both comparisons).Ninety-two patients (90.2%) were vaccinated with both doses of an mRNA vaccine (mRNA-1273 or BNT162b2), seven patients (6.9%) were vaccinated with both doses of ChAdOx1nCoV-19, and three patients (2.9%) were vaccinated with a dose of Ad26.COV2-S and then a dose of an mRNA vaccine. There were no statistically significant differences between groups in demographic, clinical, and treatment characteristics. Fifty-two patients (51%) received a third dose of an mRNA vaccine. However, in all cases, the immune response was evaluated before the third dose.Fourteen (22.6%) of the 62 patients with available pre-vaccination serological analysis were positive for IgG anti-SARS-CoV-2 antibodies as a result of previous infection, but only two had protective titers. After two doses of a SARS-CoV-2 vaccine, 84 (82.4%) patients had positive anti-SARS-CoV-2 IgG titers, and 65 (63.7%) of them reached protective titer levels. p = 0.0001). Likewise, seven of 16 (44%) patients under ocrelizumab had positive anti-SARS-CoV-2 IgG titers, and three of 16 (19%) had protective levels. The median titer was 6.0 [1.2–231.8] BAU/mL, clearly lower than those in patients with other DMTs (p = 0.0001). Similarly, eight of 12 (66.7%) patients receiving rituximab had positive anti-SARS-CoV-2 IgG titers, and five of 12 reached protective levels. The median titer of 106.3 [4.5–384.4] BAU/mL was again lower than those in patients with other DMTs (p = 0.0001).Post-vaccination anti-SARS-CoV-2 IgG titers by DMTs are represented in Thirteen (21%) of the 62 patients with available pre-vaccination IFN-γ quantification had levels ≥80 pg/mL due to a previous infection. After two doses of a SARS-CoV-2 vaccine, 81 of the 92 patients with an IFN-γ determination had levels ≥80 pg/mL, with a median value of 876.9 [270.7–2764] pg/mL.p = 0.011). There were no other significant differences between DMTs.Post-vaccination IFN-γ levels in patients classified according to their DMTs are represented in rho = 0.18, p = 0.08).The concordance between cellular and humoral responses is represented in n = 89) compared to late (n = 40) samples . Likewise, values of IFN-γ decreased from 855.6 [218.6–2549] pg/mL in the early samples to 269.7 [132.4–977.3] pg/mL in the late samples (p = 0.005).The median time between the second dose of the vaccine and sample extraction was 42 [28.5–51.5] days for the early samples and 140 [124–177] for the late samples. After two doses of a SARS-CoV-2 vaccine in the early samples, 53/80 (66.3%) patients had anti-SARS-CoV-2 IgG titers ≥ 260 BAU/mL, and 66/75 (88%) had IFN-γ levels ≥ 80 pg/mL. Twenty-six of the 41 patients (63.4%) with a late sample showed persistent protective levels of IgG anti-SARS-CoV-2 antibodies, and 18/22 (81.8%) IFN-γ levels higher than 80 pg/mL. When quantitative values were explored, we observed higher antibody titers in early (p = 0.03), alemtuzumab (p = 0.04), or FLT (p = 0.005). No significant decreases were observed in untreated patients or those treated with cladribine. Finally, levels were low in early or late samples of most patients treated with anti-CD20 antibodies or fingolimod.We next studied changes in patients classified according to DMTs. Results are shown in p = 0.04) in the late samples.Moreover, in most patients receiving induction therapies or natalizumab and in non-treated patients, anti-SARS-CoV-2 IgG titers remained above protective levels A. In therho = −0.32, p = 0.001) but not with IFN-γ levels . Disability measured with the EDSS score did not correlate with either humoral or cellular immune responses. Absolute lymphocyte counts classified into three grades  in relation to post-vaccination anti-SARS-CoV-2 IgG titers and IFN-γ levels by DMT are presented in Demographic, clinical, and laboratory data that could affect vaccines’ immune response were studied. Age weakly correlated with post-vaccination anti-SARS-CoV-2 IgG titers . When we analyzed pre-vaccination N-reactive T-cell responses, patients recovered from COVID-19 had significantly higher IFN-γ levels than patients without prior COVID-19. After receiving a full course of a SARS-CoV-2 vaccine, IFN-γ levels progressively decreased in both groups of patients, although they remained statistically higher in patients with previous COVID-19  suffering a subsequent COVID-19 and the mild disease course observed in most cases. In our study, the only patient who required hospitalization was on fingolimod treatment and had not developed humoral or cellular protective responses after SARS-CoV-2 vaccination. Moreover, 3 months after the second vaccination (when antibody and cellular immune responses were high), no patients suffered COVID-19.No association between SARS-CoV-2 titers or IFN-γ levels and subsequent COVID-19 was demonstrated in our study. This may be because most of our patients with post-vaccination COVID-19 were infected with the Omicron variant, given their symptoms and the timing of their presentation. Results from a recently published study  show thaOur study has some limitations. First, the small size of the different cohorts of DMTs restricts the comparison of vaccine-induced immune responses between groups. Second, the timing of sample collection after vaccination was not homogeneous among patients. Despite these limitations, we believe that our findings are relevant to expanding knowledge about immune responses to COVID-19 vaccination and subsequent protection against SARS-CoV-2 infection.


Autoimmune pancreatitis (AIP) is one of the recently established immunoglobulin G4-related diseases (IgG4-RD) . The det+ T cells, and eosinophils into the fibrotic lesions of pancreatic ducts [FH) cells plays an important role [+ CXCR5+ TFH cells were detected in the peripheral blood and their frequencies were increased in patients with IgG4-RD [+ SLAM7+ T cells with cytotoxic activity that clonally expanded and were associated with clinical IgG4-RD symptoms [T cell receptor alpha joining (Traj)+33 T cells with a type 2 helper T (TH2)-like phenotype contributed to autoantibody productions [AIP is characterized by abundant infiltration of IgG4-positive plasma cells, CD4ic ducts . B-cell ic ducts . Previouic ducts , indicatant role . The CD4 IgG4-RD . AnotherThe present study performed a single-cell RNA analysis in conjunction with T-cell receptor (scTCR-RNA-seq) analysis and identified T-cell clonotypes that correlated disease progression in patients with AIP.+ cells were enriched from peripheral blood mononuclear cells of patients with AIP before steroid treatment and healthy donors to examine T-cell populations that were associated with clinical AIP symptoms. These CD3+ cells underwent scTCR-RNA-seq analysis followed by t-distributed stochastic neighbor embedding (tSNE)-based clustering. The dimension reduction analysis revealed four clusters that were present in all samples  populations were present in Cluster #3 , which is a marker for mucosal-associated invariant T (MAIT) cells that recognize microbial metabolites [FH-like CD4+SLAMF7high CTLs are associated with serum IgG4 production.CD3 samples . Among tnd ITGA6 , which and ITGA6 . The clund ITGA6 . Furtheruster #3 , indicatabolites   are also known as MAIT cells [TRAJ33+ T cells, which are rearranged with the non-TRAV1-2 segment and recognize unknown metabolites presented by MR1 [Trav1−Traj33+ non-MAIT cell population is associated with disease symptoms [TRAV1-2-TRAJ33 (human counterpart of mouse Trav1–Traj33 gene segment) expression, were present -loaded hMR1 tetramers (Ac-6-FP-tet) [+ αβ T cells expressed significantly higher CD69 levels on the cell surface in patients with AIP than in healthy donors  . Hence, y donors . Furtherwith AIP . Altogetin vivo dynamic examination of individual immune cells. We used this technology to identify the correlation of T-cell clonotypes with clinical AIP symptoms. Help from particular T-cell subsets including TFH cells and TH2 cells is essential for high-affinity antibody production by B cells, which are major pathogenic drivers of many autoimmune diseases, including AIP. Following the T–B interaction, T cells undergo clonal expansion upon recognition of cognate antigens presented by B cells. Therefore, identifying clonally expanded T-cell clonotypes with TFH- and TH2-like signatures will provide a valuable insight to estimate disease progression in AIP.Recent advances in single-cell analysis technology have enabled a more detailed and goad071_Supplementary_DataClick here for additional data file.


RNA-Seq analyses revealed that germline-specific inx-14 RNAi downregulated Notch/GLP-1 signaling, while lysosome and PMK-1/p38 pathways were upregulated. Consistently, disruption of inx-14 or glp-1 in the germline enhanced resistance to PA14 infection and upregulated lysosome and PMK-1/p38 activity. We show that lysosome signaling functions downstream of the INX-14/GLP-1 signaling axis and upstream of PMK-1/p38 pathway to facilitate intestinal defense. Our findings expand the understanding of the links between the reproductive system and intestinal defense, which may be evolutionarily conserved in higher organism.Gap junctions mediate intercellular communications across cellular networks in the nervous and immune systems. Yet their roles in intestinal innate immunity are poorly understood. Here, we show that the gap junction/innexin subunit  Activate (MAPK) .C. elegans gap junctions allow direct cell-to-cell communication based on membrane voltage, nutrient and second messengers exchange , and metabolic signal transmission across cell membranes   as no viable mutants are available  mutants to pathogens during PA14 infection. We found that the percentages of occupancy on a PA14 lawn were indistinguishable between inx-14(ag17) mutants and wild-type animals at 2, 4, 6, 8, and 10 hours after exposure to a lawn of PA14 bacteria (inx-14(ag17) mutants also displayed normal enteric muscle contractions  after infection. Our results showed that GFP-labeled PA14 bacteria (PA14::GFP) accumulated less in the intestine of inx-14(ag17) mutants compared to wild-type. However, accumulation was not different in the pharynx (inx-14(ag17) mutant as assayed by colony forming units was dramatically decreased compared to wild-type  was sufficient to phenocopy this effect (inx-14(ag17) mutants to PA14 infection was fully rescued by inx-14 transgene expression in the germline under the control of germline-specific pgl-1 promoter (ges-1 promoter (inx-14(ag17) males  (inx-14(ag17) mutant background and compared their survival on PA14 bacteria. We observed that the resistance of inx-14(ag17) mutant to PA14 was almost completely suppressed by RNAi knockdown of the PMK-1/p38 pathway genes nsy-1, sek-1, and pmk-1  , insuline (MAPK) , and DBLe (MAPK) . Activate (MAPK) . To examdaf-2 (insulin/IGF receptor) and age-1  exhibit enhanced resistance to PA14 infection  mutants, we observed a significantly stronger resistance to PA14 than in either single mutant (daf-16 RNAi in wild-type or inx-14(ag17) mutant animal did not alter the sensitivity of these strains  genetic background  mutants , apx-1 and lag-2 (two Notch ligands), and arg-1 and dsl-1 (encoded Notch binding activity) (inx-14 RNAi (C. elegans germline development (glp-1 caused enhanced resistance to PA14 infection (glp-1(e2141) mutant, but not lin-12 RNAi, showed dramatically enhanced resistance to PA14 infection   were dowelopment  and lossnfection . Consistdaf-1 or sma-6, and a type II receptor encoded by daf-4. DAF-7 and DBL-1 are ligands of type I and type II TGF-β receptors, respectively, which are referred to as the Dauer pathway and the Sma/Mab pathway  mutants did not significantly alter their susceptibility to PA14 infection (C. elegans.The transforming growth factor β (TGF-β) family is a major evolutionarily-conserved signal transduction pathway that plays crucial roles in development and homeostasis , 50, 51. pathway . Our res pathway . To addrglp-1 genetically interacts with inx-14, we examined and compared the survival rate of wild-type and inx-14(ag17) mutants with vector control or glp-1 RNAi, upon PA14 infection. We observed that the resistant effect of inx-14 or glp-1 RNAi in wild-type animals to PA14 infection was stronger than that of any corresponding inx-14(ag17) or glp-1(e2141) mutants (inx-14(ag17) or of GLP-1 in glp-1(e2141) mutants for both ag17 and e2141 alleles are substitution mutations (glp-1 RNAi in inx-14(ag17) mutants exhibited no significant difference to that of glp-1 RNAi in wild-type animals (inx-14 RNAi in glp-1(e2141) mutants blocked the effect of significantly different from that of inx-14 RNAi in wild-type animals, indicating that loss function of glp-1 completely blocked the more enhanced resistance of inx-14 RNAi in wild-type than that of glp-1(e2141) mutants  (C. elegans.To address in which tissue DCL569)         mutants (inx-14 (K08D8.5 (F08G5.6 (F55G11.4 (F35E12.5 (C14C6.5 (C17H12.8 (glp-1(e2141) mutants  mutants fed with control, hlh-30 or lipl-1 RNAi. We found that the enhanced susceptibility of hlh-30 (lipl-1 (inx-14(ag17) (glp-1(e2141) (inx-14 or glp-1 by RNAi in the hlh-30(tm1978) (lipl-1(tm1987) (hlh-30(tm1978) (lipl-1(tm1987)  mutant were comparable to that of wild-type animals , present on adjacent cell. Found in almost all cells, gap junctions play a pivotal role in many physiological and cellular processes, and have even been linked to the progression of diseases, such as arrhythmia, neurosensory deafness, cancer –68. Gap d muscle , 40. To d muscle , while ad muscle .C. elegans, prevention of germline stem cell proliferation results in the extension of lifespan, which requires the activity of DAF-16, a member of the forkhead/winged-helix family of transcriptional regulators   , 73–75. To provide insight into the mechanisms underlying the regulation of intestine defense by gap junctions from the reproductive tract, we performed comprehensive transcriptome profiling and found that GLP-1/Notch specifically acts in the germline and downstream of INX-14 to suppress gut defense. Previous studies revealed that loss of heterotypic and heteromeric gap junctions INX-8/9/14/21 prevents germline development and gametogenesis , 48, 76.2+, cAMP, and IP3 by gap junctions have been implicated in the activation of innate immunity  in the ectodomain accessible to ADAM  family proteases SUP-17/ADAM10 and ADM-4/TACE , which function redundantly as S2 proteases. The second cleavage is a substrate for γ-secretase, which cleaves within the transmembrane domain at “site 3”. The intracellular domains released from its transmembrane tether translocate to the nucleus. In the nucleus, the intracellular domain forms a complex with a sequence-specific DNA binding protein of the CSL family and a co-activator of the Mastermind family to promote target gene expression  translocates to the nucleus and associates with a DNA binding protein to activate expression of downstream target genes. This core signal transduction known as the “canonical” pathway is used in most Notch-dependent processes . C. elegpression . In our C. elegans HLH-30/bHLH, a basic helix-loop-helix transcription factor orthologous to mammalian TFEB, plays an important role in longevity  or glp-1(e2141) mutants are indistinguishable from that of inx-14(ag17) or glp-1(e2141) single mutants. These data suggest that inx-14 and glp-1 are epistatic to hlh-30 and lipl-1. Since gonadal INX-14 controls many other biological processes, including peroxisome, longevity regulating pathways, or carbon metabolism, these pathways may function redundantly or in parallel to each other, and downstream of the INX-14/GLP-1 signaling axis for the intestinal defense. Hence, if one of these signaling pathways, for instance the lysosome pathway, is removed, we might not observe an obvious effect on the survival of animals upon PA14 infection, which is consistent with our findings in this study.Our RNA-Seq data revealed that lysosome pathway genes were enriched and upregulated by germline-specific loss of onyl-CoA , 86 or oonyl-CoA  that trionyl-CoA  in the gEscherichia coli OP50 as a food source using standard procedures, except glp-1(e2141) mutants that were raised at 15˚C , inx-9(gk293), inx-14(ag17), inx-21(ok2524), inx-22(tm1661), inx-19(ky634), unc-7(e5), unc-9(e101), eat-5(ad1402), che-7(ok2373), inx-6(rr5), inx-2(ok376), inx-5(ok1053),inx-7(ok2319), inx-10(ok2714), inx-11(ok2783), inx-16(ox144), inx-18(ok2454), inx-20(ok681), inx-1(tm3524), inx-15(tm3394), inx-17(tm3292), inx-14(ag17);pmk-1(km25), glp-1(e2141), DCL569 mkcSi13 [sun-1p::rde-1::sun-13’UTR+unc-119(+)];rde-1(mkc36), VP303 rde-1(ne219);kbIs7 [nhx-2p::rde-1+rol-6(su1006)], WM118 rde-1(ne300);neIs9 [myo-3::HA::RDE-1 + rol-6(su1006)], TU3311 uIs60 [unc-119p::YFP + unc-119p::sid-1] pmk-1(km25), nsy-1(ag3), sek-1(km4), glp-1(e2141), hlh-30(tm1978), lipl-1(tm1987), AY101 acIs101 [F35E12.5p::GFP + rol-6(su1006)], SAL144 pha-1(e2123);denEx22 [K08D8.5::GFP + pha-1(+)], unc-17(e245), unc-25(e156), unc-13(e51), MAH235 sqIs19 [hlh-30p::hlh-30::GFP + rol-6(su1006)], unc-17(e245), unc-13(e51), unc-25(e156), qxIs520[Pvha-7::LAAT-1::GFP].Strains were maintained at 20˚C on nematode growth medium (NGM) plate seeded with  at 15˚C . The N2 inx-14(ag17) genetic background are: inx-14(ag17);aijEx186 and inx-14(ag17);aijEx219.Transgenic strains generated for this study to express INX-14 under the control of endogenous or tissue-specific promoters in an Escherichia coli OP50, E. coli HT115, and Pseudomonas aeruginosa PA14 , the plasmid DNA of pXZ20 (Ppgl-1::inx-14::SL2::GFP::unc-54 3’ UTR), or pXZ23 (Pges-1::inx-14::SL2::GFP::unc-54 3’ UTR) was injected at 20 ng μl-1 with co-injection marker at 5 ng μl-1, and 1 kb ladder up to 100 ng μl-1.Transgenic animals were created by microinjection of plasmid DNA into the gonad of young adult eviously . For speThe plasmids were constructed by using isothermal assembly , unless Ppgl-1::inx-14::SL2::GFP::unc-54 3’ UTR. To assemble pXZ20, a 207 bp fragment of the germline-specific promoter Ppgl-1 and a 1.6 kb genomic DNA fragment of the inx-14 coding region (from initiation codon ATG to stop codon TAA) were amplified from N2 genomic DNA and fused to an SL2::GFP sequence and unc-54 3’UTR (from pNP403). This assembly was inserted into pJET1.2  using isothermal assembly  were amplified from N2 genomic DNA and fused to an SL2::GFP sequence and unc-54 3’UTR (from pNP403). This assembly was inserted into pJET1.2  using isothermal assembly (pXZ23: assembly .P. aeruginosa PA14 infection was performed as described previously (P. aeruginosa PA14 was seeded onto modified 35 mm-diameter NGM plates (0.35% instead of 0.25% peptone) to cover the entire surface of NGM agar   was added to plates to prevent the growth of progeny and “bagging”. Animals that crawled off the plate or died from vulva bursting were censored. All survival assays were performed in at least three independent replicates.The killing assay by eviously  with minNGM agar . These sE. coli strain HT115 (DE3) expressing double-strand RNA homologous to a target gene as previously described  was performed by feeding worms with escribed . RNAi clE. coli HT115 RNAi clones at 20°C until the progeny reached the L4 stage. These animals were then transferred to NGM plates with full-lawn PA14 at 25°C for survival assays.Tissue-specific gene knockdown was performed as described previously , 92 with-ΔΔCT) method and normalized to tba-1/tubulin. All experiments were performed at least three times independently. Primers used for these experiments are listed in Supporting Synchronized larval L4 worms were transferred onto NGM plates seeded with OP50 or PA14 maintained at 25°C for 24 hours. Animals were washed off and collected, quickly rinsed once with M9 solution, and then re-suspended in TRIzol  for total RNA extraction or frozen by liquid nitrogen and then stored at -80°C for subsequent experiments. Total RNA was obtained using TransZol Up Plus RNA Kit  and cDNAs were synthesized using the TransScript All-in-One First-Strand cDNA Synthesis SuperMix . cDNA aliquots were stored at -80°C for subsequent experiments. qRT-PCR was performed using SYBR PCR Master Mix  on a Bio-Rad CFX96 real-time PCR machine. Relative fold-changes for transcripts were calculated using the comparative CT (2inx-14 RNAi (prior to PA14 infection), or OP50 for 24 hours. These samples was used as input material for RNA sample preparation. Sequencing libraries were generated using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB) following manufacturer recommendations, and index codes were added to attribute sequences to each sample. Library preparations were sequenced on an Illumina NovaSeq 6000 platform using 150 bp paired-end reads.Total RNA (1 µg) was obtained as described above from the germline-specific DCL569 RNAi strain fed with L4440 empty vector control, Clean reads were mapped to the worm reference genome (Wbcel235) using HISAT2 . UniquelThe defecation cycle length was measured on synchronized young adult hermaphrodites on standard NGM plates at 20°C. Animals were transferred to fresh plates and were allowed to adapt for at least 10 min before observation. Measurements were obtained from animals with OP50 feeding and the duration of the cycle was defined as the time from one expulsion to the next. Ten defecation cycles were measured for each individual. To determine the percentage of successful enteric muscle contractions per defecation cycle, we scored for the occurence of an enteric muscle contraction following a posterior body contraction .Brood size was measured by picking single L4 worm to a 3.5 cm NGM plate, maintained at 20°C. Each worm was transferred to a new NGM plate every day and the previous plate was kept at 20°C for two more days until the number of live progeny was scored. This procedure was repeated until no eggs were laid anymore on NGM plate.Pharyngeal pumping was assayed by recording a 1 minute movie focusing on the pharynx of young adult worms using the DIC optics of Multi-Purpose Zoom Microscope . One opening and one closing of the terminal bulb (TB) was regarded as a complete pumping sequence. Pharyngeal pumping rates were reported as number of pumps per minute.P. aeruginosa PA14 for 24 hours, ten worms were transferred onto a fresh 3.5 cm NGM plate with M9 solution containing 25 mM sodium azide  to paralyze the worms. Worms were then washed once by M9 solution containing 1 mg/ml ampicillin and 1 mg/ml gentamicin  and incubated for 0.5 h to kill bacteria attached to the surface of the animals on the plate. After worms were lysed with a motorized pestle, serial dilutions of the lysates  were plated onto Luria-Bertani plates containing 100 g ml-1 rifampicin  and incubated overnight at 37°C to select for PA14 colony. PA14 colonies were counted to determine CFU per worm. At least three independent replicates were performed for each condition.The quantification procedure of intestinal PA14 colony forming units (CFU) was modified from a previous study . BrieflyF35E12.5p::GFP or K08D8.5p::GFP images were acquired using a digital automated microscope . Synchronized eggs were grown to the L4 stage by feeding on L4440 vector control or gene-specific RNAi bacteria, prior to E. coli OP50 feeding or P. aeruginosa PA14 infection for 12 to 24 h. Animals were paralyzed with 1 mM sodium azide (Sigma) in M9 buffer and mounted on 2% agarose pads. To evaluate the fluorescence level in different genetic backgrounds, each genotype was analyzed on three different days, and the data were pooled. Data are presented as a percentage of the mean fluorescence relative to that of the wild-type or vector control. All results of fluorescence quantification are presented as means and SEM.To quantitatively analyze the fluorescence intensity of PMK-1/p38-dependent gene reporters, P. aeruginosa PA14 expressing GFP (PA14::GFP) were seeded on NGM plates and incubated at 37°C for 24 h. Synchronized L4 larvae were transferred to these plates and cultured at 25°C for 24 h. Before the examination of bacterial accumulation in the intestine and the pharyngeal lumen of animals, worms were washed in M9 buffer to eliminate fluorescent bacteria attached to the cuticle of worms. The visualized fluorescence of PA14::GFP was observed by using ultra-high resolution spectral confocal microscope , and the fluorescence intensity was quantified using Image-J software .Overnight culture of P. aeruginosa PA14 cultures were mixed with the beads at a ratio of 25:1 and 100μl of mixture were placed onto the plate and incubated at 37°C overnight. Young adult animals were transferred to the plate and incubated at 25°C for 2 hours. Worms were then washed in M9 buffer with 25 mM sodium azide to remove the fluorescent beads that adhere to the body surface, and mounted on glass slides for imaging using the a multi-purpose zoom microscope . The fluorescence intensity of the area containing the GFP fluorescent beads was analyzed using Image-J software .This assay was done by feeding synchronized worms with 0.5 μm green fluorescent polystyrene beads . OvernigP. aeruginosa PA14 culture was seeded in the center of 6.0 cm NGM plates and cultured at 37°C for 24 h. Thirty L4 larva were placed in the center of each PA14 lawn on the NGM plate. The number of worms within the PA14 lawn was counted at 2, 4, 6, 8 and 10 h post-exposure.The PA14 avoidance assay was performed with minor modification as previously described . A 20 μLSynchronized L4 stage worms were transferred to the OP50 lawn of NGM plate. Animals were scored for survival daily and transferred to fresh plates every 2-3 days. Animals were considered dead if they failed to respond to the repeated touch of a platinum wire. Those that died of vulva burst or crawling off plates were censored. The assay was performed at 25°C.Worm lysates were prepared using lysis buffer in the presence of protease inhibitors  or protease and phosphatase inhibitors . Proteins were boiled for 10 min at 98°C before gel electrophoresis. For the western blotting, we used primary anti-phospho-p38 MAPK rabbit polyclonal antibody  at a 1:1,000 dilution, or anti-β-actin mouse monoclonal antibody  at a 1:3,000 dilution. A horseradish peroxidase (HRP)-conjugated goat anti-rabbit , or anti-mouse  were used as secondary antibody at a 1:2,000 or 1:5,000 dilution, respectively. Images of western blot membranes were captured using an imaging system . All western blotting experiments were repeated at least three times independently.E. coli OP50 feeding or P. aeruginosa PA14 infection for 12 to 24 h. L4 worms were transferred onto Magic Red dye-containing NGM plate seeded with OP50 or PA14 and cultured overnight at 25 ˚C. Worms were photographed with ultra-high resolution spectral confocal microscope  and quantified by Image-J.Magic Red stain  is cleaved by Cathepsin B and generates a red fluorescent substrate in functional lysosomes . Magic RData were analyzed with Prism 8.0  and SPSS® Statistics Version 25 . For statistical analyses of all killing and life span assays, Log-rank (Kaplan-Meier) method was used to calculate p-values. The exact p-values for all survival assay are listed in Supporting The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: GSE216879 (GEO). Codes are available at 10.24433/CO.2273316.v1 (Code Ocean).The manuscript presents research on animals that do not require ethical approval for their study.XZ and HT conceived ideas and designed experiments. XZ, YW, ZC, and ZW performed experiments and analyzed data. XZ and HT wrote the manuscript with input from all authors. All authors reviewed and edited the manuscript.


Experimental Hematology & Oncology (2023) 12:7410.1186/s40164-023-00436-9After online publication of the article1, the authors noticed Table The correct tables are published with this erratum.


PRDM1, encoding the BLIMP1 transcription factor, defined highly potent TCF7 [encoding T cell factor 1 (TCF1)]–expressing CD8+ CAR T cells, whereas enrichment of HAVCR2 [encoding T cell immunoglobulin and mucin-domain containing-3 (TIM-3)]–expressing CD8+ T cells with elevated PRDM1 was associated with poor outcomes. PRDM1 knockout promoted TCF7-dependent CAR T cell stemness and proliferation, resulting in marginally enhanced leukemia control in mice. However, in the setting of PRDM1 deficiency, a negative epigenetic feedback program of nuclear factor of activated T cells (NFAT)–driven T cell dysfunction was identified. This program was characterized by compensatory up-regulation of NR4A3 and other genes encoding exhaustion-related transcription factors that hampered T cell effector function in solid tumors. Dual knockout of PRDM1 and NR4A3 skewed CAR T cell phenotypes away from TIM-3+CD8+ and toward TCF1+CD8+ to counter exhaustion of tumor-infiltrating CAR T cells and improve antitumor responses, effects that were not achieved with PRDM1 and NR4A3 single knockout alone. These data underscore dual targeting of PRDM1 and NR4A3 as a promising approach to advance adoptive cell immuno-oncotherapy.Chimeric antigen receptor (CAR) T cells have not induced meaningful clinical responses in solid tumors. Loss of T cell stemness, poor expansion capacity, and exhaustion during prolonged tumor antigen exposure are major causes of CAR T cell therapeutic resistance. Single-cell RNA-sequencing analysis of CAR T cells from a first-in-human trial in metastatic prostate cancer identified two independently validated cell states associated with antitumor potency or lack of efficacy. Low expression of  Two transcription factors, BLIMP1 and NR4A3, reciprocally regulate T-cell stemness and exhaustion and can be exploited for CAR T-cell therapy. et al. investigated a strategy to reverse this phenotype based on analysis of CAR T-cells employed in a clinical trial for metastatic prostate cancer. The authors identified two transcription factors, BLIMP1 and NR4A3, that regulated CAR T-cell dysfunction. Dual knockout of these two transcription factors reduced CAR T-cell exhaustion and enhanced their antitumor activity in multiple murine models, outperforming single knockouts. These data suggest that transcription factor modulation may be a key strategy to improve CAR T-cells for solid tumors.Tuning Up CAR T-Cells. Despite their success at treating hematological malignancies, CAR T-cells have not been as effective in treating solid tumors. This is, in part, due to loss of stemness and development of an exhausted phenotype after infusion. Here, Jung  Chimeric antigen receptor (CAR) T-cells have induced unprecedented high rates of complete remission in relapsed and refractory B-cell malignancies. Despite these successes, a substantial portion of patients with B-cell leukemia, lymphoma, and myeloma fail to respond to CAR T-cell therapy and only small subsets of patients experience long-term durable responses –5. The eCAR T-cells derived from naïve and early memory subsets have been shown to robustly expand in vivo and are long-lived with a self-renewal capacity –11. We a+ T-cell transcriptional programs enriched in axicabtagene ciloleucel infusion products were strongly related to the long-term durability of response, whereas gene signatures of exhaustion correlated with early treatment failure in patients with large B-cell lymphomas (LBCL) (We previously found that sustained remissions in CD19 CAR T-cell therapy of chronic lymphocytic leukemia (CLL) were associated with an increased peak expansion after infusion and relatively longer persistence. Cell products that were particularly effective showed greater proliferative capacity prior to and during treatment. Transcriptomic analysis suggested that remission correlated with early memory T-cell signatures as well as stemness , whereas gene expression profiles from non-responders were associated with terminal differentiation and exhaustion . Similars (LBCL) . Importaclinicaltrials.govNCT03089203) (PRDM1) gene encoding the B-lymphocyte-induced maturation protein 1 (BLIMP1) transcription factor, which is upregulated in poorly functional Hepatitis A Virus Cellular Receptor 2 -expressing CD8+ T-cells and downregulated in highly potent Transcription Factor 7 -expressing CD8+ T-cells, may potentiate CAR T-cell stemness and antitumor function. This hypothesis is further supported by the results of a very recently published study showing that genetic knockout of PRDM1 in T-cells enhances persistence and therapeutic responses in cancer models of adoptive immunotherapy (PRDM1 single knockout (KO) CAR T-cells exhibited enrichment of the TCF7-expressing CD8+ phenotype in association with improved CAR T-cell persistence and expansion. However, genetic deletion of PRDM1 alone resulted in only marginal enhancement of CAR T-cell efficacy against B-cell leukemia and completely failed to rescue CAR+ tumor-infiltrating lymphocyte (TIL) function in animal models of high tumor burden. These results are consistent to some degree with previous reports of antigen-specific PRDM1 KO T-cells exhibiting comparable or even inferior control of viruses and cancer through mechanisms that remain largely elusive -dependent manner. PRDM1 and NR4A3 dual-ablation counteracted the development of T-cell exhaustion, which was not observed with single KO of either gene. This strategy also led to a marked increase in the survival and expansion of TILs; increased the capacity of the CAR TILs to produce effector cytokines after stimulation; and reduced their expression of inhibitory factors. In immunodeficient mice bearing soft tissue or bone engrafted prostate cancer cells, pancreatic tumors or aggressive B-cell leukemia, a single intravenous infusion of PRDM1/NR4A3 dual KO CAR T-cells resulted in higher antitumor efficacy, T-cell response durability upon rechallenge and overall prolongation of survival compared to conventional CAR T-cell treatment. There is considerable interest in modulating CAR T-cells to control tumors more successfully. Our identification of the reciprocal nature of BLIMP1 and NR4A3 activity as a distinct mechanism regulating the balance of T-cell differentiation and function represents an actionable and therapeutically effective strategy for achieving this desired outcome.Here we analyzed dominant negative transforming growth factor β receptor II (TGF-βRII) ‘armored’ prostate-specific membrane antigen (PSMA) CAR T-cell infusion products from patients with metastatic castration-resistant prostate cancer (mCRPC) (3089203) . Using sotherapy . Accordi elusive –27. We fin vivo CAR T-cell expansion and prostate-specific antigen (PSA) response -expressing CD8+ clusters; effector-like Granzyme A (GZMA)- and HAVCR2-expressing CD8+ sub-populations  infection .–30.TCF7-n scores . Consist8+ cells .+ T-cell clusters identified in our analysis for this battery of genes and found that HAVCR2-expressing CD8+ T-cells in mCRPC patient infusion products had significantly (P < 0.001) higher IFN response signature scores compared to the other populations  demonstrated that enrichment of an interferon (IFN) response signature is associated with poor CAR T-cell persistence . We thusulations . Accordi outcome .+ clusters associated with response to CAR T-cell therapy in LBCL (TCF7-expressing CD8+ population highly expressed a gene set enriched in anti-CD19 CAR T-cell infusion products derived from complete responders (CR), whereas the HAVCR2-expressing CD8+ T-cell population exhibited the highest scores for a gene set that characterizes non-responder (NR) anti-CD19 CAR T-cells  treated with autologous CD19 CAR T-cells. As expected, pre-infusion CAR T-cells from CR and very good partially responding (PRTD) individuals exhibited significantly lower expression of PRDM1 (P < 0.01) and elevated TCF7 (P < 0.01), compared to poorly functional products from non-responders (NR) or conventional partial responders (PR)  .+ and CD8+ T-cells within the clinical CAR infusion products profiled in this study are presented in + compartment was also performed  and Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4)-expressing CD4+ clusters. CTLA4-expressing CD4+ cells displayed the highest expression of PRDM1 whereas TCF7-expressing CD4+ T-cells exhibited low PRDM1 among the CD4+ clusters /CRISPR-associated protein 9 (Cas9) technology (Adeno-associated virus integration site 1 (AAVS1) safe harbor locus were used as a negative control for gene editing in subsequent in vitro and in vivo functional studies , B-cell lymphoma 6 (BCL6) and Inhibitor of DNA-binding 3 (ID3) -2, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. PRDM1 ablation increased frequencies of polyfunctional CAR T-cells compared to AAVS1 KO control CAR T-cells   and B (3 3 (ID3) –38. Morestemness . Consistproducts . To exam in TCF7  and subs T-cells  to H. Be T-cells , which culations , suggestPRDM1 KO CAR T-cells in the setting of repetitive antigen exposure, we first assessed cytokine concentrations 24 hours after the first and fifth rounds of in vitro PC3-PSMA tumor cell stimulation. PRDM1 KO initially increased effector cytokine production as shown in PRDM1 KO CAR T-cells exhibited dramatically reduced effector cytokine secretion  model (NALM-6), although these CAR T-cells eventually failed to eradicate tumors  transcription factors as well as Thymocyte Selection-associated HMG BOX (TOX), TOX2, and Interferon Regulatory Factor 4 . To rule out potential model-dependent effects, we took advantage of a publicly available RNA sequencing expression dataset of CD8+ TILs from B16F10 (melanoma) tumor-bearing PRDM1 conditional knockout (cKO) syngeneic mice. Concordant with our findings in CAR T-cells, PRDM1 cKO CD8+ TILs exhibited upregulated expression of NR4A3, NR4A1, and IRF4 compared to wild type counterparts (right).To elucidate the mechanism by which ulations . Intrigu 4 IRF4; , left. Tterparts , right.PRDM1 KO CAR T-cells during chronic stimulation and that deletion of these exhaustion factors will render PRDM1 KO CAR T-cells capable of maintaining antitumor effector function. PRDM1 KO CAR T-cells exhibited reduced effector function, despite inhibitory receptor downregulation  expressing cells compared to AAVS1 KO CAR T-cells  on control and PRDM1 KO CAR T-cells. PRDM1 KO increased global chromatin accessibility  CAR T-cells in mice bearing intraosseous tumors generated by PC3-PSMA cell engraftment (PRDM1/NR4A3 double KO significantly increased the absolute numbers of CAR T-cells in the tumor (P < 0.05) and peripheral blood (P < 0.001) compared to AAVS1 KO CAR T-cells PRDM1 KO  and TIM-3 expression in the peripheral blood, their inhibitory receptor expression was comparable to that of AAVS1 KO CAR T-cells in the tumor where CAR T-cells receive persistent antigen stimulation (PRDM1/NR4A3 KO CAR T-cells demonstrated a significant reduction in the proportion of PD-1+TIM-3+ CD8 T-cells in both the tumor (P < 0.05) and peripheral blood as compared to AAVS1 KO CAR T-cells  expression in CAR T-cells in vivo. Both PRDM1 single KO and PRDM1/NR4A3 double KO CAR T-cells exhibited increased stem cell-like TIM-3−TCF1+ CD8 T-cells  that normally become accessible in CD8+ TILs , type of statistical used and P values are reported in the figure legends. No statistical methods were used to pre-determine sample size. These were estimated based on preliminary experiments. All in vitro functional experiments in the main text were carried out at least three times and no outliers or other data points were excluded from our analyses. For animal experiments, NSG mice were treated with CAR T cells from one to two different healthy donors. Cages of mice were randomly assigned to treatment groups. Investigators were not blinded to group allocation during experiments and assessment of outcome.The objective of this study was to define the functional effects of ClinicalTrials.gov (NCT03089203)  of the University of Pennsylvania. All ethical regulations were followed. This study is registered at 3089203) . Correla3089203) .PC3, PSMA-expressing PC3, and NALM-6 cells engineered to express click beetle green luciferase and green fluorescent protein (CBG-GFP), were kindly provided by Carl H. June and Marco Ruella (University of Pennsylvania), respectively. AsPC1 cells were obtained from the American Type Culture Collection (ATCC). PC3 and PC3-PSMA cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetus bovine serum (FBS) and streptomycin/penicillin. NALM-6 and AsPC1 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 media supplemented with 10% FBS and streptomycin/penicillin (R10 media). HEK 293T cells, used for lentivirus production, were obtained from ATCC and cultured in R10 media. Low-passage banks of cells were tested for mycoplasma with a MycoAlert kit (Lonza), according to the manufacturer’s instructions. Authentication of cell lines was carried out by the University of Arizona Genetics Core, based on criteria established by the International Cell Line Authentication Committee. Short-tandem-repeat profiling revealed that these cell lines were above the 80% match threshold. Mycoplasma testing and authentication were routinely performed before and after molecular engineering.Vector construction and lentiviral production were conducted as previously described . In brieHealthy donor T-cells were isolated from PBMCs using the Pan T Cell Isolation Kit according to the manufacturer’s instructions (Miltenyi Biotec). T-cells were activated with anti-CD3/CD28 antibody coated Dynabeads (Thermo Fisher Scientific) at 3:1 bead:cell ratio in T-cell media (OpTmizer CTS SFM media (Gibco) supplemented with 5% human AB serum and 100u/mL human IL-2). Following a 24-hour incubation, lentivirus encoding the PSMA CAR was added to the culture at a multiplicity of infection (MOI) of 2.5. CAR T-cell expansion was carried out as previously described .6 CAR T-cells were transfected with 12 μg TrueCut S. pyogenes Cas9 (Invitrogen) and 0.2nmol of chemically-modified tracrRNA and crRNA (Integrated DNA Technologies). Following electroporation, CAR T-cells were cultured in T-cell media. The crRNA sequences used in this study were: AAVS1: 5’-CCATCGTAAGCAAACCTTAG-3’, PRDM1: 5’-CATCAGCACCAGAATCCCAG-3’, TCF7: 5’-TCAGGGAGTAGAAGCCAGAG-3’, NR4A3: 5’-CCTTGGCAGCACTGAGATCA-3’. The frequency of targeted mutations generated by double strand break were determined by Sanger sequencing and subsequent TIDE (tracking of indels by decomposition) analysis. Primers used for targeted amplification were: PRDM1-F1: tctcagaaggagccacaggaacgg, PRDM1-R1: cacccaccctatgctgcaagttgc, NR4A3-F1: gaggagaggatgacacttcctctctgtttc, NR4A3-R1: ctgcccagcacctccatgtacttcaagcag. Western blot and flow cytometric analysis were conducted to confirm KO at the protein level.Following T-cell activation as described above, beads were removed using a magnet on day 3 and electroporation was carried out with a P3 primary cell 4D-nucleofector kit (Lonza). 2 × 106 cells) were suspended in a low-salt lysis buffer  and allowed to swell on ice for 30 minutes. After centrifugation (1000 × g), supernatants obtained from cells lysates (30 μg) were analyzed by 10% SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to PVDF membranes (Millipore). The membranes were washed with phosphate-buffered saline (PBS) containing 0.1% Tween 20 (PBST), and then blocked for 1-hour in 5% skim milk in PBST. After washing with PBST, membranes were incubated overnight with one of the following antibodies: Mouse monoclonal anti-β-Tubulin  or rabbit monoclonal anti-BLIMP1  or mouse monoclonal anti-NR4A3 . Membranes were washed with PBST and treated with 1:1000 diluted horseradish peroxidase-coupled goat anti-mouse or anti-rabbit secondary antibodies (Thermo Fisher Scientific) in PBST for 1-hour. After washing, the membranes were incubated in Pierce ECL western blotting substrate (Thermo Fisher Scientific) and visualized on X-ray film.T-cells (1 × 10Cell surface anti-human antibodies were diluted in FACS buffer (PBS + 2% FBS). PSMA CAR expression was measured using an allophycocyanin (APC)-conjugated recombinant human PSMA protein . T-cell immunophenotyping was carried out using the following antibodies: PD-1-Brilliant Violet (BV) 421 , CD45-BV570 , CD8-BV650 , CD8-APC-H7 , CD4-BV785 , TIM-3-phycoerythrin (PE) , CCR7-PE-CF594 , CD62L-PE-Cy5 , LAG3-PE-Cy7, hCD45-APC , murine CD45-Peridinin chlorophyll protein (PerCP)-Cy5.5 , CD127-BV570 , human leukocyte antigen (HLA)-DR-Alexa Fluor 700 , CD25-APC . For intracellular staining, cells were first permeabilized and washed using the FoxP3 Transcription Factor Staining Buffer Kit (eBioscience) and subsequently stained with following antibodies: IL2-PE-CF594 , IFN-γ-BV570 , TNF-α-Alexa Fluor700 , Perforin-BV421 , Perforin-APC , Granzyme B-PE-Cy5.5 , TCF1-Alexa Fluor 488 , TOX-APC , NFATC1-PE  according to our previously published methods 2 fragment (Jackson ImmunoResearch #115-065-072) and anti-biotin kits (Miltenyi Biotech). Following isolation, CAR T-cell purity was confirmed by flow cytometric analysis and CAR T-cells were then serially exposed (every 2 to 5 days) to irradiated PC3-PSMA cells at an effector-to-target (E:T) ratio of 3:1 or 1:1. Restimulation assays were carried out for 25 to 30 days, since cell counts become largely unreliable beyond the fifth and sixth rounds of stimulation due to senescent proliferative arrest and a marked decrease in the viability of chronically stimulated CAR T-cells. Supernatants were collected 24-hours post-tumor challenge for cytokine analysis using the LEGENDplex human CD8 panel (BioLegend), and absolute numbers of T-cells in culture were monitored using a Luna automated cell counter (Logos Biosystems) during the assay.CAR T-cell expansion capacity and effector function were assessed using a restimulation assay, as previously described , 56, 59.4 PC3-PSMA cells were seeded in E-Plate VIEW 96 PET microwell plates. After 24-hours, PSMA CAR T-cells or control (untransduced) T-cells were added to wells containing tumor targets to achieve the desired E:T ratios. 20% Tween 20 was added to separate wells consisting of PC3-PSMA cells alone as a full lysis control. Electrical impedance was monitored in 20-minute intervals over 7 days and cytotoxicity was assessed by calculating the normalized cell index and % cytolysis.The longitudinal killing capacity of engineered CAR T-cells against PC3-PSMA cells was assessed using the xCELLigence system (ACEA Biosciences Inc.). CAR-expressing T-cells were magnetically enriched prior to the cytotoxicity assay. 2 × 10+ CAR T-cells were isolated from culture using CD8 microbeads (Miltenyi Biotec) for qRT-PCR. First, total RNA was extracted from CD8+ CAR T-cells using RNA Clean & Concentrator kits (Zymo Research). cDNA was then synthesized using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio) per the manufacturer’s protocol and qRT-PCR was conducted using Applied Biosystems TaqMan Fast Advanced Master Mix (Thermo Fisher Scientific) on the QuantStudio3 (Applied Biosystems). The primer/probe sets used for these experiments were as follows: CD3E: Hs01062241_m1, NR4A2: Hs00428691_m1 and NR4A3: Hs00545009_g1 (Thermo Fisher Scientific).After 2 to 4 rounds of restimulation, CD86 or 5 × 106 PC3-PSMA-CBG-GFP tumor cells were premixed with Matrigel (Corning) and injected into the flanks of NSG mice. When the average tumor size reached 150 to 200 mm3 or 500 mm3, 3.5 × 105 PSMA CAR T-cells were injected intravenously. Tumor growth weekly monitored weekly by taking caliper measurements (tumor volume = (length × width2)/2). Animals were euthanized when tumor volume exceeded 1500 mm3 or 2 cm in diameter. For the intraosseous PC3-PSMA model, male NSG mice were intrafemorally transplanted with 2 × 105 PC3-PSMA cells. On day 27, 1 to 2 × 105 PSMA CAR T-cells were infused intravenously, and tumor burden was measured by injecting mice intraperitoneally with luciferin and quantifying bioluminescence using an IVIS Spectrum In Vivo Imaging System (PerkinElmer). To characterize CAR T-cell phenotype and function, peripheral blood and tumor tissues were isolated from mice. Blood samples were obtained at peak CAR T-cell expansion by cheek bleeding. Tumors isolated on day 13 post-CAR T-cell injection were minced with a scalpel and treated with 100 U/mL collagenase IV and 0.25mg/mL DNase I for 1 hour at 37°C. The absolute number of human T-cells in the peripheral blood and tumors was quantified using 123count eBeads (Thermo Fisher Scientific). To assess the effector function of TILs, these ex vivo isolated T-cells were stimulated with 50 ng/mL phorbol 12-myristate 13-acetate (PMA) and 1mg/mL ionomycin in the presence of 5 ug/mL Brefeldin A for 6-hours and expression of IL-2, IFN-γ and TNF-α were assessed by intracellular staining. We used PMA/ionomycin stimulation to assess TIL effector function to eliminate potential confounding variables associated with antigen receptor expression or signaling.Mouse studies were performed with 6- to 8-week-old male NOD/SCID/IL-2Rγ-null (NSG) mice in compliance with a University of Pennsylvania Institutional Animal Care and Use Committee approved protocol. For the subcutaneous PC3-PSMA model, 106 AsPC1 cells were premixed with Matrigel and subcutaneously injected into NSG mice. On day 30, when tumor volume reached 300 to 400 mm3, mesothelin-directed CAR T-cells were intravenously injected, and tumor growth was monitored using caliper measurements. For the B-cell acute lymphoblastic leukemia model, NSG mice were intravenously injected with 1 × 106 NALM-6-CBG cells. On day 7 post-tumor injection, 3 × 105 CD19 CAR T-cells were intravenously infused, and tumor growth was assessed twice weekly using bioluminescent imaging . The mice that had exhibited initial control of NALM-6 cells and survived were rechallenged with 2 × 106 NALM-6 cells on day 40. Tumor growth was monitored using bioluminescence imaging, as described above.To assess the memory recall response of CAR T-cells, NSG mice were engrafted with 10PSMA CAR T-cell infusion products from 5 different patients were thawed in a 37°C water bath and dead cells were eliminated using the Dead Cell Removal Kit (Miltenyi Biotec). PSMA CAR-positive T-cells were enriched by magnetic cell separation using a Biotin-goat anti-mouse IgG F(ab)2 fragment (Jackson ImmunoResearch #115-065-072) and anti-biotin kits (Miltenyi Biotech). scRNA-seq libraries were prepared using Chromium Single Cell 3’ v3.1 Reagent Kits (10X Genomics) following the manufacturer’s instructions. Isolated CAR T-cells were washed and resuspended in PBS containing 0.04% BSA and about 20,000 cells were loaded per reaction to capture about 10,000 cells. Sequencing was performed on a Novaseq 6000 (Illumina) at a depth of at least 20,000 reads per cell.Reads were aligned to the human reference genome (GRCh38) using Cell Ranger version 6.0.0. Subsequent quality control and downstream analyses were performed using Seurat 4.0. Cells were filtered based on the following criteria to eliminate low-quality cells: 1) a minimum of 1000 genes and a maximum of 6000 genes detected per cell; and 2) less than 15% of mitochondrial gene counts. After quality control, 20,702 cells remained. Gene-cell matrices from five CAR T-cell products were then integrated using SelectIntegrationFeatures, PrepSCTIntegration, FindIntegrationAnchors, and IntegrateData functions in Seurat. CD4-positive and CD8-positive cells were clustered using FindNeighbors/FindClusters, followed by differential gene expression analysis using the FindClusters command. Module scores were then calculated using AddModuleScore to assess the enrichment of gene signatures in CD4 and CD8 subclusters.+ CAR T-cells were isolated using CD8 microbeads (Miltenyi Biotec) and total mRNA was extracted with RNA Clean & Concentrator kits (Zymo Research). Bulk RNA-seq was conducted by Novogene using the Novaseq6000 system (paired-end 150bp) at 40 × 106 reads per sample. Reads were pseudoaligned to the human genome (GRCh38) transcriptomes using kallisto v0.46.0.On days 0, 5 and 20 of the CAR T-cell restimulation assay, before stimulation and after the first and fourth tumor challenge respectively, CD8AAVS1 KO PSMA CAR T-cells and PRDM1 KO PSMA CAR T-cells were performed using the edgeR v3.34.0 and limma v3.48.0 packages. Briefly, expression data were normalized using a trimmed mean of log expression ratios method and transformed into log2(counts per million). Linear models were used to assess differential expression, and P values were adjusted using the Benjamini-Hochberg method. Gene set enrichment analysis was conducted using GSEABase v1.54.0, clusterProfiler v4.0.2, and msigdbr v7.4.1.Differential expression analyses between + CAR T-cells were isolated using CD8 microbeads (Miltenyi Biotec). 100,000 cells per sample were cryopreserved. Nuclei were isolated from CD8+ T-cells for each replicate, followed by the transposition reaction in the presence of Tn5 transposase  for 45 minutes at 37°C. Purification of transposed DNA was then completed with the MinElute Kit (Qiagen) and fragments were barcoded with dual indexes (Illumina Nextra). Library preparation and sequencing were performed by Novogene using NovaSeq 6000 (paired-end 150bp reads) at a depth of 30 × 106 reads per sample.After CAR T-cell manufacturing, dead cells were eliminated using the Dead Cell Removal Kit (Miltenyi Biotec) and CD8https://github.com/marcelm/cutadapt/). They were aligned to the hg19 reference genome using Bowtie2, restricting to properly aligned and properly paired reads between 10 and 1000 base pairs. Mitochondrial reads were removed (https://github.com/jsh58/harvard/blob/master/removeChrom.py). Files were sorted using samtools, and PCR duplicates were removed using Picard. BAM files were indexed using samtools in order to visualize tracks in IGV. Peak calling was performed with MACS2 with a false discovery rate (FDR) q-value of 0.01. The R package Diffbind was used to remove ENCODE blacklisted regions (https://sites.google.com/site/anshulkundaje/projects/blacklists), then to identify peaks differentially opened between the control and the PRDM1 knockout. The findMotifsGenome script from HOMER was used to map the hg19 genome for occurrences of the BLIMP1 motif (derived from ENCODE data accessible by GEO at GSE31477) and the NFAT2 motif (FASTQ files for each sample were trimmed of adapter contamination using cutadapt (T2 motif .P values <0.05 were considered significant.Raw, individual-level data are presented in Supplementary MaterialData file S1. Raw, individual-level data


Macrophages are innate immune cells and often classified as M1 macrophages (pro-inflammatory states) and M2 macrophages (anti-inflammatory states). Exosomes are cell-derived nanovesicles that range in diameter from 30 to 150 nm. Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and circular RNAs (circRNAs), are abundant in exosomes and exosomal ncRNAs influence immune responses. Exosomal ncRNAs control macrophage-linked intercellular communication via their targets or signaling pathways, which can play positive or negative roles in lung cancer and inflammatory lung disorders, including acute lung injury (ALI), asthma, and pulmonary fibrosis. In lung cancer, exosomal ncRNAs mediated intercellular communication between lung tumor cells and tumor-associated macrophages (TAMs), coordinating cancer proliferation, migration, invasion, metastasis, immune evasion, and therapy resistance. In inflammatory lung illnesses, exosomal ncRNAs mediate macrophage activation and inflammation to promote or inhibit lung damage. Furthermore, we also discussed the possible applications of exosomal ncRNA-based therapies for lung disorders. Resident tissue macrophages remain one of the most frequent immune cells and develop from embryonic progenitors or bone marrow monocytes . The funExtracellular vesicles (EVs) are membranous particles released by cells into the extracellular space. Exosomes (30 to 150 nm in diameter), shed microvesicles (MVs) (150 to 1000 nm in diameter), and apoptotic bodies (50 to 5000 nm in diameter) are the three subtypes of EVs that are separated based on size range and biogenesis . ExosomeIn this review, we discussed the functional roles of the exosomal ncRNAs in macrophage-linked intercellular communication in lung cancer and inflammatory lung diseases, including ALI, asthma, and pulmonary fibrosis. In lung TME, exosomal ncRNAs govern bilateral communication between TAMs and lung tumor cells. In inflammatory lung diseases, exosomal ncRNAs mediate the crosstalk between macrophages and lung tissue cells. Furthermore, exosomal ncRNAs have the promising potential to function as diagnostic and prognostic biomarkers in lung diseases.According to the M1/M2 paradigm, macrophages can be activated into two distinct populations: M1 macrophages  and M2 macrophages  . M1 macrLung cancer is the most frequent cancer based on current diagnoses and the leading cause of cancer-related mortality globally . Lung caCompared to M1 macrophages, miR-942 expression is greater in M2 macrophages. In lung adenocarcinoma tissues, increased miR-942 expression is correlated with strong TAM infiltration. miR-942 over-expression eliminates forkhead box subfamily O 1 (FOXO1) expression to boost β-catenin expression . miR-942miR-19b-3p expression is higher in lung tumor cell-secreted exosomes than in lung tumor cells. miR-19b-3p targets protein tyrosine phosphatase receptor type D (PTPRD). Lung cancer cell-derived exosomal miR-19b-3p raises miR-19b-3p levels in macrophages. Then, miR-19b-3p up-regulation reduces PTPRD to promote M2 polarization of macrophages through the phosphorylation of signal transducer and activator of transcription 3 (STAT3). Chen et al. indicate that miR-19b-3p knockdown abrogates lung cancer metastasis via attenuating macrophage M2 polarization . miR-181Unphosphorylated Yes-associated protein (YAP) has been shown to translocate into the cell nucleus where it binds to the TEA domain transcription factor (TEAD) family, resulting in gene transcription. Both YAP cytoplasmic retention and YAP degradation can be induced by phosphorylated large tumor suppressor kinase 1/2 (LATS1/2) . As a meExosomes obtained from A549 cells have large amounts of the lncRNA FGD5 antisense RNA 1 (FGD5-AS1). Exosomal FGD5-AS1 from A549 cells induces M2 macrophage polarization, leading to increased A549 cell migration and invasion . In comp+ T cells. Consequently, tumor exosomal circZNF451 is favorable for immune-suppressed TME and represses immunotherapy efficacy in lung cancer. Additionally, serum exosomal circZNF451 can be used as a biomarker to identify patients with lung adenocarcinoma who have a poor prognosis [Exosomal circSHKBP1 expression is up-regulated in serum from NSCLC patients. By sponging miR-1294, circSHKBP1 induces pyruvate kinase M2 isoform (PKM2) mRNA expression . A549-derognosis . Comparerognosis . In shorTaken together, exosomal ncRNAs regulate intercellular communication between TAMs and lung tumor cells in lung TME , which cALI is a severe respiratory syndrome with the existence of hypoxemia. Acute respiratory distress syndrome (ARDS) is a serious form of ALI. ALI and ARDS are prevalent among critically ill patients . In ALI,Pyroptosis, an inflammatory cell death type mediated by caspase-1 or caspase-11, can elicit strong inflammatory responses to antagonize microbe infection. Excessive pyroptosis can result in a variety of inflammatory illnesses, including sepsis and autoimmune diseases ,97. The Mesenchymal stem cells (MSCs) are defined as multipotent mesenchymal stromal cells with capacities to self-renew and differentiate into the mesodermal lineage  . Bone mamiR-92a-3p is up-regulated in exosomes released by AECs after LPS stimulation. miR-92a-3p binds to PTEN 3′UTR . Exosoma+ AMs, the regulator of G protein signaling-1 (RGS1) depletion elevates IL10, SOCS3, and IL-4-stimulated Arg-1 and TGF-β1 expression. Endothelial cell-derived exosomal miR-223 down-regulates RGS1 to promote the anti-inflammatory and profibrotic effects of Tie2+ AMs [+ AM proportion, dampen lung injury, and improve blood oxygenation [miR-155 is highly expressed in serum exosomes extracted from ALI mice. Serum exosome-derived miR-155 from LPS-treated ALI mice stimulates macrophage proliferation via attenuating Src Homology-2 (SH2) domain-containing Inositol 5′-Phosphatase-1 (SHIP1) . Serum eie2+ AMs . In LPS-genation . As mentPulmonary fibrosis is a devastating lung disease featured by progressive and irreversible destruction of lung architecture, resulting in impaired gas exchange and respiratory failure . RecentlCOL1A2, COL3A1, and TGF-β1) via reducing TGF-β receptor 1 (TGFβ-R1) [Sprouty homologue 1 (Spry1) is a negative regulator of the extracellular signal-regulated protein kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling pathway . miR-721TGFβ-R1) . MacrophTGFβ-R1) . It is iTGFβ-R1) . The expTGFβ-R1) . circRNATGFβ-R1) . Exosoma+ AMs [+ AMs via decreasing RGS1. In addition, exosomes released by type II AECs increase the Flt3+ AM proportion and suppress BLM-exposed pulmonary fibrosis [miR-27b-3p has been shown to induce synovial fibrosis in knee osteoarthritis . Feng et+ AMs . Exosomafibrosis . Recentlfibrosis ,130. miRfibrosis . ExosomaAsthma is a chronic inflammatory respiratory illness affecting an estimated 334 million people in the world . The symMacrophages are one of the most prevalent immune cells found in lung tissues. Macrophages are highly plastic cells with different phenotypes and functions, which are influenced both by their origin and resident tissue microenvironment. M1 macrophages potentiate systemic inflammatory responses and restrain tumor progression. M2 macrophages restrict inflammation and accelerate the development of tumors. The specific components of exosomes determine whether they have supportive or inhibitory functions. ncRNAs are enriched in exosomes secreted by macrophages or other cell types. Cell-to-cell communication between macrophages and target cells is governed by exosomal ncRNAs. Exosomal ncRNAs modulate macrophage polarization as well as TME reprogramming. Exosomal ncRNAs regulate macrophage-linked intercellular communication to influence lung cancer and inflammatory lung diseases. Medical applications of ncRNAs in treating human illnesses have recently gained prominence. Some miRNA-based therapies are being examined in clinical trials, such as TargomiR (miR-16 mimic-based therapy) for malignant pleural mesothelioma  and MiraIn lung cancer, exosomal ncRNAs, such as miR-19b-3p , SNHG7 , and cirAgomirs up-regulate miRNA expression in exosomes and counteract the impacts of exosomes. For instance, miR-770 agomir administration elevates miR-770 expression in A549 exosomes and reverses the effects of A549 exosomes on TAMs . AgomirsPresently, despite significant advancements in the study of lung cancer and inflammatory lung illnesses, the research based on the exosomal ncRNA-macrophage axis is still lacking. Exosomal ncRNAs are critical regulators of macrophage activation, polarization, inflammation, and recruitment. In lung cancer, studies are primarily conducted using NSCLC cell lines, and studies on SCLC cell lines and in lung cancer in vivo models are still limited. In inflammatory lung disorders, it is widely suggested that LPS-induced inflammation can mimic the injurious microenvironment. Animal models induced by LPS may be useful in investigating the pathological mechanism of lung injury.


Correction:J Exp Clin Cancer Res37, 273 (2018)https://doi.org/10.1186/s13046-018-0921-1• Figure 5e - the 24 h wound healing of shRNA1 DANCR + Vector MSI2 group was misplacedFollowing publication of the original article , the autThe correct figure is given below.


Magnaporthe oryzae forms specialized infectious structures called appressoria that breach host cells to initiate infection. Previous studies demonstrated that the regulator of G-protein signaling (RGS)-like protein MoRgs7 undergoes endocytosis upon fungal sensing of hydrophobic environmental cues to activate cAMP signaling required for appressorium formation. However, the mechanism by which MoRgs7 internalizes and its fate remains undetermined. We here show that MoSep1, a conserved protein kinase of Mitotic Exit Network (MEN), phosphorylates MoRgs7 to regulate its function. MoRgs7 phosphorylation determines its interaction with MoCrn1, a coronin-like actin-binding protein homolog that also modulates the internalization of MoRgs7. Importantly, the endocytic transport of MoRgs7 is critical for its GTPase-activating protein (GAP) function important in cAMP signaling. Together, our findings revealed a novel mechanism by which M. oryzae activates MoRgs7-mediated hydrophobic cue-sensing signal transduction involving protein phosphorylation and endocytic transport to govern appressorium formation and fungal pathogenicity.The rice blast fungus  M. oryzae forms a highly specialized infectious structure called appressorium to initiate infection. Previous studies showed that the seven transmembrane (7-TM) domain-containing regulators of G-protein signaling (RGS)-like protein MoRgs7 is vital for the perception of environmental cues and cAMP-dependent appressorium formation. However, little is known about how MoRgs7 transduces hydrophobic cues and their regulation. In this study, we have identified that MoSep1 phosphorylates MoRgs7 during appressorium formation, and phosphorylation of MoRgs7 leads to a high binding affinity with MoCrn1 and its internalization. Additionally, the internalization of MoRgs7 contributes to the activation of cAMP signaling, further governing appressorium formation and pathogenicity.During its pathogenic interaction with rice,  Magnaporthe oryzae poses the most severe threat to rice production across the world and is also an excellent model organism for studying plant–pathogen interactions  was computed in the sample from the standard curve. The relevant experimental procedures were as described previously [His-MoRgs7, His-MoRgs7eviously .M. oryzae strains and transformants were cultured in liquid CM for 48 h and harvested. Mycelia were grounded into fine powders in liquid nitrogen and total cAMP levels were quantified by HPLC following previously established procedures [ocedures .5A, MoRgs75D, and MagAG187S to MoRgs7, MoRgs75A, and MoRgs75D were determined by MST using Monolith NT.115 (Nano Temper Technologies) according to the manufacture provided protocol. MST premium-coated capillaries (Monolith NT.115 MO-K005) were used to load the samples into the MST instrument at 25°C using medium MST power. Laser on and off was set at 30 and 5 s, respectively. This experiment was repeated with three biological repetitions and three replicates under the same experimental conditions. Data were analyzed using Nano Temper Analysis software v. 1.2.101 (Nano Temper Technologies) [Bindings of MoCrn1 to MoRgs7, MoRgs7ologies) .back, His-MoSep1STK, and His were expressed in E. coli BL21-CodonPlus (DE3) cells. Samples were induced with 0.1 mM IPTG  at 20°C for 4 h. E. coli Cells were lysed in lysis buffer . Samples were centrifuged at 3,600 rpm for 10 min, and the supernatants were transferred to new 1.5-mL tubes and stored at -70°C. The GST-MoRgs7 supernatant was mixed with 30 mL of GSH Sepharose beads, incubated at 4°C for 4 h, and then incubated with His-MoSep1, His-MoSep1back, His-MoSep1STK, and His supernatants at 4°C for another 4 h. Finally, the beads were washed with buffer  5 times and proteins eluted. Eluted proteins were analyzed by immunoblot (IB) with anti-His and anti-GST antibodies [GST-MoRgs7, His-MoSep1, His-MoSep1tibodies .All transformants were observed under a confocal laser scanning microscope , and the filtered channels were set as followings: GFP . Exposure time: 800 ms. Insets highlight areas analyzed by line-scan. Bar = 10 μm.4 conidia ml-1 in sterile water. Droplets (30 μl) of conidial suspension were applied on coverslips  and incubated under humid conditions at 28°C prior to appressorial formation assessment [Conidia were harvested from 7-day-old SDC cultures and adjusted to 5×10sessment .The turgor pressure of mature appressorium was measured using an incipient cytorrhysis (cell collapse) assay. The 1.0–4.0 M glycerol solution was used to measure turgor pressure according to the previous description . Collapsp value of <0.05 or <0.01.Results are presented as the mean ± standard deviation (SD) of at least three replicates. Significant differences between replicates were statistically evaluated using SDs and one-way ANOVA in SPSS 2.0. Results of two different treatments were statistically compared by ANOVA, followed by the F-test if the ANOVA result was significant at a S1 FigIn vitro pull-down assay examines interactions between MoRgs7 and MoSep1 full-length, and MoRgs7 and two segments of MoSep1 (MoSep1STK and MoSep1BACK). His-MoSep1, His-MoSep1STK, and His-MoSep1BACK were expressed and incubated with Sepharose beads. Eluted proteins were detected with anti-His and anti-GST antibodies and analyzed by immunoblotting. Asterisks indicate the main bands. (B) Co-IP assay for the interaction between MoRgs7 and MoSep1 full-length, and MoRgs7 and two segments of MoSep1 (MoSep1STK and MoSep1BACK). Co-expression of MoRgs7-RFP and MoSep1STK-GFP, MoRgs7-RFP, and MoSep1BACK-GFP in the wild-type strain Guy11. The proteins were incubated with anti-RFP beads and detected by anti-GFP and anti-RFP antibodies, respectively. Asterisks indicate the main bands.(A) (TIF)Click here for additional data file.S2 FigMosep1 mutant expressing MoRgs7 variants.MoRgs7 phosphorylation sites in Guy11 in comparison with the Δ(TIF)Click here for additional data file.S3 Fig(A) Pathogenicity assay. The experimental procedures were conducted as the same as described for (TIF)Click here for additional data file.S4 Fig(A) The cytorrhysis assay was conducted by incubating conidia on the hydrophobic surface and treated with various concentrations of glycerol (1–4 M). Numbers of collapsed appressoria among 100 appressoria were recorded and the examination was repeated twice. Error bars represent SDs and asterisks represent significant differences (**p < 0.01). (B) Observation for penetration on the rice sheath. The penetration rate was counted 3 times. Error bars represent SDs and asterisks represent significant differences (*p < 0.05).(TIF)Click here for additional data file.S5 Fig Pathogenicity assay, diseased leaf area analysis, and infectious hyphal type assessment were conducted as the same as described for (TIF)Click here for additional data file.S6 FigG187S was co-introduced with MoRgs7 and the site-directed mutagenesis mutants MoRgs75A and MoRgs75D into the AH109 strain. The transformants were plated on SD-Leu-Trp (as control), SD-His-Leu-Trp , and SD-Leu-Trp-His-Ade (for further selection) for 5 days. (B) Co-IP assay for the interaction between MoMagAG187S, MoRgs7 and its site-directed mutagenesis MoRgs75A and MoRgs75D. Co-expression of MoMagAG187S-RFP and MoRgs7-GFP, MoRgs75A-GFP and MoRgs75D-GFP in Guy11, respectively. Proteins were incubated with anti-RFP beads and detected by anti-GFP and anti-RFP antibodies.(A) Yeast two-hybrid analysis. MoMagA(TIF)Click here for additional data file.S7 Fig5A and MoRgs75D variants into AH109 strain, respectively. Transformants were plated on SD-Leu-Trp (as control), SD-His-Leu-Trp , and SD-Leu-Trp-His-Ade (for further selection) for 5 days. (B) Co-IP assay for the interaction between MoCrn1, MoRgs7 and its site-directed mutagenesis MoRgs75A and MoRgs75D. Co-expression of MoCrn1-RFP and MoRgs7-GFP, MoRgs75A-GFP and MoRgs75D-GFP in the wild-type strain Guy11, respectively. Proteins were incubated with anti-RFP beads and detected by anti-GFP and anti-RFP antibodies.(A) Yeast two-hybrid analysis. MoCrn1 was co-introduced with MoRgs7 and its site-directed mutagenesis MoRgs17(TIF)Click here for additional data file.S8 FigMosep1/MoSEP1, and ΔMosep1/MoRGS75D mutants expressing the H1-RFP construct were stained with CFW and examined by epifluorescence microscopy. Scale bar: 10 μm. (B) Hyphae of Guy11 and the ΔMosep1, ΔMosep1/MoSEP1, and ΔMosep1/MoRGS75D mutants were stained with CFW and examined by epifluorescence microscopy. Percentages shown in all images were calculated by the observation of 100 randomly-selected hyphae, and the observation was repeated twice. Scale bar: 10 μm.(A) Hyphae of Guy11 and the ΔMosep1, Δ(TIF)Click here for additional data file.S1 TextMagnaporthe oryzae into yeast AH109 strain. Then, AH109 strain was incubated on SD-Leu-Trp and SD-Leu-Trp-His-Ade for 5 days. The names of known protein filled in the brackets. The underlined protein is mentioned in this research. Among this table, MoMagA (MGG_01818) and MoCrn1 (MGG_06389) has been proved been interacted with MoRgs7 in previous studies .MoRgs7 was inserted into vector pGBKT7, and was were co-introduced with a yeast two-hybrid cDNA library contained various stages RNS of (DOCX)Click here for additional data file.


Drosophila melanogaster extends their mating duration when surrounded by conditions enriched with rivals. Here we report a different form of behavioral plasticity whereby male fruit flies exhibit a shortened duration of mating when they are sexually experienced; we refer to this plasticity as ‘shorter-mating-duration (SMD)’. SMD is a plastic behavior and requires sexually dimorphic taste neurons. We identified several neurons in the male foreleg and midleg that express specific sugar and pheromone receptors. Using a cost-benefit model and behavioral experiments, we further show that SMD behavior exhibits adaptive behavioral plasticity in male flies. Thus, our study delineates the molecular and cellular basis of the sensory inputs required for SMD; this represents a plastic interval timing behavior that could serve as a model system to study how multisensory inputs converge to modify interval timing behavior for improved adaptation.Males have finite resources to spend on reproduction. Thus, males rely on a ‘time investment strategy’ to maximize their reproductive success. For example, male  To maximize their return on investment, male flies utilize a wide variety of sensory inputs, including memories of past sexual encounters. Therefore, when males have sufficient sexual experiences, they shorten their mating duration. The term "Shorter-Mating-Duration" (SMD) was coined to describe this behavior. SMD must be triggered by sugar and pheromone, which is detected by cells and receptors in the male forelegs. We found that these cells include male-specific sensory neurons that are tuned to collect on a male’s sexual experiences and relay that information to the brain, where it is used to determine how long to spend mating the next available mate. We hypothesize that SMD can serve as a straightforward genetic model system through which we can investigate "interval timing", the capacity of animals to distinguish between periods ranging from minutes to hours in duration. Drosophila, for instance, respond to the presence of competitors by extending the mating duration in order to guard the female and pass on their genes. Hence, female guarding has typically evolved as a tactic for males to invest their time mCD8GFP; fruFLP, UAS[stop]nsybGFP; fruFLP, UAS[stop]TNTactive; fruFLP, fru-GAL4; from Dr. Toshiro Aigaki : UAS-mSP; from Dr. Martin Heisenberg : WT Berlin, ninaE17; from Dr. Michael Gordon : Gr5a-lexA, Gr64f-lexA, ppk23-GAL4, ppk25-GAL4, ppk29-GAL4.We are very grateful to the colleagues who provided us with many of the lines used in this study. We obtained the following lines from Dr. Joel D. Levine and Joshua J. Krupp : Orco1 (#23129), Orco2 (#23130), UAS-tubGAL80ts (#7018), Df(1)Exel6234 (#7708), UAS-traF (#4590), GustDx6 (#8607), Gr66a-GAL4 (#28801), UAS-mCD8GFP (#5130), UAS-RedStinger (#8547), snmp11 (#25043), snmp12 (#25042), UAS-snmp1 (#25044), Snmp2-RNAi (#51432), UAS-TNT (#28997), UAS-dicer , tra-RNAi (#28512), lush-RNAi (#31657), tud1  (#1786), lexAop -tdTomato.nls, UAS-Stinger (#66680), Gr5a-RNAi (#31282), Gr43a-RNAi (#64881), Gr61a-RNAi (#54030), Gr64c-RNAi (#36734), ppk23-RNAi (#28350), ppk25-RNAi (#27088), ppk29-RNAi (#27241), fru-lexA (#66698), Gr64f-GAL4 (#57668), se1 , elavc155; UAS-Dcr-2 (#25750), CalexA (#66542), UAS-TrpA1 (#26264), UAS-shits (#44222), Poxn-GAL4 (#66685), Poxn-RNAi (#26238); from Vienna Drosophila Stock Center: Gr5a-RNAi (#v13730), Gr43a-RNAi (#v39518), Gr61a-RNAi (#v106007), Gr64a-RNAi (#v103342), Gr64b-RNAi (#v42517), Gr64d-RNAi (#v29422), Gr64e-RNAi (#v109176), Gr64f-RNAi (#v105084). Following transgenic stocks are available from Korea Drosophila Resource Center (KDRC): UAS[stop]TNTinactive; fruFLP (1124).The following lines were obtained from Bloomington Stock Center (#stock number): Mating duration assay was performed as previously described ,22. For Defexel6234 females which lacks SPR and shows multiple mating with males [To deplete sperm from males, 40 virgin th males  were plaCourtship assay was performed as previously described , under nFor climbing assay, individual flies were placed in a 15 ml falcon tube (Fisher Scientific) and were gently tapped to the bottom of the tube. The time taken for the flies to climb 8 cm of the tube wall was recorded. Each fly was tested 5 times. Other than a single instance, all flies were seen to reach the target height within 2 min, which was the experimental cut-off time. Velocity was obtained by dividing the lines (mm) a fly crossed  by time (sec) a fly reached the line of the tube. For horizontal (spontaneous) locomotor activities, a single fly was first brought to the middle of the column by gentle shaking and then the fly movement was constantly monitored for 5 min and recorded. Total fraction of time flies walked during 5 min was calculated and number of stops during 5 min was also counted then calculated .As described before , brains http://rsb.info.nih.gov/ij).). Fluorescence was quantified in a manually set region of interest (ROI) of the 4th or 5th tarsus. To compensate for differences in fluorescence between different ROI, GFP fluorescence for CaLexA was normalized to autofluorescence, and then the fluorescence of ROI was quantified using the measure tool of ImageJ. All specimens were imaged under identical conditions.To quantify the calcium level in leg sensory neurons, we measured fluorescence intensity using the measure tool of ImageJ  . All analysis was done in GraphPad (Prism). Individual tests and significance are detailed in figure legends.Statistical analysis of mating duration assay was described previously ,22. MoreWhen we compare the difference of mating duration in experiments without internal control built in, we always performed control experiments of wild type for each independent experiment for internal comparison. And in this case, we analyzed data using ANOVA for statistically significant differences  between the means of mating duration for all conditions. If a significant difference between the means was found by Kruskal-Wallis test, then the Dunn’s Multiple Comparison Test was used to compare the mean mating duration of each condition to determine which conditions were significantly different from condition of interest. (# = p < 0.05)t-test for statistical analysis, we added estimation statistics for all MD assays and two group comparing graphs. In short, ‘estimation statistics’ is a simple framework that—while avoiding the pitfalls of significance testing—uses familiar statistical concepts: means, mean differences, and error bars. More importantly, it focuses on the effect size of one’s experiment/intervention, as opposed to significance testing [effect size, the difference diagram encourages quantitative reasoning about the system under study [Besides traditional  testing . In comper study . Thus, wer study .We performed egg laying assay as previously described . In shorse1 or CS virgin females were introduced to se1 or CS males either as naïve or experienced condition for 24 hours to be confident of all females’ mating with the first males. Then we introduced the second males for 24 hours. After this treatment, we separated females from second males then counted the number of progenies from females. To confirm that the effect from this fecundity test was not originated from the genotype background, we performed the same experiments by reversing the genotypes of the first and second males (se1 then CS vs. CS vs. se1). We calculated the percentage of progeny either from the first male or the second male by counting the eye color of progeny.Basically, we followed the protocols previously described by other group . In shor10.1126/science.abk2432 [https://github.com/vib-singlecell-nf), the availability of raw and processed datasets for users to explore, and the development of a crowd-annotation platform with voting, comments, and references through SCope (https://flycellatlas.org/scope), linked to an online analysis platform in ASAP (https://asap.epfl.ch/fca).snRNAseq dataset analyzed in this paper is published in Li et al., doi:.abk2432  and avaihttps://flycellatlas.org [For the gene expression pattern of the 10 genes involved in SMD in each cell type of leg and other tissues, we used the single-cell RNA-seq data from tlas.org , and theThe digital expression matrices were analyzed with the Seurat 4.1.0 R package . The dotS1 FigEXPERIMENTAL PROCEDURES section for detailed methods. (D-F) MD assays of CS males after isolated from female experience. Males were reared with sufficient numbers of virgin females for 24 h to be assured having sexual experience then isolated. Assay times after isolation are below the boxes as (D) no recovery, (E) 24 h recovery, and (F) 48 recoveries. (G) The diagram of MD assays of CS males after different time of isolation after sexual experience with females. (H-K) MD assays of CS males after sperm deleted as shown in (L). (M) Histogram of SMD behavior shown in (A) Mating duration (MD) assays of Oregon-R males. (B) Locomotion of naïve and experienced male flies were quantified as velocity by locomotion activity by horizontal paradigm, and (C) stop frequency by horizontal paradigm. See (EPS)Click here for additional data file.S2 FigUAS-KCNJ2 crossed with Orco-GAL4. (B) MD assays of Poxn-RNAi crossed with Poxn-GAL4. (C) MD assay of CS males experienced with D. simulans females. (D) CS male court oenocytes-musicalized female and show orientation behavior, (E) chasing (F) licking (G) wing extension, (H) copulation attempt, and (I) can successfully mate with it. (J) CS male court feminized male and show wing extension behavior and (K) copulation attempt. (L) MD assays of CS males with oenocytes-masculinized female as a female partner to test whether genotypes of female partners affect MD.(A) MD assays of (EPS)Click here for additional data file.S3 FigDrosophila melanogaster (A) The anatomical structures of male foreleg are labeled. Ta1-Ta5 comprise fore tarsus and represents tarsomeres 1–5, respectively.The foreleg of male (EPS)Click here for additional data file.S4 FigFig 4 and the location of Gr5a-positive neurons in male foreleg (A-D) MD assays of (A) UAS-Hid/rpr (B) UAS-TNT, tub-GAL80ts (C)UAS-KCNJ2, tub-GAL80ts (D) UAS-NachBac, tub-GAL80ts crossed with CS. (E) Foreleg tarsus of male flies expressing Gr5a-lexA together with lexAOP-mCherry. White arrows indicate Gr5a-positive neurons and numbers represent the order from the distal part of the leg. (F) Foreleg tarsus (left panels) and tibia (right panels) of male (top panels) or female (bottom panels) flies expressing Gr5a-GAL4 together with UAS-RedStigner. White arrows indicate Gr5a-positive neurons. White arrows with dotted line indicate missing neurons in female leg compared to male leg.Control experiments for MD assays in (EPS)Click here for additional data file.S5 FigGr5a-GAL4 together with UAS>stop>nSybGFP; fruFLP were immunostained with anti-GFP (green) and nc82 (magenta) antibodies. Scale bars represent 100 μm in the colored panels and 10 μm in the grey panels. White boxes indicate the magnified regions of interest presented next right panels. The right panels are presented as grey scale for clearly showing the axon projection patterns of gustatory neurons in the adult subesophageal ganglion (SOG) labeled by GAL4 drivers. (B-D) Control experiments for MD assays in  MD assays of (B) UAS>stop>TNTactive; fruFLP (C) UAS>stop>TNTinactive; fruFLP (D) UAS-traF crossed with CS. (E) Proboscis of male flies expressing fru-lexA; lexAOP-tdTomato.nls, UAS-Stinger with Gr5a-GAL4 were imaged in live. Scale bars represent 100 μm.(A) Brains of male flies expressing (EPS)Click here for additional data file.S6 FigFigs 6B–6D and S6B–S6G. (B-G) MD assays of flies expressing fru-GAL4 driver together with (B) Gr61a-RNAi (C) Gr64a-RNAi (D) Gr64b-RNAi (E) Gr64c-RNAi (F) Gr64d-RNAi (G) Gr64e-RNAi. (H) Female foreleg (upper panels), midleg (middle panels), and hindleg (bottom panels) of flies expressing Gr5a-lexA and Gr64f-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Yellow arrows indicate Gr5a-positive and Gr64f-positive neurons. Scale bars represent 50 mm. (I) Male proboscis of flies expressing fru-lexA and Gr64f- GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Tested gustatory sugar receptors were selected based on previous study [(A) Control experiments for MD assays in us study .(EPS)Click here for additional data file.S7 FigFig 7A and 7B. (B-C) Control experiments for MD assays in Fig 7C–7H. (D) Female foreleg of flies expressing Gr5a-lexA and Snmp1-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (E-F) MD assays for GAL4 mediated knockdown of LUSH via different lush-RNAi using Gr5a-GAL4. The stock numbers are written at the bottom of each graph. (G-H) MD assays for GAL4 mediated knockdown of SNMP1 via different snmp1-RNAi using (G) Gr5a-GAL4 or (H) Gr66a-GAL4.(A) Control experiments for MD assays in (EPS)Click here for additional data file.S8 FigFig 8A–8F. (D) Male foreleg (upper panels), midleg (middle panels), and hindleg (bottom panels) of flies expressing Gr5a-lexA and ppk23-GAL4 drivers together with lexAOP-tdTomato and UAS-Stinger were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (H) Control experiments for MD assays in S8I–S8J Fig. (I-J) MD assays for GAL4 mediated knockdown of PPK28 via ppk28-RNAi using (I) Gr5a-GAL4 and (J) Gr66a-GAL4 drivers.(A-C) Control experiments for MD assays in (EPS)Click here for additional data file.S9 Figppk29-GAL4 together with UAS-RedStinger; UAS>stop>mCD8GFP; fruFLP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (B) Male foreleg of flies expressing ppk29-GAL4 together with UAS-RedStinger, UAS-mCD8GFP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (C) Female foreleg of flies expressing ppk29-GAL4 together with UAS-RedStinger, UAS-mCD8GFP were imaged live under the fluorescent microscope. Scale bars represent 50 μm. (D-H) MD assays for GAL4 mediated knockdown of SNMP2 via snmp2-RNAi using (D) ppk23-GAL4, (E) ppk25-GAL4, (F) ppk29-GAL4, (G) Gr5a-GAL4, and (H) Gr64f-GAL4 drivers. (I) MD assays for GAL4 mediated knockdown of SNMP1 via snmp1-RNAi using ppk25-GAL4.(A) Male foreleg of flies expressing (EPS)Click here for additional data file.S10 Fig(A) A diagram of the cells in the male legs expressing genes involved in SMD behavior.(EPS)Click here for additional data file.S11 Fig(A) Percentage of progeny originated from sepia (se) male vs. CS male. CS male was introduced to se female as first mate then followed se males as second mate. The eye color of progeny was counted and interpreted as the source of farther; for detailed methods, see EXPERIMENTAL PROCEDURES. (B-C) MD assays for GAL4 mediated knockdown of PPK29 via ppk29-RNAi using elavc155 for (B) LMD and (C) SMD behavior.(EPS)Click here for additional data file.S12 FigGAL4, lexA, and RNAi control experiments. Genotypes are labelled below the graph.(A-N) MD assay for (EPS)Click here for additional data file.S13 FigGAL4, RNAi, and UAS-dicer control experiments. Genotypes are labelled below the graph.(A-H) MD assay for (EPS)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(TIF)Click here for additional data file.S1 Box(TIF)Click here for additional data file.S1 Raw Data(ZIP)Click here for additional data file.S2 Raw Data(ZIP)Click here for additional data file.


CDX-CoV-001 was a randomized, double-blind, placebo-controlled dose-escalation study in healthy adults, that established its safety and tolerability (NCT04619628). Here, we characterize the immune response to CoviLiv in participants in the high-dose cohort of 5x10in silico mapping to TCRs with known antigen specificity.Humoral immune responses were characterized in sera  with a spike-specific IgG ELISA and both microneutralization (MNT) and pseudovirus neutralization (PVN) assays. T cell functionality was analyzed in PBMCs (days 1 and 36) using interferon-γ (IFNγ) ELISpot and intracellular cytokine staining (ICS) after restimulation with peptide pools for SNMO or spike, as well as T cell receptor (TCR) sequencing combined with + T cell subset, including induction of polyfunctional CD4+ T cells. TCR repertoire mapping revealed increases in both depth and breadth of CD4+ T cell clones specific for spike, nucleocapsid phosphoprotein and membrane glycoprotein. CD8+ T cell responses were less pronounced with CoviLiv-induced responses directed against nucleocapsid and ORF1ab.After 2 doses of CoviLiv, all participants exceeded a 2-fold increase in spike-specific IgG with a geometric mean fold rise of 19.5 (95% CI 3.4-113.8) on day 57. Neutralizing antibodies at this timepoint were induced 2.6-fold (CI 1.0-7.0) and 4.9-fold (CI 1.4-16.6) using MNT and PVN. On day 36 post-vaccination, IFNγ response by ELISpot after restimulation with the SNMO peptide pool increased 4.5-fold (CI 2.8-7.4) in the 2-dose cohort and 2.5-fold (CI 1.4-4.2) in the 1-dose cohort. No increase in IFNγ response was observed after placebo vaccination or in any group after restimulation with spike-only peptides. ICS confirmed significant regimen-dependent SNMO-specific increases in IFNγ, IL-2 and TNFα response especially in the CD4Two doses of CoviLiv induced a poly-antigenic immune response with specificity to targets beyond spike, including highly conserved viral proteins, highlighting the value of this differentiated vaccine candidate.Johanna K. Kaufmann, PhD, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Keri Wyllie, MPA, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Yiwen Zhao, PhD, Codagenix Inc.: Salaried employee Lasmy Tea, MS, MPH, Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest Sybil Tasker, MD, MPH, FIDSA, Codagenix Inc.: Advisor/Consultant|Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest|Inventprise: Salaried employee|Inventprise: Ownership Interest Leena R. Yeolekar, PhD, Serum Institute of India Pvt. Ltd.: Salaried employee Rajeev Dhere, PhD, Serum Institute of India Pvt. Ltd.: Salaried employee Steffen Mueller, PhD, Codagenix Inc.: Board Member|Codagenix Inc.: Patent owner|Codagenix Inc.: Salaried employee|Codagenix Inc.: Ownership Interest