Heterologous prime-boost immunization strategies have the potential to augment COVID-19 vaccine efficacy. We longitudinally profiled SARS-CoV-2 spike (S)-specific serological and memory B cell (MBC) responses in individuals receiving either homologous (ChAdOx1:ChAdOx1) or heterologous (ChAdOx1:mRNA-1273) prime-boost vaccination. Heterologous mRNA booster immunization induced higher serum neutralizing antibody and MBC responses against SARS-CoV-2 variants of concern (VOCs) compared to homologous ChAdOx1 boosting. Specificity mapping of circulating B cells revealed that mRNA-1273 boost immunofocused ChAdOx1-primed responses onto epitopes expressed on prefusion-stabilized S. Monoclonal antibodies isolated from mRNA-1273 boosted participants displayed overall higher binding affinities and increased breadth of reactivity against VOCs relative to those isolated from ChAdOx1-boosted individuals. Overall, the results provide molecular insight into the enhanced quality of the B cell response induced following heterologous mRNA booster vaccination. Multiple safe and effective COVID-19 vaccines have been developed at an unprecedented scale and speed. However, waning vaccine-induced immunity and the emergence of SARS-CoV-2 VOCs with increased neutralization resistance have limited the effectiveness of currently available vaccines  to angiotensin converting enzyme 2 (ACE2) triggers shedding of the S1 subunit and transition of the S2 subunit to a highly stable postfusion conformation (n = 28) or mRNA-1273 (n = 27). We collected the first blood sample on the day of booster immunization to analyze ChAdOx1-primed immune responses and a second sample 7-10 days following the second dose to study the early secondary B cell response induced by homologous or heterologous booster vaccination . None of the volunteers had a documented history of prior SARS-CoV-2 infection. Participants received one dose of ChAdOx1 and 9-12 weeks later, a second dose of either ChAdOx1  increase in serum IgG binding antibodies .Cross-reactive MBCs induced by seasonal β-CoVs are activated and expanded following primary SARS-CoV-2 infection and mRNA vaccination . HoweverWe next evaluated serum neutralizing activity against authentic SARS-CoV-2 D614G, B.1.617.2/Delta, B.1.351/Beta, and B.1.1.529/Omicron variants at both sampling timepoints. At the pre-boost timepoint, the vast majority of donors (87%) displayed weak or undetectable serum neutralizing activity against SARS-CoV-2 D614G, with 50% neutralization titers ranging from <16 to 66 , and non+ peripheral blood B cells . mRNA-1273 boost also elicited significantly higher magnitude B cell responses to individual subdomains within prefusion S , including RBDs encoding mutations found in the Beta, Delta, and Kappa variants, relative to homologous ChAdOx1 boost  and WT (unstabilized) forms of the S protein , ≥30% of the total S-specific IgGand S-2P . As expeand S-2P . In contand S-2P . CorrespTo characterize the specificities and functional properties of the WT S-specific antibodies induced by ChAdOx1 prime immunization, we cloned and expressed 33 monoclonal antibodies from single WT S-specific B cells in five donors. The antibodies utilized a diversity of variable heavy- (VH) and light-chain (VL) germline genes, and 28 out of 33 contained somatic mutations, consistent with an MBC origin (fig. S8). The avid binding affinities of the antibodies for recombinant WT S ranged from 1 to 36 nM and none displayed detectable binding to S-2P . Over 85To determine whether homologous and heterologous booster vaccination regimens induce distinct B cell responses to prefusion S, we obtained 163 and 252 paired VH and VL sequences from single S-2P-reactive B cells from 4 donors in each cohort following ChAdOx1 or mRNA-1273 booster immunization (table S2). Both booster regimens induced highly diverse B cell responses, with 0% to 8.6% of antibodies belonging to expanded clonal lineages (fig. S10). IGHV3-30 was significantly over-represented in the S-reactive MBC population in both ChAdOx1 and mRNA-1273 boosted individuals, as observed previously in antibodies isolated from naturally infected and mRNA vaccinated donors (D = 12 nM) compared to those isolated from ChAdOx1 boosted donors (median KD >100 nM)  . However>100 nM) . Thus, tTo examine how the type of booster immunization impacts the B cell immunodominance hierarchy to prefusion S, we evaluated the proportion of S-2P-reactive antibodies directed to the NTD, RBD, and prefusion-stabilized S2 subdomains in each donor from which monoclonal antibodies were isolated. We observed relatively similar proportions of antibodies targeting each subdomain within prefusion S, although S2-directed antibodies dominated the response in a subset of donors in both groups . S-2P-reKD=1.4-3.7 nM) relative to the ChAdOx1-induced antibodies (median KD=4.9-20.3 nM) . Against pre-Omicron VOCs/VOIs , 41% (12/29) of anti-RBD antibodies derived from ChAdOx1 boosted individuals displayed reduced binding activity (>2-fold) to two or more of these variants as compared to only 20% (8/40) of antibodies isolated from mRNA-1273 boosted individuals, suggesting that mRNA-1273 booster vaccination activates a larger proportion of broadly reactive B cells (20.3 nM) , potenti20.3 nM) . Strikin20.3 nM) . This reD < 50nM) with all VOC NTDs or RBDs relative to ChAdOx1 boosted donors (26%)  compared to only 31-37% of antibodies isolated from ChAdOx1 boosted donors . Overallrs (26%) . Thus, hIn conclusion, heterologous ChAdOx1:mRNA-1273 prime-boost immunization induces significantly broader and more potent serum neutralizing antibody and MBC responses against WT SARS-CoV-2 and VOCs relative to homologous ChAdOx1 vaccination, and this difference appears to be driven by both the magnitude and quality of the early secondary B cell response. Expression of WT S by ChAdOx1 appears to distract the B cell response away from neutralizing sites of vulnerability present on prefusion S, and homologous booster vaccination further expands these non-neutralizing specificities. In contrast, heterologous booster immunization with mRNA-1273, which encodes S-2P, re-directs the B cell response toward epitopes expressed on prefusion-stabilized S. Furthermore, mRNA-1273 activates B cells with higher affinity for prefusion S and greater breadth of reactivity relative to ChAdOx1. The molecular basis for this difference remains to be determined but could potentially be associated with differences in cell-surface expression of S-2P relative to WT S or the distinct innate immunostimulatory properties of mRNA versus adenoviral particles (


Scientifc Reports (2021) https://doi.org/10.1038/s41598-021-86470-1, published online 31 March 2021Correction to: The original version of this Article contained typographical errors in Figure 1b where there was erroneous spacing in the sequence alignments. The original Figure The original Article has been corrected.


Coronavirus (CoV) nonstructural protein 14 (nsp14) has exoribonuclease (ExoN) activity, responsible for proofreading and contributing to replication fidelity. It has been reported that CoVs exhibit variable sensitivity to nsp14-ExoN deficiency. Betacoronavirus murine hepatitis virus (MHV) and severe acute respiratory syndrome (SARS)-CoV were viable upon nsp14-ExoN deficiency. While betacoronavirus Middle East respiratory syndrome (MERS)-CoV and SARS-CoV-2 were non-viable with disabled nsp14-ExoN. In this study, we investigated the nsp14-ExoN deficiency of alphacoronavirus porcine epidemic diarrhea virus (PEDV) in viral pathogenesis using reverse genetics.2+-binding site was rescued using the infectious clone of PEDV PC22A strain (icPC22A). The passage no.1–3 (P1-3) of E191A grew to very low titers in Vero cells. To evaluate the pathogenesis of the E191A, 4 or 5-day-old gnotobiotic pigs were inoculated orally with 100 TCID50/pig of the E191A-P1, icPC22A, or mock. All mock pigs did not shed virus in feces or show clinical signs. All pigs inoculated with icPC22A shed high viral RNA levels, had severe diarrhea, and died by 6 days post-inoculation (dpi). In contrast, only 3 pigs  in the E191A-P1 group shed low levels of viral RNA and 2 pigs had moderate diarrhea at acute infection phase. At 22 dpi, each pig was challenged orally with 106 plaque forming unit of virulent icPC22A. All pigs in the mock group developed severe diarrhea and 2 of the 5 pigs died. Pigs in the E191A-P1 group had less severe diarrhea and no pigs died. Sanger sequencing analysis revealed that the viral genome in the fecal sample of one E191A-P1-inoculated pig and the P4 virus passaged in vitro lost the E191A mutation, suggesting the genetic instability of the E191A mutant.Eight nsp14-ExoN deficient mutants, targeting the predicted active sites and the Zinc finger or mental-coordinating sites, of PEDV were designed. Only one mutant E191A with a mutation in the MgThe recombinant PEDV variants carrying mutations at the essential functional sites within nsp14-ExoN were either lethal or genetically unstable. Our finding further confirmed the critical role of nsp14-ExoN in CoV life cycle, suggesting that it may be a target for the design of universal anti-CoV drugs.The online version contains supplementary material available at 10.1186/s13578-021-00598-1. Alphacoronavirus genus in the family of Coronaviridae. It is an enveloped virus with a single-stranded, positive sense RNA genome. PEDV genome is 5’-capped, 3’-polyadenylated and consists of seven open reading frames (ORFs): ORF1a, ORF1b, and spike (S) glycoprotein, ORF3, envelope (E) protein, membrane (M) protein, and nucleocapsid (N) protein. Upon infection, ORF1a and ORF1b of viral genomic RNA are directly translated into two polyprotein precursors, pp1a and pp1ab, respectively, via a frame shift mechanism. These polyproteins are then processed into 16 nonstructural proteins (nsp1 to nsp16) carrying various functions in viral life cycles. For examples, nsp12 is an RNA-dependent RNA polymerase (RdRp) coordinating with primases, nsp7 and nsp8. Complex of nsp12-nsp7-nsp8 was defined as the minimal core component mediating coronavirus RNA synthesis . Supernatants were titrated in 96-well plates for 50% tissue culture infective doses (TCID50/pig), icPC22A , or mock , respectively. One piglet in the icPC22A group (pig#1) and one showing positive viral RNA shedding (pig#8) from E191A-P1 group were euthanized at 3 dpi. At 22 dpi, each pig was challenged orally with 6 log10 PFU of icPC22A and housed for one more week to evaluate protection against virulent PEDV challenge. After inoculation, clinical signs, including diarrhea and vomiting were monitored twice a day. Rectal swabs were collected daily. The severity of diarrhea was scored based on the fecal consistency (FC) in individual pigs: 0, solid; 1, pasty; 2, semiliquid (moderate diarrhea); and 3, liquid (severe diarrhea) . sgmRNA-N was amplified with forward primer (5′-CTCTTGTCTACTCAATTCAACTAAACAGAAAC), reverse primer (5′-CCAGTATCCAATTTGCTGGTCC), and probe (FAM-5’-TCAGGATCGTGGCCGCAAAC-BHQ). The genomic RNA was amplified with forward primer (5’-TGAAGCCGTCTCATACTATTCTG), reverse primer (5’-AATCCCTCAACAGTGTCAGC), and probe (FAM-5’-TGCAATGCCGTTTCGTGTCCTTC-BHQ). Copy numbers of each were determined by standard curves.The statistical analyses were performed using GraphPad Prism 6.0. Data in Fig. Additional file 1: Figure S1. Pathogenicity of the E191A-P4 in Gn piglets that were initially inoculated with E191A-P1 but did not shed virus by 3 dpi and re-inoculated with E191A-P4 at 3 dpi. (A) Fecal consistency scores of pigs after the E191A-P4 inoculation. Fecal consistency was scored as follows: 0, solid, 1, pasty, 2, semiliquid; and 3, liquid. Scores of ≥ 2 and 3 were considered diarrhea and severe diarrhea, respectively. Each dot represents the score of an individual pig; each line indicates the mean scores of a group. (B) PEDV RNA (N gene) shedding titers in rectal swabs after inoculation. Each line indicates the mean values of a group. The dash line at 4.8 log10 copies/mL indicates the detection limit. (C) Survival curves of pigs after inoculated with E191A-P4.


Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.The online version contains supplementary material available at 10.1186/s13567-021-00944-8. Porcine reproductive and respiratory syndrome (PRRS) is an economically significant viral disease in the swine-producing countries of the world. The clinical symptoms are characterized by reproductive failure in sows and respiratory disease in young pigs . The cauIn China, even though PRRSV1 isolates were sporadically detected in recent years, PRRSV2 isolates were obviously predominant . In 1995Several commercial PRRS modified live virus (MLV) vaccines have been widely utilized in China, including RespPRRS MLV, CH-1R, R98, JXA1-R, HuN4-F112, TJM-F92 and GDr180. Generally, immunization with commercial PRRS MLV vaccines confers excellent homologous protection against closely related isolates but only limited cross protection against heterologous strains [Neutralizing antibodies (nAbs) are a vital component of the immune armory against viral infection, which are induced against the viral outer coat proteins or envelope proteins . The PRRMultiple strategies have been employed to increase the breadth, potency and longevity of nAbs against rapidly evolving viruses, including epitope masking, sequential vaccination and centralized envelope antigens . To overHere we hypothesized that a chimeric virus containing the consensus sequence encoding envelope proteins of PRRSV may induce broader nAbs. Therefore, a consensus sequence of ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins of PRRSV was designed and synthesized. An infectious clone of avirulent HP-PRRSV2 isolate JSTZ1712-12 was first generated (named as rJSTZ1712-12) as we previously described , 25, andPRRSV strains used in this study were all stored in our laboratories including the following: PRRSV1: HLJB1 strain ; JXA1-liThe approach so called “centralized envelope antigens” was adopted to overcome the extraordinary genetic diversity of PRRSV isolates . To desiTo generate an infectious clone carrying the ORF2-6-CON sequence, a full-genome cDNA clone of the avirulent HP-PRRSV2 isolate, JSTZ1712-12, was first constructed as we previously described . Three oTo construct the chimeric virus containing ORF2-6-CON, overlap extension PCR was performed to generate a recombined F3 fragment carrying the ORF2-6-CON. In details, the synthetic ORF2-6-CON sequence (in pUC57 vector) was amplified with the PRRSV2-AscI-ORF2-CON-F3/PRRSV2-ORF6-CON-R primer pairs to produce a fragment PRRSV2-AscI-ORF2-6-CON. In addition, the rJSTZ1712-12 clone was amplified with the PRRSV2-ORF6-CON-F2/JSTZ1712-12-NotI-2R3 primers to generate the JSTZ1712-12-ORF6-3ʹUTR-NotI fragment. Then an overlap PCR was performed with PRRSV2-AscI-ORF2-CON-F3/JSTZ1712-12-NotI-2R3 primers using the PRRSV2-AscI-ORF2-6-CON and JSTZ1712-12-ORF6-3ʹUTR-NotI fragments as templates to generate chimeric F3 containing ORF2-6-CON (ORF2-6-CON-F3). The recombined ORF2-6-CON-F3 was double-digested with AscI and NotI and then ligated into the rJSTZ1712-12 clone, which was linearized with the same restriction enzymes to generate the rJSTZ1712-12-ORF2-6-CON clone (abbreviated as rJS-ORF2-6-CON). The rJS-ORF2-6-CON genome sequence has been submitted to GenBank (MW460545).50) of rJS-ORF2-6-CON, rJSTZ1712-12 or parental JSTZ1712-12 isolate, respectively. The multiple-step growth curves within 96 h post-infection (hpi) were determined by real-time RT-PCR assay [The rJSTZ1712-12 and rJS-ORF2-6-CON clones obtained were transfected into BHK-21 cells using Lipofectamine 3000 Reagent  according to the manufacturer’s instructions. The culture supernatants collected at 48 h post-transfection (hpt) were serially passaged in Marc-145 cells. The recovery of the infectious viruses was confirmed by indirect immunofluorescence assay (IFA) . PRRSV-sCR assay . The plaCR assay .5.0 TCID50/mL rJS-ORF2-6-CON (5th passage). Piglets in the second group were injected with one dose of the commercial TJM-F92 vaccine . Piglets in the other two groups were inoculated with RPMI-1640 to serve as the control. At 42 days post-inoculation (dpi), in addition to one RPMI-1640 inoculated group that was mock-infected again, the other three groups of pigs were challenged with 2 mL 105.0 TCID50/mL of the virulent NADC30-like SD17-38 isolate [To evaluate the safety and immunogenicity of rJS-ORF2-6-CON, pig inoculation and challenge studies were performed. All animal experiments in this study were approved by the Animal Welfare and Ethics Committee of Yangzhou University with the Reference number of 202010001. Twenty 4-week-old PRRSV-free piglets were randomly divided into four groups (five piglets in each group). Piglets in the first group were intranasally and intramuscularly inoculated with 2 mL 10 isolate .® PRRS×3 ELISA Kit . The threshold for seroconversion was set at a sample-to-positive (s/p) ratio of 0.4. In addition, IFN-γ in the sera were detected using the commercial Porcine IFN-gamma ELISA kit  according to the manufacturer’s instructions. The weekly collected sera since 28 dpi were submitted to virus neutralization test as we previously described [Rectal temperature was recorded daily during the first 2 weeks both after inoculation and after challenge. The body weight was measured weekly and clinical signs were assessed daily. Serum samples were collected weekly for the analyses of virus load, antibody and IFN-γ levels. The dynamics of viremia were analyzed by PRRSV real-time RT-PCR assay . PRRSV-sescribed , 17. The6 cells each well) and stimulated with or without HP-PRRSV2 MLV JXA1-R strain at a multiplicity of infection (MOI) of 0.01 for 20 h. Brefeldin A  was added during the last 5 h of incubation. After incubation, the cells were harvested and stained for cell surface marker and intracellular cytokine as we previously reported [Swine periphery blood mononuclear cells (PBMC) were isolated from blood samples by density-gradient centrifugation as reported previously . The isoreported . BrieflyTo characterize the swine VDJ repertoires associated with different PRRSV nAbs, in this study, we amplified the immunoglobulin heavy chain VDJ region from representative pigs producing distinct nAbs with a single pair of primers (pVDJ-FR1-F: 5ʹ-ATGGAGTTTCGGCTGAACT-3ʹ and pVDJ-FR4-R: 5ʹ-TGAGGACACGACGACTTCA-3ʹ) modified from our previous report . The samp value < 0.05 was considered statistically significant.The data of virus load, antibody level, rectal temperature, weight gain, IFN-γ concentration and percentage of IFN-γ secreting cells are shown as means ± standard deviations (SD). The differences between groups were determined by Mann–Whitney U test using Graphpad Prism version 6.07 . A p valA set of 30 representative Chinese PRRSV isolates was obtained from GenBank. The phylogenetic tree was constructed based on ORF2-6 genes of mainly Chinese PRRSV2 and some representative PRRSV1 strains. There are four lineages  of PRRSV2 isolates co-existing in Chinese swine herds. The synthetic PRRSV2 ORF2-6-CON sequence was located at the center of the phylogenetic tree  to the cloned rJSTZ1712-12 and rJS-ORF2-6-CON viruses. The titers of all three viruses peaked at 96 hpi  have significantly higher rectal temperatures , the frequencies of PRRSV-specific IFN-γ secreting cells was low and no significant difference was detected among all the groups. After challenge with the virulent SD17-38 isolate, the frequencies of PRRSV-specific IFN-γ secreting cells increased from 0 to 14 dpc. However, no difference was noticed between TJM-F92 vaccinated and rJS-ORF2-6-CON inoculated pigs  sera. No nAbs was found in any mock-infected pigs. One out of five pigs (pig 6) immunized with TJM-F92 vaccine induced homologous nAbs against HP-PRRSV2. Three out of five pigs  inoculated with rJS-ORF2-6-CON virus induced heterologous nAbs against different lineages of PRRSV2 isolates . Remarkably, pig 12 serum could neutralize not only distinct PRRSV2 isolates but also PRRSV1 isolate  and a mock infected pig (pig 3) as the control. A total of 271 swine VDJ sequences were obtained, including 55, 68, 56, 52 and 40 sequences from pig 3 that produced no nAbs, pig 6 for homologous nAbs, pig 12 for bnAbs, and pigs 13 and 14 for heterologous nAbs, respectively. All the swine VDJ sequences from this study have been submitted to GenBank (MW460272–MW460542). The alignment of VDJ sequences indicates that the divergence of swine VDJ genes was mainly due to high variation of the CDR , two DH segments and a single JH gene to account for nearly its entire (>90%) VDJ pre-immune repertoire [H genes that comprise the pre-immune repertoire. The same VH genes comprise the adaptive repertoire but ~90% of them are somatically mutated [Pigs that produce homologous nAbs, heterologous nAbs and bnAbs provide an ideal opportunity to explore swine B cell repertoires associated with different nAb responses. To elicit immune response to unlimited numbers of foreign antigens, the immune system must recognize countless numbers of antigens. The unlimited numbers of unique antigen receptors are achieved by creating variation in the antigen-recognition regions . The mecpertoire . Further mutated . These u mutated . In this mutated –57. TherCross protection is extremely important due to the coexistence of distinct PRRSV isolates. However, by which mechanisms to confer protection by PRRSV are poorly understood . PRRSV-sEven though protection efficiency is a vital criterion for breeding next generation PRRS vaccines, safety is another major concern that is at least as important as protection efficiency. Persistent MLV infection in vaccinated pigs and transmission of vaccine strains to naïve pigs have been confirmed , 51, 61.In conclusion, an ORF2-6 consensus sequence was computationally designed based on a large amount of representative Chinese PRRSV isolates in this study. The chimeric virus containing this consensus sequence is not pathogenic to piglets, may induce bnAbs and confers better cross protection against heterologous NADC30-like isolate than a commercial MLV vaccine. The chimeric virus may serve as a promising candidate for developing broadly protective PRRS vaccine. In addition, this chimeric virus can be used to investigate the mechanisms involved in inducing bnAbs or conferring cross protection.Additional file 1. Alignment of 271 porcine VDJ amino acid sequences from this study. Overall, the variation of swine VDJ genes is concentrated in the CDR (A). The detailed alignment of 271 VDJ (B). The identical sequences from each pig are highlighted in red.


Programmed death-ligand 1 (PD-L1) is an important immune checkpoint protein that can be regarded as a pan-cancer antigen expressed by multiple different cell types within the tumor. While antagonizing PD-L1 is well known to relieve PD-1/PD-L1-mediated T cell suppression, here we have combined this approach with an immunotherapy strategy to target T cell cytotoxicity directly toward PD-L1-expressing cells. We developed a bi-specific T cell engager (BiTE) crosslinking PD-L1 and CD3ε and demonstrated targeted cytotoxicity using a clinically relevant patient-derived ascites model. This approach represents an immunological ‘volte-face’ whereby a tumor immunological defense mechanism can be instantly transformed into an Achilles’ heel for targeted immunotherapy.The PD-L1 targeting BiTE comprises an anti-PD-L1 single-chain variable fragment (scFv) or nanobody (NB) domain and an anti-CD3 scFv domain in a tandem repeat. The ability to activate T cell cytotoxicity toward PD-L1-expressing cells was established using human carcinoma cells and PD-L1-expressing human (‘M2’) macrophages in the presence of autologous T cells. Furthermore, we armed oncolytic herpes simplex virus-1 (oHSV-1) with PD-L1 BiTE and demonstrated successful delivery and targeted cytotoxicity in unpurified cultures of malignant ascites derived from different cancer patients.PD-L1 BiTE crosslinks PD-L1-positive cells and CD3ε on T cells in a ‘pseudo-synapse’ and triggers T cell activation and release of proinflammatory cytokines such as interferon-gamma (IFN-γ), interferon gamma-induced protein 10 (IP-10) and tumour necrosis factor-α (TNF-α). Activation of endogenous T cells within ascites samples led to significant lysis of tumor cells and M2-like macrophages (CD11b+CD64+ and CD206+/CD163+). The survival of CD3+ T cells  was unaffected. Intriguingly, ascites fluid that appeared particularly immunosuppressive led to higher expression of PD-L1 on tumor cells, resulting in improved BiTE-mediated T cell activation.The study reveals that PD-L1 BiTE is an effective immunotherapeutic approach to kill PD-L1-positive tumor cells and macrophages while leaving T cells unharmed. This approach activates endogenous T cells within malignant ascites, generates a proinflammatory response and eliminates cells promoting tumor progression. Using an oncolytic virus for local expression of PD-L1 BiTE also prevents ‘on-target off-tumor’ systemic toxicities and harnesses immunosuppressive protumor conditions to augment immunotherapy in immunologically ‘cold’ clinical cancers. Oncolytic viruses (OVs) replicate selectively within tumor cells and lyse them. They can also be armed to provide tumor-selective expression of therapeutic biologicals, including antibodies recognizing tumor antigens to promote antibody-dependent cellular cytotoxicity or checkpoint inhibitor antibodies designed to modulate the local immune microenvironments.BiTEs provide a powerful strategy to mediate tumor-targeted cytotoxicity and are ideally suited for expression from OVs locally within the tumor microenvironment (TME), since intravenous delivery of free BiTEs may trigger ‘on-target, off-tumor’ cytotoxicity. The first generation of BiTEs targeted antigens associated with the surface of tumor cells, thereby recognizing the same cell population as the OV.We explore approaches to direct T cell cytotoxicity toward PD-L1 to mediate broad cytotoxicity in the TME. We develop a BiTE targeting PD-L1 and encode it within oncolytic herpes simplex virus-1 (oHSV-1).HEK293A, DLD-1 and Vero cells (ATCC) were cultured and maintained in Dulbecco’s modified Eagle medium  supplemented with 10% (v/v) heat-inactivated fetal bovine serum . Viral infections and propagations were performed in DMEM with 2% (v/v) FBS.4-S)3. Control BiTEs were generated similarly and contained scFv or NB fragments targeting irrelevant antigens (scFv recognizing filamentous hemagglutinin adhesin of Bordetella pertussisPD-L1-targeted BiTE was constructed by linking an scFv or nanobody (NB) recognizing human PD-L110.1136/jitc-2020-001292.supp1Supplementary data10.1136/jitc-2020-001292.supp2Supplementary data‘Armed’ oHSV-1 were constructed by insertion of the BiTE cassette into the parental oHSV-1 )Human leukocyte cones from consenting healthy volunteers  were used for PBMC isolation by density-gradient centrifugation using Ficoll-Paque Plus . CD3+ cells were isolated using the Pan T-cell Isolation Kit (Miltenyi Biotech).Primary human malignant ascites samples were obtained from consented cancer patients at the Churchill Hospital . Ascites samples were processed immediately, with cells and fluid separated by centrifugation as described previously.10.1136/jitc-2020-001292.supp3Supplementary dataMonocytes were isolated from leukocyte cones by double density-gradient centrifugation.ex vivo experiments, unpurified total ascites cells were seeded in normal serum (NS) medium ) or autologous acellular ascites fluid mixed (50% v/v) with NS medium. Cultures were treated with free BiTEs (40 nM) or infected with virus. Where appropriate, CD3/CD28 Dynabeads  were included as positive controls for T cell activation. T cells were harvested after 48 hours (free BiTE) or on day 4 (virus infection), and activation was assessed using fluorescently labeled antibodies against CD3, CD4, CD8, CD25 and CD69  using an Attune NxT Flow Cytometer .For PBMC-based experiments, CD3+ T cells were isolated and cocultured with DLD-1 human colorectal carcinoma cells at a ratio of 5:1. For Cytotoxicity was measured by lactate dehydrogenase release , MTS viability assay  or in real-time by measuring cell impedance using xCELLigence (Acea Biosciences). Cell viability was determined by flow cytometry using a live-dead stain .Cytokines and chemokines were measured by commercial ELISA kits  or multiplex LEGENDplex Human Macrophage/Microglia panel kit .M2 macrophages were stained with CellTrace Far-Red Cell Proliferation Kit (ThermoFisher Scientific) and cocultured 1:2 with tumor cells (DLD-1) in NS media. After 24 hours, cells were infected with parental or oHSV-1 expressing PD-L1 BiTEs at MOI 1. After 3 hours of infection, T cells stained with CellTrace Violet Cell Proliferation Kit (ThermoFisher Scientific) were added to the coculture. Uninfected cells were used as a negative control. Also, 2 µM NucView 530 Caspase-3 Substrate (Biotium) was added to each well to visualize apoptosis. Brightfield and fluorescence images were captured on a Nikon Ti-E Microscope fitted with Andor Zyla 4.2 sCMOS camera  at intervals of 15 min for 4 days. Time-lapse movies (14 frames/s) were generated using Fiji software. Caspase-3 substrate signal was detected in Cy3 channel of fluorescence microscope (excitation/emission wavelength of 528/563 nm) and was undisturbed by viral EGFP cassette . Green colour for Caspase was selected to display better contrast during image/movie processing using Fiji software .Statistical analyses were performed using two-way analysis of variance with Bonferroni post hoc analysis. All data are presented as mean±SEM. The significance levels used were p=0.01–0.05 (*), 0.001–0.01 (**) and 0.0001–0.001 (***). Experiments were performed in biological triplicate, unless otherwise stated.The ability of PD-L1 BiTEs to mediate activation of human lymphocytes and target cell toxicity was assessed by incubating them at increasing concentrations with PD-L1-positive DLD-1 cells  with or 10.1136/jitc-2020-001292.supp4Supplementary dataInterestingly, both CD4+ and CD8+ T cells demonstrated increased expression of CD69 and CD25 with significant increases in the generation of interferon-gamma (IFN-γ)-producing T cells . PD-L1 B10.1136/jitc-2020-001292.supp5Supplementary dataAscites is a pathological accumulation of fluid, usually in the peritoneal cavities of patients with ovarian, breast, gastrointestinal, pancreatic and uterine cancer.10.1136/jitc-2020-001292.supp6Supplementary data10.1136/jitc-2020-001292.supp7Supplementary data10.1136/jitc-2020-001292.supp8Supplementary data10.1136/jitc-2020-001292.supp9Supplementary dataA multiplex immunoassay was used to assess the impact of BiTE treatment on cytokine release. Ascites cells (patient #7) were treated with PD-L1 BiTEs, and supernatants were harvested after 72 hours. Robust increases in IFN-γ (1000-fold), IP-10 (355-fold) and TNF-α (220-fold) levels were observed in samples treated with PD-L1 BiTEs compared with untreated samples. A strong fold increase in thymus and activation-regulated chemokine (TARC), IL-4, IL-10 and IL-1β was also observed . The hig10.1136/jitc-2020-001292.supp10Supplementary dataThese data indicate that PD-L1 scFv and PD-L1 NB BiTE mediate activation of endogenous T cells in ascites samples, triggering a dramatic shift in the inflammatory milieu.Similar to previous studies, we observed PD-L1 expression on surface of activated T lymphocytesMalignant ascites fluids contain various soluble factors  that inhibit immune activation and accentuate an immunosuppressive environment while providing a supportive environment for cell growth and tumor spread.In contrast, there was no suppressive effect of any ascites fluid on PD-L1 scFv or PD-L1 NB BiTE activity. Robust T cell activation was achieved in all tested fluids , even inWe also assessed the impact of ascites fluids on PD-L1 expression in target cells. DLD-1 cells were cultured in the presence of NS media or ascites fluid. After 48 hours, cells were harvested and stained for PD-L1 expression. We observed a significant increase in the percentage of PD-L1-positive cells  from cells infected with oHSV-1 expressing PD-L1 BiTEs but not from cultures infected with oHSV-1 expressing control BiTE or the parental oHSV-1 . We obseNext, we monitored oncolysis mediated by BiTE-armed oHSV-1 in real-time using xCELLigence. DLD-1 cells were infected with parental and BiTE-armed oHSV-1 (MOI 1), then 3 hours later, human PBMC-derived T cells were added. In the absence of T cells, no cytotoxicity of target cells was observed with any of the BiTE-armed viruses  except f10.1136/jitc-2020-001292.supp13Supplementary dataWe measured the replication kinetics of the viruses and secretion of PD-L1 BiTE in the presence of malignant ascites fluids . In para10.1136/jitc-2020-001292.supp14Supplementary data10.1136/jitc-2020-001292.supp15Supplementary data10.1136/jitc-2020-001292.supp16Supplementary video10.1136/jitc-2020-001292.supp17Supplementary video10.1136/jitc-2020-001292.supp18Supplementary video10.1136/jitc-2020-001292.supp19Supplementary videoPrevious studies have shown that TAMs promote cancer cell proliferation, invasion, metastasis and production of immune-suppressive molecules.10.1136/jitc-2020-001292.supp20Supplementary dataNext, we tested whether PD-L1 BiTE mediates simultaneous cytotoxicity of tumor cells and TAMs. Cocultures of tumor cells , human PBMC-derived M2-like macrophages (red) and T cells (blue) were infected with parental oHSV-1 or PD-L1 BiTE-armed oHSV-1. Cultures were monitored for 4 days using time-lapse microscopy . Dramati10.1136/jitc-2020-001292.supp22Supplementary data10.1136/jitc-2020-001292.supp23Supplementary dataWe next tested the cytotoxic activity of virus-encoded PD-L1 BiTE in a human malignant ascites-based cell model. Whole malignant ascites cells were infected for 4 days with recombinant or parental oHSV-1 (MOI 1) in NS media or in the presence of autologous ascites fluid (50% v/v). PD-L1 BiTE-armed oHSV-1 triggered activation of endogenous T cells as measured by significant induction of CD25 . CD25 induction was markedly higher in the presence of autologous ascites fluids for most patients than in NS medium  can be repurposed for BiTE-mediated cytotoxicity.Unexpectedly, the activation of endogenous T cells by PD-L1 BiTE, present as free protein or expressed by oHSV-1, was seen to be greater in the presence of autologous ascites fluid compared with NS media. This was coupled with the observation that the presence of ascites fluids led to significantly greater PD-L1 expression on the surface of tumor cells . This, iin vitro and ex vivo to determine any differences in T cell activation or tumor targeting. ScFv comprise two variable domains (VL and VH), while NB is composed of a single VH domain. Compared with scFv, NBs are smaller, have superior solubility and stability at pH extremes and achieve excellent tissue penetration in vivo.In our study, we compared the scFv and NB versions of the PD-L1 BiTE Arming oHSV-1 with PD-L1 BiTE can target cytotoxicity toward tumor cells but also kills TAMs, critical contributors to tumor progression and immune suppression. The BiTE-mediated cytotoxicity was able to overcome immunosuppressive ascites fluids, and cytotoxicity was augmented, not inhibited, by the immunosuppressive milieu. We believe the mechanism involves upregulation of PD-L1 expression on target cells within ascites, facilitating improved activation of endogenous T cells leading to greater target-mediated cytotoxicity. To our knowledge, this concept has not been demonstrated before and represents a simple approach to exploit immune-suppressive tumor escape strategies and converting them to opportunities for targeted immunotherapy.10.1136/jitc-2020-001292.supp21Supplementary data


Antibody responses are crucial for the control of virus infection. Understanding of the mechanism of antibody induction is important for the development of a vaccine eliciting effective anti-virus antibodies. Virus-specific B cell receptor (BCR)/antibody repertoires are different among individuals, but determinants for this difference remain largely unclear. We have recently reported that a germline BCR immunoglobulin (IgG) gene polymorphism (VH3.33_ET or VH3.33_VI) in rhesus macaques is the determinant for induction of potent B404-class anti-simian immunodeficiency virus (SIV) neutralizing antibodies in neutralization-sensitive SIVsmH635FC infection. In the present study, we examined whether neutralization-resistant SIVsmE543-3 infection can induce the anti-SIV neutralizing antibodies associated with the germline VH3.33 polymorphism. Anti-SIVsmE543-3 neutralizing antibodies were induced in all the macaques possessing the VH3.33_ET allele, but not in those without VH3.33_ET, in the chronic phase of SIVsmE543-3 infection. Next generation sequencing analysis of BCR VH genes found B404-class antibody sequences only in those with VH3.33_ET. These results indicate that anti-SIVsmE543-3 neutralizing antibody induction associated with the germline BCR IgG gene polymorphism can be triggered by infection with neutralization-resistant SIVsmE543-3. This animal model would be useful for the elucidation of the mechanism of potent antibody induction against neutralization-resistant viruses. Development of an effective vaccine is a key for global control of infectious diseases. An antibody is a major acquired immune effector to prevent and/or control virus infection. Efficiency of antibody induction by vaccination is affected by both vaccine-related and host-related factors ,2. RegarPotent antibodies are produced from plasma cells differentiated from mature B cells with higher antigen-B cell receptor (BCR) binding affinity ,7. MaturWe previously reported a potent monoclonal anti-simian immunodeficiency virus (SIV) B404, and related B404-class neutralizing antibodies (NAbs), induced in rhesus macaques infected with SIVsmH635FC, a NAb-sensitive SIV strain obtained by passage from NAb-resistant SIVsmE543-3-infected macaques ,13. The In the present study, we examined the effect of the germline Ig VH3.33 polymorphism on antibody induction in neutralization-resistant SIVsmE543-3 infection. Anti-SIVsmE543-3 NAb responses were induced only in rhesus macaques possessing the VH3.33_ET allele. Next-generation sequencing (NGS) analysis of BCR VH genes detected B404-class antibody sequences only in those macaques with VH3.33_ET. These results indicate that NAb induction associated with the germline Ig VH3.33 polymorphism can occur in NAb-resistant SIVsmE543-3 infection.50 (50% tissue culture infective dose) of SIVsmE543-3. Viruses were obtained from COS-1 cells transfected with the molecular clone SIVsmE543-3 DNA (Genbank accession number U72748) [This study was performed using frozen samples obtained in our previous study. In that study, seven Burmese rhesus macaques were intravenously infected with 100 TCID U72748)  and prop U72748) ,19 appro U72748) . Blood cGermline VH3.33 polymorphisms were determined by direct sequencing of PCR amplified VH3.33 genes as described before . VH3.33 TRIM5 polymorphisms were determined as described previously . Briefly50 of SIVs  at 37 °C for 1 h and added into 1 × 104 TZM-bl cells in a 96-well plate. Two days later (or three days later for anti-SIVsmH635FC NAb assay), cells were lysed, and the luciferase activity was measured using Beta-Glo assay system .The neutralizing activity of plasma samples was measured by examining the reduction in infection-induced luciferase activity in TZM-bl cells by addition of plasma as described before ,16. SIVs6 PBMCs or submandibular lymph nodes (LNs)-derived lymphocytes using RNeasy Mini kit and subjected to RT using QuantiTech RT kit  and random primers. BCR IgG VH-coding cDNAs were amplified using a VH3 specific 5′-primer (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACA GAAGGTGTCCAGTGTGARGTGCG-3′), and an IgG-specific 3′-primer (5′-GTCTCGTG GGCTCGGAGATGTGTATAAGAGACAGGCCCTTGGTGGAGGCTGAGGAGACGGT GAC-3′). PCR amplification was performed with a set of unique 8 bp Illumina barcodes to obtain DNA libraries, which were subjected to 2 × 300 bp paired-end indexed sequencing using Illumina MiSeq.NGS analysis of BCR IgG VH3 cDNAs was performed as described previously . Total RPCR amplicon deep sequencing of BCR VH region was performed by the Illumina MiSeq reagent kit v3 on the MiSeq instrument  according to the manufacturer’s instructions (the methods for 2 × 300-mer paired-end). Sequencing reads were analyzed by Cutadapt to remove the adaptors and Trimmomatic to remove low-quality reads (QV <20) ,24. Trimn = 28) and cynomolgus (n = 24) macaques  in our previous study. Four  of the seven macaques received a DNA-prime/SeV-Gag vaccine ,19 approWe also determined TRIM5 polymorphisms, which are known to have influence on susceptibility to SIVsmE543-3 infection . Five #. These fWe examined post-infection plasma NAb titers against NAb-sensitive SIVmac316 and SIVsmH635FC and NAb-resistant SIVsmE543-3 in the chronic phase of SIVsmE543-3 infection . All macTo examine B404-class Ab induction, we performed NGS analysis of BCR VH3.33 genes in the seven macaques. IgG VH cDNAs derived from PBMCs and LNs at month 24 post-infection were amplified by using a VH3-specific forward primer and an IgG-specific reverse primer, and subjected to NGS analysis. Amplicon sequences, which were determined as VH3.33 by IgBLAST and B404-class VH controls obtained in the previous study  were subThe B404-class NAbs have heavy chains using VH3.33 with long CDR3 . We founOur previous study reported a germline VH3.33 polymorphism-dependent induction of B404-class antibodies that can neutralize NAb-resistant SIVsmE543-3 in NAb-sensitive SIVsmH635FC infection . In the In our previous study, analysis using germline-reverted B404 mutants revealed that the 38E in VH3.33 is responsible for B404-class antibody binding to SIVsmH635FC Env . A germlFive macaques possessing TRIM5α_TFP showed lower plasma viral loads, whereas two macaques without TRIM5α_TFP had higher viral loads . Higher Transmitted founder viruses in HIV infection are known to be NAb-resistant ,30. NAb-In summary, the present study indicates anti-SIVsmE543-3 NAb induction associated with germline VH3.33 polymorphisms in NAb-resistant SIVsmE543-3 infection. Further analysis of B-cell maturation in the process of NAb induction may help us to reveal the mechanism of potent NAb induction.


Treatment with Menin inhibitor (MI) disrupts the interaction between Menin and MLL1 or MLL1-fusion protein (FP), inhibits HOXA9/MEIS1, induces differentiation and loss of survival of AML harboring MLL1 re-arrangement (r) and FP, or expressing mutant (mt)-NPM1. Following MI treatment, although clinical responses are common, the majority of patients with AML with MLL1-r or mt-NPM1 succumb to their disease. Pre-clinical studies presented here demonstrate that genetic knockout or degradation of Menin or treatment with the MI SNDX-50469 reduces MLL1/MLL1-FP targets, associated with MI-induced differentiation and loss of viability. MI treatment also attenuates BCL2 and CDK6 levels. Co-treatment with SNDX-50469 and BCL2 inhibitor (venetoclax), or CDK6 inhibitor (abemaciclib) induces synergistic lethality in cell lines and patient-derived AML cells harboring MLL1-r or mtNPM1. Combined therapy with SNDX-5613 and venetoclax exerts superior in vivo efficacy in a cell line or PD AML cell xenografts harboring MLL1-r or mt-NPM1. Synergy with the MI-based combinations is preserved against MLL1-r AML cells expressing FLT3 mutation, also CRISPR-edited to introduce mtTP53. These findings highlight the promise of clinically testing these MI-based combinations against AML harboring MLL1-r or mtNPM1. MLL1 KMT2A) is a large (3696 amino acids) transcriptional regulator and a histone-lysine-N-methyltransferase A is a la. Menin bSNDX-50469, venetoclax, gilteritinib, cobimetinib, etoposide, and abemaciclib were obtained from MedChem Express . DTAG-13 (Cat. No. 6605) was obtained from Tocris/Bio-Techne . SNDX-5613 was obtained from Syndax Pharmaceuticals Inc.  under a material transfer agreement. All compounds were prepared as 10 mM stocks in 100% DMSO and frozen at −80 °C in 5–10 µL aliquots to allow for single use, thus avoiding multiple freeze-thaw cycles that could result in compound decomposition and loss of activity. Anti-c-Myc [RRID:AB_1903938], anti-MCL1 [RRID:AB_2799149], anti-BIM [RRID:AB_1030947], anti-BAK [RRID:AB_10828597], anti-BAX [RRID:AB_10557411], anti-PUMA [RRID:AB_2797920], anti-Bcl-xL [RRID:AB_10695729], anti-Menin [RRID:AB_10858216], anti-β-Tubulin [#86298], anti-MEF2C [RRID:AB_10548759], anti-p-ERK1/2 [RRID:AB_331772], ERK1/2 [RRID:AB_390779], p-S6 [RRID:AB_916156], total S6 [RRID:AB_331355], p-Rb [RRID:AB_11178658], total Rb [RRID:AB_823629], and p16 [RRID:AB_2799960] antibodies were obtained from Cell Signaling Technologies . Anti-HA-tag [RRID:AB_444303], Anti-MEIS1 [RRID:AB_776272], anti-FLT3 [ab245116], anti-HOXA9 [ab140631], anti-PBX3 [RRID:AB_10858991], and anti-CD11b [RRID:AB_2650514] antibodies were obtained from Abcam . Anti-BFL1 [# ABC490] antibody was obtained from Millipore/Sigma . Anti-CDK6 [RRID:AB_10610066], anti-BCL2 [RRID:AB_626733], anti-NOXA [RRID:AB_784877], anti-GAPDH [RRID:AB_627679] and anti-β-Actin [RRID:AB_626630] antibodies were obtained from Santa Cruz Biotechnologies . Anti-p27 [RRID:AB_397636] antibody was obtained from BD Transduction Labs .MOLM13  and OCI-AML3  cells were obtained from the DSMZ. MV4-11 , cells were obtained from the ATCC . MOLM13 cells with isogenic TP53 mutations  were a gift from Dr. Benjamin L. Ebert . HEK-293T cells were obtained from the Characterized Cell Line Core Facility at M.D. Anderson Cancer Center, Houston TX. All experiments with cell lines were performed within 6 months after thawing or obtaining from ATCC or DSMZ. The cell lines were also authenticated in the Characterized Cell Line Core Facility at M.D. Anderson Cancer Center, Houston TX. MOLM13 and OCI-AML3 cells were cultured in RPMI-1640 media with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin. MV4-11 cells were cultured in ATCC-formulated IMDM media with 20% FBS and 1% penicillin/streptomycin. HEK293T cells were cultured in high-glucose-formulated DMEM media with 10% FBS, 1% penicillin/streptomycin, and 1% glutamine. Logarithmically growing, mycoplasma-negative cells were utilized for all experiments. Following drug treatments, cells were washed free of the drug(s) prior to the performance of the studies described.The cell lines utilized in these studies were authenticated in the Characterized Cell Line Core Facility at M.D. Anderson Cancer Center, Houston TX utilizing STR profiling.http://synergyfinder.fimm.fi/). Delta synergy scores were determined by the ZIP method.Following designated treatments, cultured cell lines or PD-AML cells were washed with 1× phosphate-buffered saline, stained with TO-PRO-3 iodide , and analyzed by flow cytometry on a BD Accuri CFlow-6 flow cytometer . We used matrix dosing of agents in combinations to allow synergy assessment utilizing the SynergyFinder V2 online web application tool  and reverse transcribed with a High-Capacity Reverse Transcription kit from Life Technologies . Quantitative real-time PCR analysis for the expression of target genes was performed on cDNA using TaqMan probes and a TaqMan Universal PCR Mastermix from Applied Biosystems . Relative mRNA expression was normalized to the expression of GAPDH and compared to the untreated cells.F36V in MOLM13 cells, following near-complete KO of the endogenous Menin, to assess biologic effects in MOLM13 cells ectopically transduced with and expressing Menin-FKBP12F36V [We first determined the effects of CRISPR-Cas9-mediated KO of Menin on MLL1/MLL1-FP targets in AML MOLM13 cells that harbor MLL1-r, FLT3-ITD, and two copies of wild-type TP53. Figure BP12F36V . A near-We next determined effects of treatment with the pre-clinical MI drug candidate SNDX-50469 (VTP50469) and the closely related clinical drug SNDX-5613  Fig.  in MOLM1cis-regulatory regions of MEIS1 and FLT3. A similar log2-fold reduction in H3K27Ac peak density was also observed at BCL2, CDK6, and PBX3 loci  in TP53 were managed in the Leukemia Department of MD Anderson Cancer Center. Of these, 17 patients (~9%) harbored mutations in TP53 Table . These i/− cells . As showod Figs. . This agod Figs. .Fig. 6CoP < 0.005)   cells . Compare05) Fig. . The dos05) Fig. C. Notabl01) Fig. . NeitherFindings presented here demonstrate for the first time that, by either utilizing the dTAG system to degrade Menin or CRISPR-Cas9 to edit and KO Menin, depletion of Menin is associated with a reduction in expression of MEIS1, FLT3, CDK6, and BCL2, sensitizing MOLM13 cells harboring MLL1-r and FLT3-ITD to BCL2 inhibitor venetoclax and CDK6 inhibitor abemaciclib-induced apoptosis. Five days after transduction of gRNA to Menin, protein levels of HOXA9, MEF2C, PBX3, and p27 were also notably reduced in MOLM13 cells. A previous report had documented that loss of occupancy of Menin, MLL1 (N-terminus), DOT1L, and H3K79Me2 from selective chromatin targets induced concordant repression of a subset of MLL1/MLL-FP target genes . UtiliziPresent studies also show that MI-mediated reduction in expression of protein levels of BCL2 and CDK6 could be therapeutically exploited. They demonstrate that co-treatment with SNDX-50469 and venetoclax or abemaciclib induced synergistic lethality in not only AML cell lines but also PD AML cells that harbor either MLL1-r or mtNPM1c along with FLT3-ITD and/or mtFLT3 (ITD or TKD). These findings were associated with SNDX-50469-mediated downregulation of not only BCL2 and CDK6, as noted above, but also of MCL1 and to a lesser extent Bcl-xL in PD, CD34+ phenotypically characterized  AML stem/progenitor cells. These perturbations might further explain the synergistic interaction between SNDX-50469 and venetoclax, since overexpression of MCL1 and Bcl-xL have been documented as important mechanisms dampening the pro-apoptotic activity of venetoclax . A recen+/+ cells [−/− cells are almost as sensitive as MOLM13-TP53+/+ cells to MI-induced loss of viability. Additionally, co-treatment with MI and venetoclax is synergistically lethal against MOLM13 TP53-R175H or -R248Q cells or against MOLM13-TP53−/− cells. Furthermore, consistent with a previous report, the combination of MI with the FLT3 kinase inhibitor gilteritinib exerted synergistic lethality against MOLM13-TP53+/+ as well as MOLM13 TP53-R175H or -R248Q cells or MOLM13-TP53−/− cells [Although observed in more than 50% of epithelial cancers, somatic missense mutations and/or allelic loss of TP53 is sub-clonal and occurs in approximately 10% of patients with MLL1-r AML, and even less common in AML with mtNPM1 , 25, 37./+ cells . However/− cells , 40. OveSupplemental Figures and TablesSupplemental Figure LegendsSupplemental Materials and Methods


Surveillance of Non-Ventilator Hospital-Acquired Pneumonia (NV-HAP) is limited by the ambiguity in diagnosing pneumonia. We implemented electronic surveillance criteria for NV-HAP across the VA healthcare system and tested for reliability, validity and meaning of the electronic criteria vs manual chart review.We defined NV-HAP surveillance criteria as oxygen deterioration concurrent with fever or abnormal WBC count, ≥3 days of antibiotics, and orders for chest imaging. We applied these criteria to EHR data from all patients hospitalized ≥3 days at all VA acute care facilities from 1/1/2015-12/31/2020 and calculated NV-HAP incidence and inpatient mortality. Clinician reviewers used a consensus review guide to independently review and adjudicate 47 cases meeting NV-HAP surveillance criteria for 1) clinical deterioration, 2) CDC-NHSN pneumonia criteria, 3) treating clinicians’ assessment, and 4) reviewer’s diagnosis. All reviewers subsequently adjudicated all cases and conducted an error analysis to identify sources of discordance.Among 2.3M hospitalizations, 14,023 met NV-HAP surveillance criteria (0.6 per 100 admissions). Inpatient mortality was 26% . Among 47 hospitalizations flagged by surveillance criteria, 45 (97%) had a confirmed clinical deterioration, (the other 2 were immediate post-operative cases), 20 (43%) met CDC-NHSN pneumonia criteria, 21 (47%) had possible pneumonia per treating clinicians, and 25 (53%) had possible or probable NV-HAP per reviewers. Agreement among the 3 reviewers before adjudication was 51% (Fleiss’ κ 0.43) for CDC-NHSN and 58% (Fleiss’ κ 0.33) for NV-HAP. The most common source of discordance between reviewers was chest imaging classification (15/19 discordant cases).NV-HAP electronic surveillance criteria demonstrated high precision for identifying clinical deterioration and moderate concordance with CDC-NHSN pneumonia criteria or reviewer diagnosis. Agreement between electronic surveillance criteria vs manual chart review was low but similar to agreement amongst manual reviewers applying NHSN criteria. Electronic surveillance may provide greater consistency than human review while facilitating wide-scale automated surveillance.Chanu Rhee, MD, MPH, UpToDate  Michael Klompas, MD, MPH, UpToDate


The adverse effect of cisplatin administration causes acute kidney injury (AKI) following renal inflammation and nephrotoxicity, characterized by proximal tubular cell apoptosis and necrosis. Pro-apoptotic and pro-inflammatory roles of glycogen synthase kinase (GSK)-3β have been reported. This study investigated the therapeutic blockade of GSK-3β in cisplatin-induced AKI. A renal cisplatin nephrotoxicity model showed activation of GSK-3β in vivo, particularly in proximal tubular epithelial cells. Pharmacologically inhibiting GSK-3β abolished cisplatin nephrotoxicity, including proximal tubular injury, cell cytotoxicity, and biochemical dysfunction. Additionally, GSK-3β inhibitor treatment ameliorated renal inflammation by reducing immune cell infiltration, cell adhesion molecule expression, and pro-inflammatory cytokine/chemokine production. Cisplatin treatment caused GSK-3β activation in vitro in the human renal proximal tubular epithelial cell line HK-2, whereas either pharmacological administration of GSK-3β inhibitors or genetic transduction of GSK-3β short-hairpin RNA impeded cisplatin-induced cytotoxicity. These results indicate that cisplatin activates GSK-3β followed by GSK-3β-mediated renal inflammation and nephrotoxicity, contributing to AKI. Cisplatin, a highly effective chemotherapeutic drug, is widely used to treat various cancers, including sarcomas, lymphomas, small-cell lung cancer, head and neck cancer, ovarian cancer, and testicular cancer . FollowiThe mechanisms that underlie cisplatin-induced nephrotoxicity are complicated. Current studies have demonstrated that the exposure of tubular cells to cisplatin activates multiple signaling pathways, including DNA damage and ATR/ATM/p53 activation ; reactivGlycogen synthase kinase (GSK)-3β, a serine/threonine kinase, facilitates apoptotic signaling and cellular inflammatory activation in response to various stimuli, such as growth stress, pathogens, anticancer agents, and cytokines . In renahttps://www.rcsb.org/3d-view/1UV5, accessed on 20 July 2021) , the secondary structure of the GSK-3β:BIO complex was modified and drawn according to the previous works [https://pubchem.ncbi.nlm.nih.gov/substance/833555#section=3D-Conformer, accessed on 20 July 2021) .Antibodies against phospho-glycogen synthase (GS) (Ser641) (Cat.# 3891), GS (Cat.# 3886), GSK-3β (Cat.# 9832), and GAPDH (Cat.# 2118) were purchased from Cell Signaling Technology . Anti-phospho-GSK-3β (Tyr216) (Cat.# NB10081946) antibody was purchased from Novus Biologicals . Alexa Fluor 488-labeled anti-mouse CD3 (Cat.# 100210), Gr-1 (Cat.# 108417), CD11b (Cat.# 101217), and CD54 (Cat.# 116112) were obtained from BioLegend . Horseradish peroxidase (HRP)-conjugated goat anti-mouse (Cat.# 62-6520), goat anti-rabbit (Cat.# 65-6120), goat anti-rat (Cat.# 31470), and donkey anti-goat (Cat.# A15999) IgG antibodies were obtained from Invitrogen . GSK-3 inhibitors 6-bromo-indirubin-3′-oxime , SB216763 (Cat.# S3442), and SB415286 (Cat.# S3567) were purchased from Sigma-Aldrich . All drugs were assessed for their cytotoxic effects in cells using cytotoxicity assays before the experiments were performed. Non-cytotoxic dosages were used in this study. By utilizing the online tools, adapted from the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) 1UV5  and fed standard laboratory chow. To establish a murine model of cisplatin-induced AKI, mice were intraperitoneally (i.p.) injected with 30 mg/kg of cisplatin  in a total volume of 200 µL diluted in sterile phosphate-buffered saline (PBS) [2O2 in methanol. Antibody diluent  was used to dilute primary antibodies, including phospho-GS (pGS Ser641), phospho-GSK-3β (pGSK-3β Tyr216), CD3, Gr-1, CD11b, and CD54. Tissue sections were then stained with diluted antibodies at 4 °C overnight. Samples were washed twice in PBS and stained with HRP-labeled secondary antibodies 1 h at room temperature. Sections were developed with 3-amino-9-ethylcarbazole (AEC)-based staining. Sections were washed with PBS and visualized under a microscope  or a fluorescence microscope . We used the appropriate standard RGB intensity range for each cell marker to count the numbers of cells stained positively for CD3, Gr-1, CD11b, and CD54.The deparaffinized and rehydrated tissue sections were quenched with 3% HMice sera were collected from peripheral blood via retro-orbital bleeding under pentobarbital anesthesia (40 mg/kg i.p.). An automatic biochemical analyzer  was utilized for detecting serum levels of BUN and creatinine. For histopathological analysis, mice kidney tissues were harvested under a lethal overdose of pentobarbital (200 mg/kg i.p.) treatment. The kidney tissues were fixed, dehydrated, embedded in paraffin, and sliced, following deparaffinized in xylene, rehydrated in decreasing ethanol concentrations, stained with H&E, and viewed by light microscopy. The pathological changes, including renal cell necrosis, apoptosis, and cytoplasmic vacuolization, were validated accordingly .2.Human renal proximal tubular epithelial HK-2 cells (ATCC Cat# CRL-2190), which Dr. Y. Y. Chiou, Department of Pediatrics, National Cheng Kung University Hospital, kindly provided, were routinely grown on plastic in Dulbecco’s modified Eagle’s medium  and Minimum Essential Medium , supplemented with L-glutamine, HEPES, antibiotics, and 10% heat-inactivated fetal bovine serum , and cultivated in a humidified atmosphere containing 95% air and 5% COWe utilized a colorimetric LDH activity assay , as diagnosed by a microplate reader , to evaluate cell damage according to the manufacturer’s instructions. To measure apoptotic cells, TUNEL  staining was performed using a TUNEL assay kit  according to the manufacturer’s instructions. Furthermore, we also used Annexin V Apoptosis Detection Kit (BioLegend) to identify apoptotic and necrotic cells. Fluorochrome-labeled Annexin V can specifically target and identify apoptotic cells by interacting with phosphatidylserine in a calcium-dependent manner. Additionally, a counterstaining dye propidium iodide (Sigma-Aldrich) was used to distinguish between the necrotic and apoptotic cells as measured by fluorescent microscopy (BX51) and flow cytometry  and FlowJo software . Hoechst 33258 (Sigma-Aldrich) was used as a nuclear stain.https://www.sigmaaldrich.com/TW/en/applications/protein-biology/western-blotting, accessed on 20 July 2021). The proteins were subjected to standard SDS-PAGE and then transferred to a PVDF membrane . The protein membrane was blocked with 5% skim milk and then stained with diluted primary antibodies at 4 °C overnight. After being washed twice in PBS-Tween 20, the membrane was stained with diluted horseradish peroxidase (HRP)-conjugated secondary antibodies 1 h at room temperature. The blots were developed using the enhanced chemiluminescence solution  according to the manufacturer’s instructions.Western blotting was carried out to measure protein expression according to the online support of Immunoblotting Protocol  (A lentivirus-based shRNA was performed to silence the expression of human GSK-3β (TRCN0000040001: targeting sequence 5′-GCTGAGCTGTTACTAGGACAA-3′). In addition, targeting luciferase (shLuc) was used as a negative control of shRNA. All shRNA clones obtained from the National RNAi Core Facility, Institute of Molecular Biology/Genomic Research Center, Academia Sinica, Taipei, Taiwan, were prepared for cell transfection. Following 24 h post-transfection, cells were selected in a culture medium containing puromycin  for 3 days.According to the manufacturer’s instructions, the concentrations of cytokines/chemokines in the murine sera were determined using ELISA kits .t test; significance was set at p < 0.05.The values are given as the mean ± standard deviation (SD). The groups were compared using Student’s two-tailed unpaired p < 0.05) increase in serum BUN . Drug treatment caused severe changes in histopathology on proximal tubular injury, including vacuolization, necrosis, immune cell infiltration a, and therum BUN e and creerum BUN f at 24 hp < 0.05) attenuated by treatment with the GSK-3 inhibitor BIO.In this study, a synthetic ATP-competitive cell-permeable derivative BIO was used. The RCSB PDB 1UV5 and PubChem SID 833555, respectively, showed the secondary structure of the GSK-3β:BIO complex a and thep < 0.05) inhibited the cisplatin-induced increase in serum BUN  increase in the infiltration of CD3-positive T cells (p < 0.05) caused upregulation of CD54 (p < 0.05) reduced cisplatin-induced immune cell infiltration and CD54 expression. Thus, in addition to renal cell protection, pharmacologically inhibiting GSK-3 effectively diminishes cisplatin-induced immune cell infiltration in cisplatin-induced AKI. We next studied the involvement of infiltrated inflammatory immune cells, such as neutrophils, monocytes/macrophages, T lymphocytes, and adhesion molecule CD54, which all are critical mediators of experimental cisplatin nephrotoxicity, in the animal model of cisplatin-induced AKI. In renal tissue slices of cisplatin-induced AKI, immunofluorescent staining showed a significant  decreased levels of serum TNF-α  reduced cisplatin-induced cytotoxicity as detected by LDH release (p < 0.05) induced cytotoxicity in a GSK-3β-regulated manner /Hoechst 33258 followed by fluorescence microscopic observation showed distinct types of cell death, including apoptosis and necrosis, in cisplatin-treated HK-2 human renal proximal tubular epithelial cells a. Annexi release d and apo release e. Knocki release f confirmd manner g. These In this study, inhibiting GSK-3β prevented nephrotoxicity in vivo and in vitro and suppressed inflammatory responses and renal dysfunction in cisplatin-induced AKI. Consistent with our findings, pharmacologically and genetically targeting GSK-3β confers cytoprotection against renal injury in several different in vivo models of AKI ,20,21,22Our in vivo study showed substantially increased expression of active GSK-3β in proximal tubular epithelial cells around the glomerulus under cisplatin treatment. Further in vitro studies confirmed that cisplatin directly activated GSK-3β in proximal tubular epithelial cells. All of the data showed that cisplatin treatment activates GSK-3β in vivo and in vitro; however, the possible molecular mechanisms of GSK-3β activation initiated by upstream factors and regulated by downstream signaling pathways remain unclear. Although inactivation of GSK-3β by PI3K/Akt signaling generally causes cisplatin resistance ,28, it iIn general, cisplatin-based chemotherapy causes nephrotoxicity directly by triggering tubular cell death and indirectly by exacerbating renal inflammation ,33. DespWe proposed that GSK-3β signaling pathways may mediate cellular activation of cisplatin stimulation, such as DNA damage, oxidative stress, MAPK activation, and cell responses to cisplatin treatment, including renal cell death and inflammation. Consistent with other studies showing that GSK-3 is involved in several nephrotoxicity models, including endotoxemia, ischemia, and non-cisplatin drugs ,19,20,22Cisplatin-induced renal inflammatory responses, such as immune cell infiltration and cytokine/chemokine production, may marginally exacerbate nephrotoxicity with the synergistic reaction of cisplatin ,45. ConsA current study also reported BIO treatment, used as Wnt/β-catenin agonist, as a therapeutic strategy against cisplatin-induced AKI . As consIn conclusion, this study demonstrated GSK-3β activation caused by cisplatin in vivo in kidneys and in vitro in proximal tubular epithelial cells. Accompanied by active GSK-3β in cisplatin-induced AKI, increased inflammatory immune cell infiltration and cytokine/chemokine production were identified with the induction of renal cell cytotoxicity all highly associated with renal injury. Pharmacological inhibition of GSK-3 effectively ameliorated renal inflammation and nephrotoxicity in vivo in cisplatin-induced AKI. For the basis of anti-AKI, inactivation of GSK-3β rescued renal cell survival from cisplatin-induced renal cell cytotoxicity.


Arabidopsis.The miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) transcription factors play key roles in regulating plant development, but little is known about their function in abscisic acid (ABA) signaling. Here, we report that the miR156-targeted SPLs enhance ABA responses and contribute to the inhibition of pre-harvest sprouting. We find that SPL9 directly activates the expression of ABA responsive genes through binding to their promoters. SPL9 was further shown to physically interact with ABSCISIC ACID INSENSITIVE 5 (ABI5), a master transcription factor in ABA signaling, thus promoting its association with the promoters of ABA responsive genes. Furthermore, we reveal that the protein kinases SnRK2s interact with and phosphorylate SPL9, which is essential for its role in the activation of ABA responses. Together, our results disclose a SnRK2s-SPLs-ABI5 regulatory module in ABA signaling in  Arabidopsis. Among them, ABA-INSENSITIVE1 (ABI1) (The stress-related phytohormone abscisic acid (ABA) inhibits seed germination and seedling growth to adapt various environmental challenges . Molecul1 (ABI1)  and ABI21 (ABI1)  are clad1 (ABI1) . Several1 (ABI1) .Since the identification of ABA receptors, PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) , a core Arabidopsis, SPL genes are divided into two subgroups, represented by SPL3  which encodes a small protein, and SPL9  which encodes a much larger protein, respectively  -like (SPL) belongs to plant-specific transcription factors and contains a highly conserved SBP-box domain , which wectively . Among t(miR156) . The miRansition ; SPL9 anranching . In addiand rice .Arabidopsis. We demonstrate that SPLs interact with the master transcription factor ABI5 to promote ABA signaling. Furthermore, we show that SnRK2s physically interact with and phosphorylate SPLs. Importantly, the ABA-induced SPL9 phosphorylation is required for its function in the activation of ABA responses.In this study, we uncover that miR156-targeted SPLs transcription factors positively regulate ABA responses and inhibit pre-harvest sprouting (PHS) in Arabidopsis thaliana ecotype Col-0 was used as the wild type. Some of the plant materials used in this study were previously described: GFP-rSPL9  solid medium containing 2% sucrose at 22°C in a light incubator with 16-h-light/8-h-dark photoperiod. N. benthamiana plants were grown under a 16-h-light/8-h-dark cycle in a greenhouse at 22°C for 1 month before infiltration.SPL9 or SPL3 was cloned into pQBV3 vector (pEarleygate202-YN (cYFP) (ABI5 was introduced into the pEarleygate201-YN (nYFP) vector  was used. The full-length coding sequence (CDS) of 3 vector  and subsN (cYFP) . Similar) vector .SPL9 was cloned into p1300-35S-nLUC vector or p1300-35S-cLUC vector  using the Site-Directed Mutagenesis Kit . Similarly, the full-length CDS of ABI5 was inserted into pGEX4T-1 vector to generate ABI5-GST. All the ligations above were performed based on ligation free cloning master mix  according to the manufacturer’s instruction.For pull-down assays, the full-length CDS of pro:LUCEm6 and pro:LUCEm1 constructs, the ∼1.5-kb promoter of Em6 and 800-bp promoter of Em1 were separately ligated into the entry vector pQBV3, and then introduced into the vector pGWB35 (35S:rSPL9-MYC was generated based on the destination vector pGWB17 (rSPL9-YFP and rSPL9(2A)-YFP were generated based on the destination vector pEarly-101 driven by the 35S promoter.For r pGWB35 . The conr pGWB17 . The conpro:GFP-rSPL9SPL9, pro:GFP-rSPL9(2A)SPL9 and GFP-rSPL9/SnRK2.6-Flag transgenic plants, 2-kb promoter of SPL9 was ligated into p1305-35S-GFP to produce pro-GFPp1305-SPL9, next the full length CDS of SPL9 or SPL9(2A) was introduced in it to generate pro:GFP-rSPL9SPL9 or pro:GFP-rSPL9(2A)SPL9 construct, respectively. SnRK2.6 gene was amplified and inserted into the p1300-35S-Flag vector. Agrobacterium strain GV3101 carrying the construct was then transformed into the Col-0 or GFP-rSPL9 plants to generate pro:GFP-rSPL9SPL9, pro:GFP-rSPL9(2A)SPL9 or GFP-rSPL9/SnRK2.6-Flag transgenic plants using the floral-dip method , and the expression levels of ACT7 were used as the internal control. The primer sequences are listed in Total RNA was extracted using Trizol (Invitrogen) reagent according to the manufacture’s instruction. About 2 μg of total RNA were used for reverse transcription with the 5× All-In One RT MasterMix system . Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed using SYBRFor ABA responses, seeds of different genotypes were harvested at the same time for the germination and cotyledon greening assays as described before . Seeds oN. benthamiana leaves as described previously .The luciferase complementation imaging (LCI) assays for the protein interaction detection was performed in eviously . The indArabidopsis seedlings grown on 1/2 MS medium were treated with or without 50 μM ABA for 2 h and then collected for chromatin immunoprecipitation (ChIP) assays as previously described -YFP construct was injected into N. benthamiana leaves. For BiFC assays, the indicated vectors were co-transformed into Agrobacterium GV3101 and then co-expressed in N. benthamiana leaves as described previously  transgenic plants using the extracted buffer . For the immunoblotting detection of GFP-SPL9, we used anti-GFP  antibody. ACT  was employed as a loading control.The GFP-SPL9 fusion proteins were extracted from the 6-day-old GFP-rSPL9 transgenic plants and anti-ABI5 antibody were used in the Co-IP assays for the interaction of SPL9 and ABI5. Total proteins were extracted from the 6-day-old seedlings treated with 50 μM ABA for 2 h using the lysis buffer  with freshly added PMSF (0.6 mM) and 1× protease inhibitor. The extracts were centrifuged for 20 min and the supernatant was incubated with anti-GFP magnetic beads  overnight. Next, the beads were washed five times with the lysis buffer and eluted samples were analyzed by immunoblotting with anti-GFP and anti-ABI5  antibodies.The Col-0, GFP-rSPL9 and GFP-rSPL9/SnRK2.6-Flag transgenic plants treated with 50 μM ABA plus 30 μM MG132 for 4 h were used in the Co-IP assays for the interaction of SnRK2.6 and SPL9. Total proteins were extracted as described above. The supernatant was incubated with anti-Flag magnetic beads  overnight. Proteins were detected with anti-GFP and anti-Flag  antibodies, respectively.The 6-day-old Escherichia coli transetta. The fusion proteins were induced with 4 mM isopropyl β-D-thiogalactopyranoside (IPTG) at 18°C overnight. For the pull-down assays of SPL9 and ABI5, the fusion proteins were incubated with glutathione resin (GenScript) overnight in column buffer ]. For the pull-down assays of SPL3 and ABI5, the fusion proteins were incubated with amylose resin (New England Biolabs) overnight in column buffer. Next, the GST bind resin or MBP bind resin was washed five times with column buffer, resolved by SDS-PAGE, and detected using anti-GST  and anti-MBP  antibodies.The constructs  were separately transformed into GFP-rSPL9 seedlings treated with 50 μM ABA for 2 h and SnRK2.6-His fusion proteins were used for the semi-in vivo pull-down assays. The GFP-SPL9 proteins were extracted with lysis buffer with freshly added PMSF (0.6 mM) and 1× protease inhibitor. Then SnRK2.6-His fusion proteins were incubated with the GFP-SPL9 protein extracts overnight and added Ni-NTA resin  for a further 2 h. The His bind resin was washed five times with PBS buffer , resolved by SDS-PAGE, and detected using anti-His  and anti-GFP  antibodies, respectively.The 6-day-old in vitro phosphorylation assays, 1 μg MBP-SPL9, MBP-SPL9(2A) or MBP-SPL3 fusion proteins were incubated with 1 μg SnRK2.6-His in 20 μl kinase reaction buffer  at 37°C for 1 h. The reactions were boiled with 5× SDS loading buffer then separated by phos-tag SDS-PAGE  and immunoprecipitated with anti-GFP magnetic beads. Then the IP products were separated by phos-tag SDS-PAGE and analyzed with anti-GFP antibody.For the N. benthamiana leaves as previously described (pro:LUCEm and effector 35S:rSPL9-MYC were separately introduced into Agrobacterium GV3101 to perform the con-infiltration in N. benthamiana leaves. The N. benthamiana leaves after infiltrating 24 h were injected with 50 μM ABA and incubated for a further 24 h. The luciferase luminescence was observed using NightSHADE LB 985 (Berthold), and quantification of luciferase activities were carried out with IndiGO software (version 2.03.0).The transcriptional activity assays were carried out in escribed . In brieSPL9 (At2g42200), SPL3 (At2g33810), ABI5 (At2g36270), ABI3 (At3g24650), ABI4 (At2g40220), SnRK2.2 (AT3G50500), SnRK2.3 (AT5G66880), SnRK2.6 (AT4G33950), Em1 (AT3G51810), and Em6 (AT2G40170).Sequence data from this article can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: GFP-rSPL9 line is identical to a gain-of-function mutant of SPL9 gene, in which a miR156-resistant version of SPL9 is expressed from its native promoter (MIM156 line has elevated expression of SPL9 and other SPLs (GFP-rSPL9 and MIM156 seedlings were much lower than the wild-type Columbia-0 (Col-0) under ABA treatment  analyses showed that ABA-induced expression levels of the representative ABA-responsive genes 0 plants . In contT plants , demonstEm6 (∼1.5-kb) promoter sequence and identified seven putative SBP-binding motifs with positions of −197/−200, −229/−232, −285/−288 (labeled as A), −427/−430 (labeled as B), −626/−629 (labeled as C), −1043/−1046 (labeled as D), and −1220/−1223 (labeled as E), respectively, (GFP-rSPL9 seedlings treated without (control) or with 50 μM ABA for 2 h. The results showed that the enrichment of SPL9 at Em6 promoter was relatively low in the absence of ABA, whereas ABA treatment substantially increased the enrichment of SPL9 at the Em6 promoter (Em1 promoter (800-bp) with positions of −382/−385 (labeled as A) and −675/−678 (labeled as B)  of target genes . We firsctively, . Next, wpromoter . Similared as B) . The ChIwith ABA , implyinNicotiana benthamiana to determine the effect of SPL9 on the transcription of target genes. The Agrobacterium strains harboring different constructs, including the pro:LUCEm6 reporter and the effector 35S:rSPL9-Myc, were co-infiltrated into N. benthamiana leaves. The results showed that transient expression of SPL9 could intensely elevate the expression of pro:LUCEm6 reporter  assays in N. benthamiana leaves. SPL9 was fused with the C-terminal part of yellow fluorescent protein (cYFP), and ABI5 was fused with the N-terminal part of YFP (nYFP) to generate cYFP-SPL9 and nYFP-ABI5, respectively. The results illustrated that co-expression of cYFP-SPL9 and nYFP-ABI5 produced strong YFP fluorescence in the nucleus, whereas no YFP signal was observed in negative controls  assays in LUC-SPL9 , indicateedlings , suggestcontrols . Finallycontrols , suggestith ABI5 .N. benthamiana. SPL9 was divided into two truncated parts , according to the position of the highly conserved SBP domain , ABI5-M (221-349 aa), and ABI5-C (350-442 aa) , based oGFP-rSPL9/abi5-7 plants via genetic crossing and examined their seed germination and cotyledon greening phenotypes in response to ABA treatment. Consistent with the above results ] for in vitro phosphorylation assays. As shown in in vitro SPL9 transgenic plants. We chose the pro:GFP-rSPL9SPL9 and pro:GFP-rSPL9(2A)SPL9 transgenic lines with similar SPL9 expression levels for further phenotypic analyses (pro:GFP-rSPL9SPL9 seedlings was higher than that in the wild type (pro:GFP-rSPL9(2A)SPL9 seedlings was lower than that in the pro:GFP-rSPL9SPL9 seedlings SPL9 seedlings was lower than that in the pro:GFP-rSPL9SPL9 seedlings under ABA treatment  gene, encoding an OsSPL14 transcription factor in rice, plays an important role in regulating plant architecture (Magnaporthe oryzae infection can induce the phosphorylation of OsSPL14, consequently alter its DNA binding specificity (M. oryzae infection remains to be identified. Significantly, we here found that ABA treatment can induce the phosphorylation of SPL9 by SnRK2s to amplify ABA responses in Arabidopsis. Notably, the ABA-induced phosphorylation of SPL9 was reduced in the snrk2.2/2.3/2.6 triple mutants rather than completely abolished as shown in the wild type seedlings treated with a general Ser/Thr-kinase inhibitor staurosporine, indicating that there might be other protein kinases could phosphorylate SPLs in vivo. Thus, we propose that the plant-specific transcription factors SPLs may be phosphorylated and functionally modulated by different protein kinases in response to endogenous cues and external challenges.Previous studies reported that the itecture . In addicificity . UnfortuThe original contributions presented in the study are included in the article/JS designed the research. HD, SY, and YJ performed the experiments. JS, HD, SY, RY, YZhang, YZhou, and YZhu analyzed the data. JS, HD, and SY wrote the manuscript. JS and YZhu revised the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Ischemic preconditioning (IPC) is the most powerful endogenous cardioprotective form of cellular adaptation. However, the inhibitory or augmenting mechanism underlying cardioprotection via IPC remains largely unknown. Suppressor of cytokine signaling-3 (SOCS3) is a cytokine-inducible potent negative feedback regulator of the signal transducer and activator of transcription-3 (STAT3) signaling pathway. Here, we aimed to determine whether cardiac SOCS3 deficiency and IPC would synergistically reduce infarct size after myocardial ischemia reperfusion injury. We evaluated STAT3 activation and SOCS3 induction after ischemic conditioning (IC) using western blot analysis and real-time PCR, and found that myocardial IC alone transiently activated myocardial STAT3 and correspondingly induced SOCS3 expression in wild-type mice. Compared with wild-type mice, cardiac-specific SOCS3 knockout (SOCS3-CKO) mice showed significantly greater and more sustained IC-induced STAT3 activation. Following ischemia reperfusion, IPC substantially reduced myocardial infarct size and significantly enhanced STAT3 phosphorylation in SOCS3-CKO mice compared to in wild-type mice. Real-time PCR array analysis revealed that SOCS3-CKO mice after IC exhibited significantly increased expressions of several anti-apoptotic genes and SAFE pathway-related genes. Moreover, real-time PCR analysis revealed that myocardial IC alone rapidly induced expression of the STAT3-activating cytokine erythropoietin in the kidney at 1 h post-IC. We also found that the circulating erythropoietin level was promptly increased at 1 h after myocardial IC. Myocardial SOCS3 deficiency and IPC exert synergistic effects in the prevention of myocardial injury after ischemia reperfusion. Our present results suggest that myocardial SOCS3 is a potent inhibitor of IPC-induced cardioprotection, and that myocardial SOCS3 inhibition augment IPC-mediated cardioprotection during ischemia reperfusion injury. Among patients with acute myocardial infarction (MI), prognosis has been improved by the development of reperfusion therapy via percutaneous coronary intervention or thrombolysis; however, the incidence of post-MI heart failure has been increasing –6. AfterMurry et al  first dejanus kinase (JAK)–signal transducer and activator of transcription-3 (STAT3) pathway is a potent pro-survival signaling pathway during stress-induced myocardial injury, including acute MI [The acute MI –17. The acute MI , 8, 18. acute MI , 18–23. acute MI  reportedacute MI  showed tOur group and others have identified suppressor of cytokine signaling (SOCS) family proteins as cytokine-inducible highly specific and potent inhibitors of JAK–STAT signaling pathways –27. We pAll experimental protocols involving animal were approved by the Animal Experiments Review Boards of Kurume University . All procedures on the mice were performed under general anesthesia with isoflurane  using an animal anesthesia machine , and all efforts were made to minimize suffering. At the end of experimental periods, mice were euthanized through deep isoflurane anesthesia.The mice were maintained with normal chow and freely available drinking water. This study utilized 8- to 12-week-old male mice in a Balb/c background. To determine the tissue-specific roles of SOCS3, we generated SOCS3-flox mice as previously described . To inveWe used an open-chest in situ model to study I/R injury, as previously described , 37. BriAs was done for the in vivo myocardial I/R injury protocol, we anesthetized mice using inhaled isoflurane, provided positive-pressure ventilation, opened the thoracic cavity, and visualized the LAD coronary artery. Next, IC or IPC was induced by three cycles comprising 5 min of coronary artery occlusion followed by 5 min of reperfusion. IC and IPC were performed following the exact same experimental procedure. Throughout the manuscript, we use the term IC when describing experiments without 1-hour ischemia, and IPC when describing experiments including 1-hour ischemia.At 24 hours post-reperfusion, each mouse was anesthetized as described above. Then chest was re-opened, and the LAD coronary artery re-occluded. The heart was perfused with 5% Evans blue dye, which stained the normally perfused area, such that an absence of staining indicated the ischemic area—i.e., the area at risk (AAR). Next the heart was excised, and the left ventricle (LV) was cut into five transverse slices from apex to base. These slices were incubated in 1% triphenyltetrazolium chloride (TTC) solution at 37°C for 10 min, photographed with a digital camera , and weighed. On each image, we measured the infarct area  and the AAR and LV areas, using a planimeter with Image-Pro PLUS software (version 7.0J). For each slice, we determined the ratios of infarct to LV area and AAR to LV area, and multiplied these ratios by the slice weight to calculate net infarct area and AAR weights, respectively. We then summed these values for all slices. The total infarct area weight was divided by the total AAR weight (infarct area/AAR) to obtain infarct size, and total AAR weight was divided by the total LV weight (AAR/LV) to obtain ischemic size , 37. We At specific time-points after IC or after I/R, tissues were collected and homogenized in lysis buffer containing 25 mM Hepes (pH 7.5), 1% Triton X100, 150 mM NaCl, 10% glycerol, 1 mM sodium orthovanadate, 50 mM NaF, 10 mM sodium pyrophosphate, and protease inhibitor cocktail . Equal amounts of proteins were separated by denaturing SDS-PAGE, and then transferred onto nitrocellulose membrane . These membranes were probed using primary antibody, and then incubated with HRP-conjugated secondary antibody. Protein signals were detected using the ECL plus system . Expression levels were determined from band intensities using Bio-ID software , and values were expressed relative to GAPDH signals , 31, 37.Total RNA was isolated from heart and kidney tissues using TRIzol , as previously described , 31, 37,Mouse serum samples were analyzed for EPO using an enzyme-linked immunosorbent assay , following the manufacturer’s instructions . We compFreshly isolated hearts were fixed in 4% paraformaldehyde (PFA), dehydrated, embedded in paraffin, and sectioned. Five-micrometer sections were stained with hematoxylin and eosin or Mallory-AZAN staining.Mice were placed under light anesthesia with isoflurane and subjected to echocardiography as previously described , 31. TraP value of less than 0.05 was considered to indicate statistical significance.Data are expressed as the mean ± standard error of the mean (SE). Statistical analyses were performed using JMPpro12. We performed multiple group comparisons using the Kruskal–Wallis test, followed by the Dunn’s test, and comparisons between two groups were performed using the Wilcoxon rank-sum test. A Before and during IC alone, we performed western blot analysis to assess the phosphorylation of cardioprotective signaling molecules downstream of JAK in WT mice . MyocardWe next performed western blot analysis to compare the activation of cardioprotective signaling pathways—including STAT3, ERK1/2, and AKT—during IC in WT mice and SOCS3-CKO mice . At 0 h Next we investigated how myocardial SOCS3 deficiency affected IPC-induced cardioprotection during myocardial I/R injury. Myocardial IPC was elicited by three cycles of 5-min LAD coronary artery occlusion and 5-min reperfusion, and myocardial injury was induced by 1-h ligation of the LAD coronary artery. At 24 h post-reperfusion, heart tissues were double-stained with Evans blue and TTC to determine the infarct area, AAR (indicating the ischemic area), and normally perfused area of the LV . The fouWe performed western blot analysis for phosphorylated STAT3, ERK1/2, and AKT in protein samples collected at 3 h after I/R, from WT and SOCS3-CKO mice, with or without IC . We deteTo elucidate the events downstream of STAT3, we performed real-time PCR array analysis for apoptosis-related genes in heart tissue before and after IC. Before IC, the expressions of most genes were comparable between SOCS3-CKO mice and WT mice . Only thWe also evaluated RISK pathway-related genes in heart tissue from the pre-IC and 5 h after IC time-points, using PCR array analysis for apoptosis-related genes. RISK pathway-related genes include target molecules of ERK1/2 and AKT, such as Bax (Bcl2-associated X protein), Bcl-2 (B-cell leukemia/lymphoma 2), Bad (BCL2-associated agonist of cell death), Bcl-xl (Bcl2-like 1), Casp9 (Caspase 9), Dapk1 (Death associated protein kinase 1), Mcl-1 (Myeloid cell leukemia sequence 1). At 5 h after IC, only the Mcl-1 gene, which is also a SFAE-related gene, exhibited significantly altered expression in heart tissue from SOCS3-CKO mice compared to WT mice .We previously reported that remote IPC via repeated transient limb ischemia rapidly activates the renal hypoxia-inducible factor-1α (HIF1α)-EPO pathway, leading to EPO production and release from the kidney, and that this released EPO activates cardioprotective signals, including STAT3, thus reducing infarct size . Based oIn the present study, we have first investigated the effects of myocardial SOCS3 deficiency on IPC-mediated cardioprotection during myocardial I/R injury. In WT mice, IC alone transiently activated myocardial STAT3, and correspondingly induced SOCS3 expression. IC-induced STAT3 activation was significantly greater and more sustained in SOCS3-CKO mice than in WT mice. Following I/R, IPC substantially reduced infarct size and significantly enhanced STAT3 phosphorylation in SOCS3-CKO mice compared to in WT mice. Moreover, in WT mice, myocardial IC alone rapidly induced mRNA expression of the STAT3-activating cytokine EPO in the kidney, and increased the circulating EPO level. Thus, myocardial SOCS3 deficiency and IPC exerted synergistic effects in the prevention of myocardial injury after I/R. Our present results suggest that myocardial SOCS3 is a potent inhibitor of IPC-induced cardioprotection, and that myocardial SOCS3 inhibition augments IPC-mediated cardioprotection during I/R.The STAT3 signaling pathway plays important roles in myocardial hypertrophy, apoptosis, and fibrosis –14. αMHCMany lines of evidences indicate that the STAT3 signaling pathway has potent pro-survival activity against myocardial injury during acute MI –17, 40. Negative-feedback regulation through SOCS3 tightly regulates the duration and intensity of STAT3-activating cytokine activities –27. As aWe previously proposed a novel remote IPC mechanism through the kidney . BrieflyPromising experimental results in animal models support a real cardioprotective effect of STAT3-activating cytokines, including EPO and G-CSF; however, clinical trials have not yet demonstrated benefits in clinical settings –48. ImpoThere is currently no clinical strategy for SOCS3 suppression. In animal studies, SOCS3 has been inhibited using adeno-associated virus 9 (AAV9)-mediated RNA interference or administration of the anti-diabetic drug pioglitazone , 53. UsiWe previously reported the molecular mechanism through which SOCS1 and SOCS3 inhibit JAK kinase activity , 29. TheIn this study, it considered that STAT3 phosphorylation is a key target for IPC and SOCS3 inhibition to induce cardioprotection during myocardial I/R injury. Therefore, it will be important to confirm whether cardioprotection during myocardial IR injury is mediated by myocardial STAT3 phosphorylation induced by IPC and SOCS3 inhibition. To this end, future studies should investigate whether cardiac STAT3 inhibition abrogates the cardioprotective effect of IPC and SOCS3 ablation during myocardial IR injury. Optimal experiments would be performed using cardiac-specific STAT3 and SOCS3 double-knockout mice.Our present study showed that cardiac IC alone transiently activated myocardial STAT3 and correspondingly induced SOCS3 expression in WT mice, and that IC-induced STAT3 activation was significantly greater and more sustained in SOCS3-CKO mice than in WT mice. Following I/R, IPC substantially reduced myocardial infarct size and significantly enhanced STAT3 phosphorylation in SOCS3-CKO mice compared to in WT mice. Moreover, myocardial IC alone rapidly induced renal EPO mRNA expression and increased the circulating EPO level in WT mice. Our present results suggest that myocardial SOCS3 is a potent inhibitor of IPC-induced cardioprotection and that myocardial SOCS3 inhibition augment IPC-mediated cardioprotection during I/R. We propose that the combination of myocardial SOCS3 downregulation and STAT3 activation via IPC may be a novel strategy for reducing myocardial injury after I/R.S1 FigA) Graph shows the ratio of heart weight-to-body weight based on intact hearts . (B) Sections from intact hearts of WT mice and SOCS3-CKO mice were stained with hematoxylin and eosin and Mallory-Azan (n = 5 per group).((TIF)Click here for additional data file.S2 FigEchocardiography was performed in intact WT mice and SOCS3-cKO mice . IVST, interventricular septum thickness; PWT, posterior left ventricular wall thickness; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; FS, fractional shortening.(TIF)Click here for additional data file.S3 Figp < 0.05 (Wilcoxon rank-sum test). Card10 , Cideb , Diablo .We prepared mRNA from heart tissue of WT or SOCS3-CKO mice, obtained pre-IC and 5 h after IC. This mRNA was subjected to real-time PCR analysis. Values are expressed as fold change relative to the values from WT mice of pre-IC (n = 5–6 for each group). *(TIF)Click here for additional data file.S4 Figp < 0.05 (Wilcoxon rank-sum test). Bax (Bcl2-associated X protein), Bcl-2 (B-cell leukemia/lymphoma 2), Bad (BCL2-associated agonist of cell death), Bcl-xl (Bcl2-like 1), Casp9 (Caspase 9), Dapk1 (Death associated protein kinase 1), Mcl-1 (Myeloid cell leukemia sequence 1).We prepared mRNA from heart tissue of WT or SOCS3-CKO mice, obtained pre-IC or 5 h after IC. This mRNA was subjected to real-time PCR analysis. Values are expressed as fold change relative to the values from WT mice of pre-IC (n = 5–6 for each group). *(TIF)Click here for additional data file.S5 FigBlots were probed using antibodies against tyrosine- phosphorylated STAT3 (P-STAT3), STAT3, phosphorylated AKT (P-AKT), AKT, phosphorylated ERK (P-ERK1/2), and GAPDH.(TIF)Click here for additional data file.S6 FigBlots were probed using antibodies against tyrosine- phosphorylated STAT3 (P-STAT3), STAT3, phosphorylated AKT (P-AKT), AKT, phosphorylated ERK (P-ERK1/2), and GAPDH.(TIF)Click here for additional data file.S7 FigBlots were probed using antibodies against tyrosine- phosphorylated STAT3 (P-STAT3), STAT3, phosphorylated AKT (P-AKT), AKT, phosphorylated ERK (P-ERK1/2), and GAPDH.(TIF)Click here for additional data file.