Immune checkpoints are a set of costimulatory and inhibitory molecules that maintain self-tolerance and regulate immune homeostasis. The expression of immune checkpoints on T cells in malignancy, chronic inflammation, and neurodegenerative diseases has gained increasing attention.+ T cells, CD3+CD56+  T cells, CD4+/CD8+ T cells, and CD4+/CD8+CD25+  T cells in this study. The expression of PD-1 on T cells was similar between the AD patients and healthy volunteers, but increased expression of PD-L1 on CD3+CD56+ T cells , CD4+ T cells , CD4+CD25+ T cells, and CD8+ T cells  was detected in the AD patients. In addition, we found negative correlations between the AD patients’ cognitive performance and both CD8+ T cells and CD8+CD25+ T cells. To identify CD8+ T-cell phenotypic and functional characteristic differences between the healthy volunteers and AD patients in different stages, a machine learning algorithm, t-distributed stochastic neighbor embedding (t-SNE), was implemented. Using t-SNE enabled the above high-dimensional data to be visualized and better analyzed. The t-SNE analysis demonstrated that the cellular sizes and densities of PD-1/PD-L1 on CD8+ T cells differed among the healthy, mild AD, and moderate AD subjects.To characterize immune checkpoints in neurodegenerative diseases, we aimed to examine the expression of the immune checkpoint PD-1/PD-L1 in peripheral T cells in different Alzheimer’s disease (AD) patients. To achieve this aim, sixteen AD patients and sixteen age-matched healthy volunteers were enrolled to analyze their CD3Our results suggest that changes in PD-1/PD-L1-expressing T cells in AD patients’ peripheral blood could be a potential biomarker for monitoring disease and shed light on the AD disease mechanism. Moreover, these findings indicate that PD-1/PD-L1 blockade treatment could be a novel choice to slow AD disease deterioration.The online version contains supplementary material available at 10.1186/s13578-022-00897-1. Alzheimer’s disease (AD) is characterized by cerebral lesions, such as amyloid plaque and phosphorylated tau. Both lesions exhibit a specific cellular and regional vulnerability pattern. Regarding the cause of the cerebral pathological changes, recent studies focusing on the postinfectious model , mitocho+ T cells, including type 1 helper T (Th1), Th17, GM-CSF-producing CD4+ T cells, and γδ T cells, may recognize central nervous system (CNS) antigens and contribute to neuroinflammation and the progression of neurodegenerative diseases  and CD8+ T cells [typically considered cytotoxic T lymphocytes (CTLs)] but also CD4+CD25+ (IL-2RA) T cells and CD8+CD25+ (IL-2RA) CTLs. CD25 belongs to the IL-2 receptor alpha chain (IL-2RA), and IL-2 induces T-cell growth and activity. IL-2/IL-2R signaling enhances CD8+ CTLs to complete the terminal differentiation of effector cells, stimulates memory cells in a growing state, and helps signal transduction important for the development and survival of regulatory T cells (Tregs) [+ T cells and natural killer T cells  [+ CTLs recognize protein antigens, and NKT cells recognize lipid antigens via nonclassical class I major histocompatibility complex (MHC) molecules (CD1d) [Among the PBMC populations, T cells play an essential role in adaptive immunity and can be divided into many subsets by the expression of different surface markers. Changes in the cell number and activity of each T-cell subset are involved in infection, cancer, neurodegenerative disease, and tissue repair , 39–41.  (Tregs) . Once IL (Tregs) . This pr (Tregs) . MoreoveT cells) –47. CD8+s (CD1d) . The surs (CD1d) . Hence, + T cells , CD8+ T cells, CD8+CD25+ T cells, CD4+ T cells, CD4+CD25+ T cells, and CD3+CD56+ T cells, were profiled using flow cytometry  were similar to those in the healthy volunteers, and only a higher PD-1 expression trend was found in CD4+CD25+ T cells  T cells, CD4+ T cells, and CD4+CD25+ (IL-2RA) T cells in the AD patients  T cells, and CD8+CD25+ (IL-2RA) T cells was noted in the AD patients  PD-1 in the moderate AD patients was significantly higher than that in the healthy group   , hepatitis B virus (HBV), and the recent outbreak of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) [+ T cells decline with cognitive impairment, implying that CD8+ T cells might be overstimulated, thereby reaching an exhausted state with dysfunctional metabolism and a related reduction in pivotal cytotoxic functions [+CD25+ (IL-2RA) T cells in PBMCs were negatively correlated with the MMSE-minus scores    , 63. CD8unctions . Moreove+ T-cell (CTL) subsets. Furthermore, the commonly used flow cytometric indicators (median/mean fluorescence intensity and positive population) cannot thoroughly elucidate the PD-1 status in CD8+ T-cell subsets in our study. Notably, CD8+ T-cell subsets share mutual flow cytometric parameters, including cell size, granularity, and CD8 expression. The data points lie in a high-dimensional space because of the multiple parameters; therefore, the data are challenging to analyze and visualize. To overcome the limitations of high-dimensional data, we adopted an unsupervised dimensionality reduction algorithm, i.e., t-distributed stochastic neighboring embedding (t-SNE).Due to this study lacking cell surface markers, we cannot distinguish different CD8+ CTLs with t-SNE as follows:+ CTLs.Gather a series of information on phenotypic and functional features, including cell size, granularity, CD8 expression, and PD-1 or PD-L1 expression on each cell from all volunteers’ CD8+ CTLs with similar features.Cluster CD8+ CTL clusters with different phenotypic features.Separate CD8+ CTL clusters with different densities on a two-axis panel (t-SNE1 and t-SNE2).Visualize CD8The t-SNE algorithm maps high-dimensional data points to a low-dimensional space; data points with similar characteristics are clustered on a two-dimensional (2D) plane with a high probability . We gate+ T-cell subsets with the following various variables: FSC (cell size), SSC (granularity), CD8 expression, and PD-1/PD-L1 expression. As shown in Fig. high-expressing CTLs, and more CD8high CTLs , and each group had its own characteristic features in terms of the cellular size, granularity, CD8 expression, and PD-1/PD-L1 expression  T cells decreased with progression, possibly resulting from T-cell exhaustion, which causes or affects AD deterioration. AD patients are infected by various viruses and/or bacteria during aging. Each infection episode exhausts the CD8+ T cells of AD patients, which correlates with cognitive decline. The above findings might also suggest a possible mechanism in delirium, which is common in elderly and demented patients during acute inflammation and infection. CD8+ T-cell exhaustion, in addition to the previous neurotransmitter dysregulation hypothesis, could provide newer insight into the mechanism of delirium in elderly individuals [Our study also uncovered an upregulation of PD-L1 expression with disease progression. PD-L1 can be induced by inflammatory molecules and intracellular metabolites , 71. Sucividuals .+ T-cell subset with AD progression, which was not discovered using a conventional flow cytometric analysis [+ T cells are involved in the late stage of T-cell activation since PD-1 acts as a senescence marker of T-cell differentiation and is prone to be inhibited by the PD-1/PD-L1 pathway in moderate AD patients. It will be necessary for future work to further verify this hypothesis by observing the T-cell activation status  after treatment with an anti-PD-1 monoclonal antibody in AD patients and comparing it with cognitive changes. In addition, our t-SNE computational results might serve as preliminary references for PD-1/PD-L1 blockade therapies for the treatment of patients at different AD stages. Moreover, an upregulation of CD8 expression and a larger cell size were found in the mild AD stage. CD8 expressed on T cells acts as a coreceptor for the T-cell receptor (TCR), which is required for a stable complex between the TCR and MHC class I, enhancing peptide sensitivity by one million-fold or more [+ T-cell sensitivity for antigens and the inefficiency of antigen-specific cytotoxicity [+ T cells have a significantly larger diameter than resting cells [+ T cells might be active in patients with mild AD.We additionally implemented the t-SNE algorithm and identified, to the best of our knowledge, for the first time that PD-1 expression is reshaped in the CD8analysis . With th or more . Depletitoxicity . In adding cells . These fThe T-cell-expressing PD-1/PD-L1 reshaping observed in this study suggests a negative immune regulation status in AD progression. This status change in AD patients might indicate concurrent inflammation states, such as viral/bacterial infections, postinfectious inflammation, and probable metabolic syndrome. Mediation of the PD-1/PD-L1 pathway might restore T-cell activity, thereby triggering immune responses and alleviating AD pathology.negative CD8+ T cells and the numerical decline in CD8+ T cells with AD progression. Here, we propose that the cytotoxicity of CD8+ T cells destroys neuronal cells in AD, thereby exacerbating the disease. In early-stage AD, peripheral immunity is shaped to an immunosuppressive-like environment by upregulating PD-L1 on immune cells. However, the downregulation of PD-1 on CD8+ T cells enables the evasion of PD-L1 inhibition, thereby attacking neurons. Similarly, the activation of specific T cells was related to the mild disease stage in AD patients in a previous study [+ T cells declines due to exhaustion. Concurrently, the negative feedback in patients’ peripheral blood suppresses CD8+ T cells with high PD-L1 expression and restores PD-1 expression in CD8+ T cells. Despite T-cell suppression, the CNS is damaged, leading to cognitive decline/impairment. The proposed model can be preliminarily validated by identifying the clonal expansion of AD-associated antigen-specific T cells and examining the cytotoxicity mediated by the antigen expressed on neuron cells. Following the preceding validation, we can further confirm this model by applying anti-CD3 antibodies in an AD murine model, which selectively depletes pathogenic T cells while preserving regulatory T cells [Cytotoxic T cells have been delineated as participants in developing neurodegenerative diseases, such as Parkinson’s disease and multiple sclerosis , 76, 77.us study . In late T cells  and obse+ T-cell subsets might maintain such a steady state. Nevertheless, with the diminished number of CD8+ T cells and the upregulation of PD-L1, the peripheral blood reshapes into an immunocompromised environment and causes neurogenesis malfunction. With disease progression, CD8+CD25+ (IL-2RA) T cells undergo exhaustion accompanied by increased PD-L1-expressing CD4+ or CD8+ T cells. Without normal neurogenesis, damaged neurons exhibit an aberrant transmission of electrical signals [In contrast, our findings provide another AD pathogenesis model, i.e., dysfunctional immunity cannot preserve neuroregeneration. Neurons undergo damage for several reasons, including oxidative stress, inflammation , mitocho signals , 90 and  signals , which sFrom the perspective of practical neurology, we enrolled outpatients who met the clinical NIA-AA 2011 criteria/DSM-5 criteria for probable Alzheimer’s disease with a typical clinical course, brain MRI findings, and neurocognitive tests. However, the lack of potential biomarkers (amyloid/tau) in cerebrospinal fluid (CSF) and blood was our study limitation, especially while we attempted to bridge the clinical setting to basic research.U test and Kruskal‒Wallis test). Finally, long-term follow-up data would be mandatory to observe temporal relationships.As a cross-sectional observational study, this study precludes causal inference, which is required to strengthen the connection between our data and clinical practice. To mitigate this limitation, a combination of in vitro and/or in vivo functional assays, medical imaging analyses , and medical records could be adopted in further research. Moreover, we had a small sample size and uneven sex distribution due to difficult enrollment. Our study was subject to sampling errors, leading to incorrect data evaluation and interpretation. Despite this limitation, this study revealed significant alterations in the PD-1/PD-L1 expression status in AD patients by utilizing nonparametric statistical methods , and these high-dimensional data points were visualized with the t-SNE algorithm. Our t-SNE analyses found a characteristic change in CD8+ T cells at different AD stages and identified a decreased PD-1negative CD8+ T-cell population in moderate AD. This finding implies that the sensitivity of T cells to the inhibitory ligand PD-L1 is higher in moderate AD. The findings in our study provide new evidence that the regulation of immune homeostasis is connected to AD and its different stages. Furthermore, PD-1/PD-L1 on peripheral T cells might be a potential factor interacting with AD; it could be rational to consider immune checkpoint blockade as a potential treatment for AD.We demonstrated an alteration in PD-1/PD-L1 (L2) expression in AD patients’ peripheral T-cell subsets, which changed the regulatory mechanism of immune homeostasis in different disease stages. The upregulation of PD-L1 (L2) expression causes probable immunosuppression in AD. In addition, the CD8Peripheral blood samples were collected from 16 AD patient volunteers  and 16 age-matched healthy volunteers  from the National Taiwan University Hospital Hsin-Chu Branch. This study was approved by the National Taiwan University Hospital Hsin-Chu Branch Institutional Review Board (IRB number: 108-008F) and performed in accordance with all relevant ethical regulations. All participants or their legal representatives provided written informed consent. All AD patients met the criteria for “probable AD” by the National Institute on Aging and Alzheimer’s Association (NIA-AA) and “dementia” criteria by the Diagnostic and Statistical Manual of Mental Disorders, fifth edition (DSM-V). Brain magnetic resonance imaging or computed tomography of the AD patients was reviewed, and patients with destructive lesions, such as brain tumors, cerebral infarcts, cerebral hemorrhage, abnormal thyroid function, vitamin B12 deficiency, and folic acid deficiency, were excluded from this study.The Clinical Dementia Rating (CDR) was rated by clinicians to evaluate the AD patients’ cognitive performance and daily function. The CDR global score is a summary of 6 cognitive domains, including memory, orientation, judgment and problem solving, community affairs, home and hobbies, and personal care. Mild AD was defined as CDR = 1, moderate AD was defined as CDR = 2, and severe AD was defined as CDR = 3. To track general changes across different stages of AD, we utilized the Clinical Dementia Rating Scale Sum of Boxes (CDR-SOB). CDR-SOB was derived by adding six CDR domain box scores with a sum minimum of 0 and maximum of 18.The Mini-Mental State Examination (MMSE) is used worldwide to evaluate cognitive function with a total score of 30. A score less than 23 on the MMSE is considered cognitive impairment. However, the educational level is a significant bias in evaluating AD patients’ cognitive states. In our study, the average years of education was only 4.35 years. The Taiwanese population-based cutoff value score for MMSE by education years was defined as 17 for illiterate, 19 for less than 4 years, 24 for 6–8 years, and 27 for 9–12 years of education . Hence, 4Cl, 10 mM NaHCO3, 1 mM EDTA) at room temperature. After removing the RBCs, peripheral blood mononuclear cells (PBMCs) were harvested, washed, and resuspended in phosphate-buffered saline (PBS).Whole blood was drawn with a VACUETTE® blood collection tube coated with lithium heparin anticoagulant . Red blood cells (RBCs) were lysed using RBC lysis buffer  included fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD3 (OKT3), CD4 (OKT4), CD8 (HIT8a), phycoerythrin (PE)-conjugated mouse anti-human CD3 (OKT3), CD4 (OKT4), goat anti-mouse IgG Fc (Poly4053), allophycocyanin-conjugated mouse anti-human CD25 (BC96), CD45 (2D1), PE/Cy5-conjugated mouse anti-human CD56 (NCAM) (5.1H11), mouse anti-human PD-L1 (29E.2A3), PD-L2 (24F.10C12), and PD-1 (NAT105).PBMCs were stained with monoclonal antibodies for membrane labeling and incubated at 4 °C in the dark for an hour. Following staining and centrifugation, the cell pellets were washed, resuspended in PBS, and fixed with 0.5% paraformaldehyde/PBS. PBMCs were acquired on an Accuri C6 Flow Cytometer  equipped with a diode laser and a solid-state laser operating at 640 nm and 488 nm. The samples were first run using single fluorochrome staining for color compensation. Singlet events were first gated on the forward-scatter area (FSC-A) and forward-scatter height (FSC-H) and then gated on FSC-H and side-scatter height (SSC-H) properties of lymphocytes and monocytes. Lymphocyte populations were gated on CD3, CD4, CD8, CD56, and CD25 expression. FlowJo software  was used to analyze the flow cytometric results. Since the distributions of the flow cytometric fluorescence intensity of immune checkpoints expressed on PBMCs rarely align with a normal distribution, we utilized the median fluorescence intensity (MFI) in the subsequent analysis.t-distributed stochastic neighbor embedding (t-SNE) is a machine learning algorithm developed by Laurens van der Maaten and Geoffrey Hinton and is a technique used for dimensionality reduction . The t-SIn this study, t-SNE was implemented using the ‘t-SNE’ plugin in FlowJo software. The Flow Cytometry Standard (FCS) 3.0 files from both AD patients and healthy volunteers were imported, concatenated to a single FCS file, and visualized with the t-SNE plugin in FlowJo. The parameters were used at the default values  in all analyses.FlowSOM is an algorithm used for clustering and visualizing high-dimensional flow cytometry datasets. The FlowSOM algorithm uses a self-organizing map (SOM), an unsupervised technique for clustering and dimensionality reduction . In thisU test  and Kruskal–Wallis test  due to small-size clinical data. The correlations between the immunological difference and the clinical neuropsychiatric indicators were analyzed with a Pearson correlation analysis. The normality of the data and autocorrelation in residuals were confirmed by the Shapiro‒Wilk test and detected by the Durbin-Watson test, respectively, before the linear regression analysis. All statistical tests and p values are two-tailed. All figures with statistical analyses were generated with GraphPad Prism 9 .The statistical analyses were performed using SPSS statistical software . The immunological difference between the AD patients and healthy volunteers was analyzed with nonparametric tests as follows: an independent-samples Mann‒Whitney Additional file 1: Figure 1. Age-matched elderly human volunteers. (A) All subjects in this study were age-matched and included 16 healthy volunteers and 16 AD patients. (B) Patients with mild AD  and moderate AD  were age-matched. The data are shown as the mean ± SD. A Mann‒Whitney U test was used for the comparison. ns: not significant. Figure 2. PD-1-expressing T-cell subsets in healthy volunteers and AD patients. The percentage of the PD-1+ population in its subset in healthy volunteers and AD patients. A Mann‒Whitney U test was used to compare healthy volunteers (n = 16) and AD patients (n = 16); median values are indicated by thick black lines in the scatter plots. Figure 3. PD-L2-expressing T-cell proportions in healthy volunteers and AD patients.  The percentage of the PD-L2+ population in its subset and PBMCs in healthy volunteers and AD patients. A Mann‒Whitney U test was used to compare healthy volunteers (n = 16) and AD patients (n = 16); median values are indicated by thick black lines in the scatter plots. Figure 4. PD-1 expression on T-cell subsets in different AD stages. The median fluorescence intensity of PD-1 expressed on T-cell subsets in healthy volunteers, mild AD patients, and moderate AD patients. A Mann‒Whitney U test was used to compare healthy volunteers (n = 16), mild AD patients (n = 10) and moderate AD patients (n = 6); median values are indicated by thick black lines in the scatter plots. Figure 5. PD-L2 expression on T-cell subsets in different AD stages. The median fluorescence intensity of PD-L2 expressed on T-cell subsets in healthy volunteers, mild AD patients, and moderate AD patients. A Mann‒Whitney U test was used to compare healthy volunteers (n = 16), mild AD patients (n = 10) and moderate AD patients (n = 6); median values are indicated by thick black lines in the scatter plots.


The combination of urolithin B with first-line therapeutic drugs improved the colorectal intestinal hematochezia by shaping gut microbiota, providing a strategy for the treatment of immunotherapy treatment for CRC treatments. UB combined with anti-PD-1 antibody could inhibit the growth of colon cancer. Urolithin B may thus contribute to anticancer treatments and provide a high immune response microenvironment for CRC patients' further immunotherapy.Colorectal cancer has risen to the third occurring cancer in the world. Fluorouracil (5-Fu), oxaliplatin, and cisplatin are the most effective chemotherapeutic agents for clinical chemotherapy. Nevertheless, due to chemotherapeutic drug resistance, the survival rate of patients with CRC remains very low. In this study, we used the inflammation-induced or mutation-family-inherited murine CRC models to study the anticancer and immunotherapy effects of urolithin B (UB), the final metabolite of polyphenols in the gastrointestinal tract. The label-free proteomics analysis and the gene ontology (GO) classifications were used to test and analyze the proteins affected by UB. And 16S rDNA sequencing and flow cytometry were utilized to uncover gut microbiome composition and immune defense improved by UB administration. The results indicated that urolithin B prevents colorectal carcinogenesis by remodeling gut microbial and tumor immune microenvironments, such as HLA-B, NK cells, regulatory T cells, and  Colorectal cancer (CRC) is the third most prominent cause of cancer-associated mortality worldwide . The Worγδ T cell, is mainly distributed in the intestinal epithelium and has a protective effect on the intestine [Alloprevotella is the pathogenic bacteria in CRC development [Akkermansia muciniphila [Akkermansia muciniphila was related to the favorable therapeutic efficacy of immunotherapy, and Bacteroides has been considered an unfavorable bacteria [Notably, in the tumor microenvironment of colorectal cancer, the increased proportion of regulatory T (Treg) cells of colorectal patients is the key factor of tumor immune escape , 12. Besntestine , 16. In elopment , 18, andiniphila –22 is thbacteria , 24.Dietary supplements, such as polyphenols, provide multiple favorable benefits, including anticarcinogenic benefits , 26. UroTherefore, in this study, we investigated the cancer-preventive properties of urolithin B on colorectal carcinogenesis. The regulations of gut microbiota, tumor immune microenvironment, and PD-L1 were also investigated.Male C57BL/6 mice, 8 weeks, weighing 18-20 g, purchased from Shanghai Model Organisms Center (Inc) were used, maintained in Chinese Experimental Animal Resources Research Institute for food and drug control . All groups were fed with a regular diet of standard feedstuff freely. The studies were approved by the Experimental Animal Management Committee at Capital Medical University (AEEI-2021-254).min/+ mice with a C57BL/6 background were obtained from Shanghai Model Organisms Center (Inc) and maintained in a standard environment in Chinese Experimental Animal Resources Research Institute for food and drug control. The progeny of Apcmin/+ intercrosses were genotyped by PCR analysis with DNA isolated from the tail with the Genomic DNA Mini Preparation Kit with Spin Column  using the following two primers: 5′-GTGCAGCAGCTTTAAGGAA-3′ and 5′-AATGGAACTCGGTGGTAGA-3′. The DNA sequencing was performed with the former primer: GACATGATGATAGTAGGTCAGACAATTTCAATACTGGAAACATGACTGTTCTTTCACCATATTTAAATACTACGGTATTGCCCAGCTCTTCTTCCTCAAGGGGAAGTTTAGACAGTTCTCGTTCTGAGAAAGACAGAAGTT (T/A) GGAGAGAGAGCGAGGTATTGGCCTCAGTGCTTACCATCCAACAACAGAAAATGCAGGAACCTCATCAAAACGAGGTCTGCAGATCACTACCACTGCAGCCCAGATAGCCAAAGTTATGGAAGAAGTATCAGCCATTCATACCTCCCAGGACGACA (wild type-T and mutant-A).ApcMale C57BL/6 mice were used at the age of 8 weeks, administrated with 12.5 mg/kg AOM . One week later, 2.5% DSS  was given in drinking water for one week and then regular drinking water for two weeks. The DSS cycle was repeated three times, and the experiment lasted for 70 days.min/+ mice were used at the age of 8 weeks, with 2.5% DSS  given in the drinking water for one week followed by regular drinking water for two weeks. The experiment cycle was repeated twice and lasted for 50 days.APCμg anti-PD-1  once a week with or without 20 mg/kg UB.Treatment groups of low, middle, and high or positive control were given a gavage of 10 mg/kg, 20 mg/kg, and 40 mg/kg urolithin B or 30 mg/kg capecitabine dissolved in 0.25% CMC-Na, and the combination group was given both 30 mg/kg capecitabine and 20 mg/kg UB, while the model group was given the same volume of distilled water, with no administration to the control group. For adjuvant immunotherapy, the mice were administrated with 100 All mice were weighed regularly. In the end, collect the colorectum of the mice after sacrifice and wash with PBS, to calculate tumor load and prepare them for further experiments.Tumor load was calculated according to the diameter of the tumors. The tumor load of each mouse is equal to the sum of the average diameters of all tumors on its large intestine.μM urolithin B. Next, cells were lysed, and the concentration of extracted protein was tested by the BCA protein assay kit . Lyophilizate of the proteins was redissolved, centrifuged, and isolated through the C18 column, tested by LC-MS/MS, followed by STRING network analysis showing proteins regulated by UB.SW480 cells were treated with 0.15 or 15 Feces of the mice were collected in the last few days of the test for 0.2-0.4 g/tube (about 3 grains) and stored at -80°C for further detection. Gut microbiota was analyzed by 16S rDNA sequencing, and the data were analyzed on the online platform of the Majorbio Cloud Platform . The operational taxonomic units (OUT) were used for alpha diversity analysis and beta diversity analysis and linear discriminant analysis (LDA).The colon and rectum with adenoid tumor were collected and digested with the Lamina Propria Dissociation Kit, mouse (130-097-410), according to the manufacturer's direction.γ/δ , NK1.1 , F4/80 , FOXP3 , CD25 , granzyme B , CD16/32 , CD11b , CD11c , Ly-6C , and Ly-6G . After being blocked with TruStain FcX (anti-mouse CD16/32) antibody, cells were stained with antibodies for cell surface markers and then the intracellular biomarker. True-Nuclear™ Transcription Factor Buffer Set  and fixation buffer  were used according to the protocol recommended by BioLegend. Before that, BD Compensation Beads  were used to make the best fluorescence compensation settings for multicolor flow cytometric analysis.The spleens were ground into single cells and lysed with red blood cell lysis buffer  on ice after centrifugation; the followed steps were done according to the instruction. All the procedures were carried out according to BD procedure steps with anti-mouse antibody for CD45 , CD3 , CD4 , CD8a , TCRMouse CRC tissues were embedded in paraffin and made into slicers. Then, the slicers were deparaffinized in xylene and hydrated into water with graded ethanol .2O2 in methanol at 37°C for 20 minutes. After being blocked with goat serum  in PBST for 1 h, tissue slides were incubated at 4°C overnight with primary antibodies as follows: anti-PD-L1/CD274 antibody . The sections were then washed with PBST for 3 times and incubated with goat anti-rabbit IgG antibody (HRP)  at 25°C for 30 minutes. Then, the slicers were treated with the DAB Substrate Kit (20x)  and then counterstained with hematoxylin. All the slides were imaged at the end for 40x amplification.For HE or PCNA staining, procedures were performed according to instructions  provided by the manufacturer. For the immunohistochemical experiment, slicers were pretreated with antigen retrieval with citrate (G1202-250ML) or Tris-EDTA (G1203-250ML) and then incubated with PBST (PBS containing 0.3% Triton X-100) for 20 minutes and incubated with 3% Hμg) were separated with 10% SDS-PAGE and transferred onto a PVDF membrane (Millipore). The membrane was then blocked with 5% skimmed milk powder  dissolved in 1x PBST  for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. The prime antibodies including anti-HLA-B antibody  was diluted in blocking solution . And β-actin  was used as an internal control. After that, HRP-labeled goat anti-rabbit IgG  was used as the secondary antibody to be incubated with the membrane. Finally, the Immobilon Western HRP Substrate  was used for the chemiluminescence detection of the bounded antibody.Colon tissues were isolated and dissected from mice, homogenized, and lysed with ice-cold RIPA Lysis Buffer  by a high-speed low-temperature tissue grinding machine, then centrifuged at 8000g for 10 min at 4°C. The protein concentrations were measured using the BCA protein assay kit (Beyotime) according to the instructions. Then, the total proteins  or capecitabine (p < 0.001) could reduce the adenoma burden of AOM/DSS CRC mice  model  and the RC model , the furRC model . CompareRC model . In addiRC model  and Apcmin/+ CRC  models. CRC mice . AlthougCRC mice , the UB CRC mice .min/+ CRC model group, urolithin B could obviously reduce the tumor load and effectively inhibit colorectal carcinogenesis.The colons with adenoma from AOM/DSS mice were sliced and stained with hematoxylin and eosin  or staint-test (p < 0.05) between samples from two groups were used to identify differentially expressed proteins. A total of 914 and 960 upregulated proteins in response to 0.15 and 15 μM UB treatments, respectively, were identified by label-free proteomics  and genus (p < 0.05) levels; after the combination of capecitabine and urolithin B, the abundance of Verrucomicrobiota (p < 0.05) and Desulfobacterota (p < 0.01) was increased on the phylum level, the abundance of Akkermansiaceae (p < 0.05) was increased at the family level, and the abundance of Alloprevotella was decreased significantly (p < 0.05) at the genus level.A comparison of gut microbial diversity indexes was carried out by 16S rDNA sequencing and bioinformatic diversity analysis. g_Dubosiella, f_norank_o_Clostridia UCG-014, o_Clostridia UCG-014, g_norank_f_orank_o_Clostridia UCG-014, and g_Desulfovibrio. Compared with the AOM/DSS group, UB decreased the abundance of Bacteroidaceae and Bacteroides, and so did the UB combined with capecitabine group  was analyzed by flow cytometry.Natural killer cells (NK cells) are one of the primary cells of the innate immune system. NK cells do not need to bind to related antigens and can directly kill tumor cells with missing protomolecules. p < 0.05, γδ TCR expression was significantly increased from 2.98 ± 2.14 to 8.05 + 3.94  are immunosuppressive in the tumor microenvironment of colon cancer. UB is a high content of multiple intestinal metabolites in food, natural drugs, and traditional Chinese medicine. It is produced by intestinal metabolism in the gastrointestinal tract. This study shows that UB can regulate the tumor microenvironment of colon cancer, upregulate NK cells and Akkermansia muciniphila is immunomodulatory bacteria [Akkermansia muciniphila was related to the favorable therapeutic efficacy of immunotherapy, and Bacteroides has been considered unfavorable bacteria. The high abundance of those immunomodulatory bacteria in the intestine can upregulate the immune response and improve the treatment rate. Akkermansia muciniphila (A. muciniphila), one of the members of immunomodulatory bacteria, is an anaerobic gram-negative strain that colonizes the mucus layer of mucin-rich ileum and colon. A. muciniphila with appropriate abundance provides nutritional support for intestinal epithelial cells, which can protect an intestinal mucosal barrier, enhance intestinal epithelial integrity, reduce inflammatory response, improve glucose and lipid metabolism, and participate in immune response [A. muciniphila in tumor patients was correlated with the immune response, and the abundance of A. muciniphila increased in patients who responded to immunotherapy [A. muciniphila can enhance the immune response to chemotherapeutic drugs [A. muciniphila preclinical tumor model and anticancer immune response in cancer patients [A. muciniphila level and downregulated Bacteroidales and Alloprevotella. So, in the primary site of intestinal metabolism, urolithin may upregulate the abundance of immunomodulatory bacteria and further regulate the immune response. Upregulating the abundance of immunomodulatory bacteria and thus regulating the immune response may be one of the mechanisms of urolithin's anti-colon cancer effect.Intestinal flora in the tumor microenvironment of colon cancer is related to immune response. The immunogenicity of colon cancer is weak, and the pathogenic intestinal bacteria increase after colon cancer surgery and chemotherapy –24. The bacteria –22. Akkeresponse –51. The otherapy –22. It iic drugs , 22. Roupatients . In the A. muciniphila, a member of immunomodulatory bacteria, is related to immune response, some studies have reported that A. muciniphila in the feces exerted specific effects on colorectal cancer [A. muciniphila regulates immune response in tumor patients [Akkermansiaceae was significantly lower in the CRC group [A. muciniphila significantly inhibited the tumorigenic effect of another flora [A. muciniphila in mice after oral administration of urolithin B by using two animal models, AOM/DSS chronic inflammation induced and APCmin/+ adenoma colon cancer model. The tumor decreased significantly after the administration of urolithin B, followed by the downregulation of PD-L1 and increase of the expression of A. muciniphila. These results support the enhanced immune response of A. muciniphila in colon cancer.Although the abundance of l cancer , 53. Howpatients , 54–59. RC group , and theer flora . In the Alloprevotella [Alloprevotella not only is related to the development of colon cancer but also participates in the immunotherapy of PD-1 of colon cancer. Our present results show that the combination of urolithin B with first-line therapeutic drugs can downregulate the expression of Alloprevotella and inhibit the expression of PD-L1. Notably, UB combined with anti-PD-1 antibody could inhibit the growth of colon cancer, which further provide a strategy for the treatment of immunotherapy for CRC treatments.Alloprevotella, which is correlated with higher levels of chromosomal aberrations , might cevotella . Those rOverall, the prevention of colorectal carcinogenesis of urolithin B is attributed to the shape of gut microbial and tumor immune microenvironment. The anticancer concentration of urolithin B is consistent with the GI concentration of polyphenols, suggesting the protective effect of polyphenols against diseases.In summary, we showed that urolithin B, the final metabolite of polyphenols in the GI tract, prevented colorectal carcinogenesis by remodeling the gut microbial and tumor immune microenvironment . UB play


Sphingosine-1-phosphate (S1P) is involved in inflammatory signaling/s associated with the development of respiratory disorders, including cancer. However, the underlying mechanism/s are still elusive. The aim of this study was to investigate the role of S1P on circulating blood cells obtained from healthy volunteers and non-small cell lung cancer (NSCLC) patients. To pursue our goal, peripheral blood mononuclear cells (PBMCs) were isolated and stimulated with S1P. We found that the administration of S1P did not induce healthy PBMCs to release pro-inflammatory cytokines. In sharp contrast, S1P significantly increased the levels of TNF-α and IL-6 from lung cancer-derived PBMCs. This effect was S1P receptor 3 (S1PR3)-dependent. The pharmacological blockade of ceramidase and sphingosine kinases (SPHKs), key enzymes for S1P synthesis, completely reduced the release of both TNF-α and IL-6 after S1P addition on lung cancer-derived PBMCs. Interestingly, S1P-induced IL-6, but not TNF-α, release from lung cancer-derived PBMCs was mTOR- and K-Ras-dependent, while NF-κB was not involved. These data identify S1P as a bioactive lipid mediator in a chronic inflammation-driven diseases such as NSCLC. In particular, the higher presence of S1P could orchestrate the cytokine milieu in NSCLC, highlighting S1P as a pro-tumor driver. The sphingosine-1-phosphate (S1P) is a bioactive lipid mediator that acts either as an intracellular second messenger or as a ligand for its membrane receptors (S1PRs), carrying out biological effects in both physiological and pathological conditions [S1P is generated by ceramidase, an enzyme that converts ceramide into sphingosine, and then phosphorylated in S1P by sphingosine kinases (SPHKs), SPHK I and/or SPHK II . In partIn our recent study, we demonstrated that the activation of Toll-like receptor 9 (TLR9) induced the synthesis of endogenous S1P in lung adenocarcinoma cells . The endn = 29) did not have any pathologies or haematological alterations. The lung cancer patients’ group (n = 54) consisted of a comparable number of male and female patients diagnosed with adenocarcinoma or squamous cell carcinoma. The age of enrolled volunteers and patients had a mean of 50 ± 10 years old. Blood samples were collected and used within 24 h. PBMCs were isolated as described below.Healthy volunteers (H) and lung cancer patients (LK) were recruited at the “Monaldi-Azienda Ospedaliera (AORN)-Ospedale dei Colli” Hospital in Naples, Italy, according to the Review Board which approved the project and the patients’ informed consent. The experimental protocol was performed in accordance with the guidelines and regulations provided by the Ethical Committee of the hospital (protocol no. 1254/2014). The healthy volunteers’ group  and treated for 8 h. PBMCs were incubated with sphingosine-1-phosphate . The concentration of S1P was chosen according to our preliminary data. We tested a concentration-dependent curve of S1P (0.1 nM up to 3 µM) and evaluated cytokine release. We found that the concentration-dependent administration of S1P described a bell-shaped curve, showing that the optimal concentration of S1P was 10 nM.PBMCs were isolated according to Ficoll’s protocol as already reported  and cultIn another set of experiments, PBMCs were also treated with ceramidase inhibitor , TY52156, a S1PR3 antagonist , SKI II, a selective inhibitor of sphingosine kinases , PF-543, a sphingosine-competitive inhibitor of sphingosine kinase I, SPHK I , ABC294640 (Opaganib), a selective inhibitor of sphingosine kinase II, SPHK II , FTI-276, a K-Ras inhibitor , Rapamycin, a potent and specific mTOR inhibitor , MG132, a proteasome inhibitor . The concentration of all substances was chosen on the basis of the existing literature and on our previous experiments/data [TNF-α and IL-6 were measured in cell-free supernatants, obtained after 8 h of cell treatment, by means of a commercially available enzyme-linked immunosorbent assay kit (ELISAs) . The absorbance wavelength was 450 nm. Cytokine levels were expressed as pg/mL.http://imagej.nih.gov/ij . The expression of S1PR3  and of ceramidase active form  were evaluated in healthy volunteers (H)- and lung cancer (LK)-derived PBMCs. Furthermore, ceramidase, phospho-NF-κB p65 , phospho-IkBα , phopho-Erk  and phospho-Akt  expression were evaluated after one hour treatment of PBMCs with S1P (10 nM). Glyceraldehyde-3-Phosphate Dehydrogenase  was used as loading control. Data were analysed by means of ImageJ software 1.53a p values less than 0.05 were considered significant. The statistical analysis was performed by using GraphPad prism 9.3.0 version .Data are reported as median and represented as scatter dot plots. Statistical differences were assessed with two-tailed Wilcoxon matched-pairs signed rank test. m) of increment, in terms of cytokine release after S1P stimulation, was of 9.7 for TNF-α  associated with the development of lung disorders , althougor TNF-α E and of or TNF-α F. In addBecause S1P carries out its biological effect by interacting with its surface receptors (S1PRs) . WhereasThe ceramidase is an enzyme that converts ceramide into sphingosine, which is then phosphorylated into S1P by SPHK I/II [p = 0.0037) (p = 0.0087) (p = 0.018) (Sphingosine kinase I and II (SPHK I/II) are biological lipid kinases catalyzing ATP-dependent phosphorylation of sphingosine to S1P. SPHK I localizes mostly in the cytoplasm and migrates to the plasma membrane upon phosphorylation. It is involved in cancer cell proliferation and invasion and is correlated with severity and poor prognosis of cancers, and chemotherapy resistance . SPHK II 0.0037) C and IL- 0.0087) D. Instea= 0.018) F. TogethIt is known that the interaction of S1P with its receptors activates oncogenic kinases, strengthening pro-survival signaling, implying the activation of PI3K/Akt/mTOR, NF-κB and/or K-Ras/Erk pathway ,17. FurtAlthough S1P was involved in the activation of NF-κB signaling, in that it was able to induce an overexpression of phospho-IkBα A and phoAltogether, these data show that S1P-induced IL-6 release from LK-derived PBMCs is mTOR and K-Ras-dependent, differently than TNF-α, which release is likely regulated by other(s) mechanism(s), which need further investigation.S1P exacerbates the pro-inflammatory milieu by inducing IL-6 and TNF-α release from LK-derived PBMCs in a S1PR3-dependent manner;The activation of S1PR3 by the exogenous S1P induces the release of TNF-α in a SPHK I-dependent manner, and of IL-6 via SPHK I/II;S1P-induced IL-6, but not TNF-α, release from PBMCs of lung cancer patients is mTOR- and K-Ras-, but not NF-κB-dependent .The involvement of S1P in cancer is currently known , howeverThese results highlight a molecular mechanism exploited by S1P in circulating cells of lung cancer patients. In our previous studies, we found that lung cancer epithelial cells were responsive to exogenous S1P, but the major source of TNF-α was related to the activation of TLR9. In particular, TLR9 activation in lung cancer epithelial cells increased the release of TNF-α, but not of IL-6, through an imbalance of the ceramide/S1P rheostat in favor of S1P . The actTNF-α and IL-6 are two key inflammatory cytokines linked to chronic inflammatory diseases and cancer . A largeAnother important issue in this study is that the outer S1P can lead to the production of endogenous S1P in LK-derived PBMCs, increasing IL-6 release in a ceramidase, SPHK II-dependent manner. On the contrary, ceramidase and SPHK I were involved in TNF-α release. In support, Beckman and colleagues and others proved that ceramidase is able to induce oncogenic Akt/mTOR and K-Ras/Erk signaling in different types of cancers ,24,25. IOur findings open up new scenarios on the physiological and pathological role of S1P and shed light on a novel and never investigated signaling network between lung oncogenesis and lung cancer-associated inflammation in a S1P-dependent manner. We believe that the identification of S1P-induced inflammatory pathways in circulating cells of lung cancer patients together with its role in structural tumor cells can open new perspectives for drug discovery for lung cancer patients.


The primary objective of the study was to assess safety, secondary objectives included pharmacokinetics of GC007F cells in patients with R/R B-ALL and preliminary efficacy. We were able to demonstrate in preclinical studies that GC007F cells exhibited better proliferation and tumor killing than conventional CAR-T (C-CAR-T) cells. In this investigator-initiated study all 18 efficacy-evaluable patients achieved a complete remission (CR)  by day 28, with 17 of the patients (94.4%) achieving CR with minimal residual disease (MRD) negative. Fifteen (83.3%) remained disease free at the 3-month assessment, 14 patients (77.8%) maintaining MRD negative at month 3. Among all 21 enrolled patients, the median peak of CAR-T cell was on day 10, with a median peak copy number of 104899.5/µg DNA and a median persistence period of 56 days (range: 7–327 days). The incidence of cytokine release syndrome (CRS) was 95.2% (n = 20), with severe CRS occurring in 52.4% (n = 11) of the patients. Six patients (28.6%) developed neurotoxicity of any grade. GC007F demonstrated superior expansion capacity and a less exhausted phenotype as compared to (C-CAR-T) cells. Moreover, this first-in-human clinical study showed that the novel, next-day manufacturing FasTCAR-T cells was feasible with a manageable toxicity profile in patients with R/R B-ALL.Chimeric antigen receptor-engineered T (CAR-T) cells have shown promising efficacy in patients with relapsed/refractory B cell acute lymphoblastic leukemia (R/R B-ALL). However, challenges remain including long manufacturing processes that need to be overcome. We presented the CD19-targeting CAR-T cell product GC007F manufactured next-day (FasTCAR-T cells) and administered to patients with R/R B-ALL. A total of 21 patients over 14 years of age with CD19 Chimeric antigen receptor-engineered T cells (CAR-T cells) represent a major advance in cancer treatment –6. To da+ relapsed/refractory B cell acute lymphoblastic leukemia (R/R B-ALL).In this study, patients were treated with a novel type of CAR-T cells manufactured on a novel manufacturing platform (FasTCAR) with next-day manufacturing - GC007F cells. Through a series of in vitro and in vivo preclinical experiments, we compared the novel GC007F cells to C-CAR-T cells in regard to expansion capability, phenotype, and potency in eliminating B-ALL. Based on the results of these preclinical studies, a multicenter clinical study was initiated to determine the safety and efficacy of CD19-directed GC007F cells in treating patients with CD19The mouse FMC63 anti-CD19 single-chain variable fragment (scFv) was inserted into a second-generation CAR cassette containing a CD8α signal peptide, a CD8α hinge region, a CD8α transmembrane domain, a CD28 costimulatory domain, and a CD3ζ activation domain. This CAR construct is herein called CD19-CAR. A FLAG tag (DYKDDDDK) was inserted into CD19-CAR between the scFv and the hinge region for detection by flow cytometry.2.T cells were isolated, and GC007F cells and C-CAR-T cells were generated as previously reported . Periphe™ Automated Cell Counter according to the manufacturer’s instructions. The absolute number of CAR-T cells during expansion was calculated based on the percentage of CAR-positive cells determined using flow cytometry three times. To compare the stemness and memory features of GC007F cells and C-CAR-T cells, flow cytometry was used to detect the expression of CD45RO and CD62L. Central memory T cells (TCM cells) were defined as CD62L+ CD45RO+  cells, and effector memory T cells (TEM cells) were defined as CD62L-CD45RO+  cells. To detect the exhaustion of GC007F cells and C-CAR-T cells, flow cytometry was used to detect the expression of PD-1, Tim3 and LAG3.To evaluate the proliferation of GC007F cells, frozen GC007F cells and C-CAR-T cells were thawed, cultured for 2 days in porous (Corning) plates or flasks and then stimulated with irradiated K562-CD19 cells every 3 days. During expansion, cultured CAR-T cells were split, and the cell viability and number were examined using the NC-200A cytotoxicity assay was performed on an xCELLigence RTCA instrument (ACEA Biosciences) according to the manufacturer’s instructions. Briefly, HeLa-CD19 cells were cocultured with GC007F cells and C-CAR-T cells at various effector-to-target (E/T) ratios in triplicate, and growth inhibition was monitored; the experiment was repeated three times. In a second cytotoxicity assay, luciferase-expressing Nalm6 cells were used as target cells. CAR-T cells and target cells were cocultured in 96-well plates at various effector-to-target ratios for 6 h, and the coculture supernatant was harvested and frozen for further cytokine assays. Surviving target cells were quantified using the Promega One-Glo Luciferase Assay System according to the manufacturer’s instructions. The percent killing was calculated as [(Target cell only wells-CAR-T cell treatment wells)/Target cell only wells] × 100%.The production of the cytokines interferon-γ (IFN-γ) and interleukin (IL)-2 by cultured cells was quantified. The levels of serum cytokines, chemokines and growth factors in clinical samples were measured according to the manufacturer’s instructions. Protocol details are provided in the supplementary material.6/mouse), followed by intravenous infusion of various doses of CD19-targeted CAR-T cells 7 days later. Tumor growth and mouse survival were monitored.Animal studies were performed under protocols approved by the Institutional Animal Care and Use Committees (IACUCs) of the service providers. NOG mice were injected intravenously (i.v.) with Nalm6-Luciferase cells  in patients over 14 years with CD19+ R/R B-ALL. Major inclusion criteria included (1)Male and female patients aged 14 to 70 years were eligible; (2) Patients were diagnosed with R/R B-ALL with any one of the following conditions: (a) relapse, including two or more relapses or relapse after allogeneic hematopoietic stem cell transplantation  with 5% blast cells; (b) refractory disease, including no complete remission (CR) or continuous minimal residual disease (MRD) positivity after two cycles of chemotherapy with gene mutation associated with unfavorable prognosis or with complex karyotype; (c) Philadelphia chromosome (Ph)-positive B-ALL with any one of the following conditions: failure to tolerate tyrosine kinase inhibitors (TKIs), progression after the first and second TKI treatments or unfit for allo-HSCT; (3) Participants with an ECOG performance status ≤1 at study entry were eligible. (4) Patients with an estimated survival time of over three months and (5) adequate main organ function were eligible. The study was approved by the Ethics Committee of the Xinqiao Hospital, and it was conducted according to Good Clinical Practice China and the principles of the Declaration of Helsinki (ChiCTR1900023212). All patients provided written informed consent prior to participation in any study procedures.The clinical study was designed to evaluate the safety and efficacy of three different doses of CAR-T cells (dose level (DL) 1: 0.5 × 109) collected by leukapheresis, the eligible enrolled patients would receive GC007F cell infusion following the FC conditioning regimen (30 mg/m2/day fludarabine and 300 mg/m2/day cyclophosphamide over 2 through 5 days), and then pharmacokinetics were examined. The conditioning regimens were used depending on the blasts percentage, with two days for <5%, three days for 5%−20%, four days for 20%−50%, and five days for over 50%. Bone marrow was collected to evaluate blasts by flow cytometry. Patient response after treatment was assessed according to the National Comprehensive Cancer Network (NCCN) guidelines “Acute Lymphocytic Leukemia Remission Criteria” 2016 V2 edition for treatment response in B-ALL.After peripheral blood mononuclear cell (PBMC) . A bone marrow blast level below 0.01% assessed using multiparameter flow cytometry or 0% BCR/ABL detected by qPCR for Ph-positive B-ALL was defined as minimal residual disease (MRD) negative. The reappearance of MRD positivity or blasts in the blood, bone marrow or an extramedullary site after CR was defined as relapsed disease. Bone marrow was evaluated every month after infusion of GC007F cells.The severity of cytokine release syndrome (CRS) in patients presenting with fever, myalgia, hypotension, hypoxia or organ toxicity was analyzed using the American Society for Blood and Marrow Transplantation (ASBMT) consensus grading system . CRS wasData are presented as mean ± standard deviation or median (minimum-maximum) for continuous variables and number (%) of patients for categorical variables as appropriate. Analysis of variance or Wilcoxon rank sum test was used to compare the characteristics between GC007F cells and C-CAR-T cells. The Kaplan-Meier survival curves were drawn for progression free survival (PFS) and overall survival (OS), respectively. PFS is defined as the time interval from the beginning of GC007F infusion to disease recurrence or death from any cause. OS is defined as the time interval from the beginning of GC007F infusion to death of any cause. The primary objective of the study was to assess the safety and pharmacokinetics of GC007F cells in patients with R/R B-ALL. The secondary objective was to assess efficacy, including CR and duration of response. The median follow-up time was 8 months .P values less than 0.05 at two-sided were considered statistically significant. All the statistical analyses were performed using R 4.0.3 (R Development Core Team), Stata 16.0 (Stata Corp), or GraphPad Prism 9.0 (GraphPad Software).A swimmers plot was provided to display effects of the study treatment on tumor response for individual patients. For serum cytokine data, univariate linear regression model was used to explore their relationship with tumor load or CAR-T cell count or Wilcoxon rank sum test with CRS or neurotoxic effects of grade 3 or above. Additional bar plots or line graphs over nominal time were provided as appropriate. The demographic and baseline characteristics, efficacy data was analyzed based on full analysis set and safety data using safety set. P = 0.0005)  and effector memory T cells . In contrast, TCM cells (73.47% ± 2.85%) predominated among GC007F cells, followed by TEM cells (18.8% ± 1.77%). GC007F cells had a higher TCM cell frequency (P < 0.05) and a lower TEM cell frequency (P < 0.01) than C-CAR-T cells, which indicated that GC007F cells were generally in an earlier stage of T cell differentiation and had a younger phenotype.GC007F cells showed significantly greater expansion than C-CAR-T cells (05) Fig. . We furtP = 0.0104)  Fig. .GC007F cells and C-CAR-T cells selectively killed HeLa-CD19 cells but not HeLa cells, exhibiting comparable cytotoxicity Fig. . This reGC007F cells showed a lower in vitro killing capacity than C-CAR-T cells at a ratio of 5:1 Fig.  and a siThe tumor burdens in the high-dose and medium-dose groups were diminished 11 days after infusion, and the mice in these groups remained in CR with further decreases in their tumor burdens observed at 21 days after infusion, but the medium-dose group exhibited a slightly higher tumor burden than the high-dose group Fig. . When GCP = 0.0479). On day 40 after CAR-T cell infusion, no C-CAR-T cells were detected, but GC007F cells persisted in the peripheral blood.GC007F cells also expanded robustly after infusion into tumor-bearing mice Fig. . On day A total of 21 patients were screened, enrolled and infused with a single infusion of GC007F cells. The median CAR positive rate was 39.5% (15.1% ~ 60.4%). The release criteria for GC007F cells was that the CAR positive rate was ≥10%. Three patients gave up early within 2 months after CAR-T cell infusion. Eighteen patients were available for response assessment. One patient gave up on day 6 due to septic shock and respiratory failure, and another patient gave up on day 9 due to intracranial hemorrhage. A third patient was noncompliant with treatment and experienced severe myelosuppression and infection after CAR-T cell infusion; he gave up on day 44. By day 28, all efficacy-evaluable patients  had achieved CR, and 17 of the 18 patients (94.4%) achieved CR with MRD negative. Sixteen of the patients (83.3%) maintained CR at day 84, while 14 patients (77.8%) maintained MRD negative. Patient characteristics are shown in Table GC007F cell numbers detected by qPCR peaked on day 10 (range: days 7–27) after infusion. The median persistence period of GC007F cells in the peripheral blood was 56 days (range: 7–327 days) after infusion. The longest persistence period was 11.7 months and was ongoing at the last follow-up time point. The median peak CAR copy number was 104899.5/µg DNA (range: 614–504158/µg DNA) Fig. .Fig. 4ThGC007F cell numbers detected by flow cytometry peaked on day 10 (range: days 7–28) after infusion. The median persistence period of CAR-T cells in the peripheral blood was 56 days (range: 7–327 days) after infusion. The longest persistence period was 11.7 months and was still ongoing at the last follow-up time point. The median peak cell number was 158 cells/µl blood (range: 0.32–1031 cells/µl blood) Fig. .For all patients, the peak levels of IL-6, IL-10, tumor necrosis factor (TNF)-α, IFN-γ and granulocyte-macrophage colony-stimulating factor (GM-CSF) were 209.56 pg/mL, 147.485 pg/mL, 16.51 pg/mL, 656.48 pg/mL, and 24.89 pg/mL, respectively Fig. . All levOverall, CRS of any grade occurred in 20 of the 21 patients (95.2%), and CRS grade ≥3 occurred in 11 patients (52.4%). Six patients (28.6%) developed neurotoxicity, with 3 patients (14.3%) grade ≥3. All patients recovered after treatment with Standard of Care. The abnormal results of laboratory tests are shown in Table P = 0.197 and P = 0.312, respectively). Severe CRS was also not related to the CAR-T cell number or disease burden . We did not observe a relationship between neurotoxic effects and the CAR-T cell number or disease burden . Severe neurotoxic effects were also not related to the CAR-T cell number or disease burden .The incidence of CRS was not related to the CAR-T cell number or disease burden (P = 0.037), IL-2 was related to the infused dose of CAR-T cells (P = 0.002), and TNF-α, IL-6, IL-10, IFN-γ, IL-2, GM-CSF, and granzyme B were related to severe manifestations of CRS . No cytokines were related to immune effector cell-associated neurotoxicity syndrome. Multivariate analysis showed that no cytokines were related to severe manifestations of CRS.Univariate analysis showed that the TRAIL level was related to the disease burden . B cells became undetectable in all patients achieving CR regardless of their MRD status. Intravenous immunoglobulin repletion dosed according to patient immunoglobulin levels was used to manage hypogammaglobulinemia. B cell aplasia was sustained for all patients with continuous CR, further indicating that the persisting GC007F cells remained functional.All 18 efficacy-evaluable patients were followed for at least 3 months; 15 of these patients (83.3%) were disease free at the 3-month assessment, and 14 (77.8%) maintained MRD negative at month 3  after GC007F cell treatment. Of the patients who underwent allo-HSCT, 2 patients relapsed at months 4.8 and 6, 1 gave up treatment due to severe infection and respiratory failure during transplant period, 1 was diagnosed with posttransplant lymphoproliferative disorder (PTLD) and received other treatment, 1 gave up treatment due to graft-versus-host disease (GvHD) caused by transplant, and the other 3 patients still maintained MRD- CR. The longest time of disease-free survival following allo-HSCT was 27.3 months.For the 10 patients who did not proceed to transplant, 8 patients relapsed , 1 died of severe infection and septic shock on day 83, and 1 maintained MRD- CR up to the last follow-up. The longest time of disease-free survival among the patients who did not proceed to transplant was 29 months.The clinical application of CAR-T cells is undoubtedly a milestone in the history of cancer treatment. The CR rate of CD19-directed CAR-T cells in the treatment of R/R B-ALL has reached 67–93%. HoweverCM, memory and naïve T cells in culture are related to long-term persistence in vivo [CM cell proportion, and the abilities to proliferate, secrete effector cytokines and antitumor activity were enhanced in our studies [A cellular product with a shorter manufacturing time and larger proportion of younger T cells may significantly enhance effector function and proliferative capacity. Previously, some methods have been used to prevent the differentiation that occurs during T cell expansion, such as Akt inhibition or blockade of Fas-FasL interactions , 22. How in vivo –30. Only in vivo –33. Nota studies . The inh5) also exhibited enhanced proliferation, effector function, and persistence in our clinical study. The median persistence time was 56 days, with a peak copy number of 104899.5/µg DNA in patients, and this time is longer than that reported for C-CAR-T cells [Long-term C-CAR-T cell culture can result in an increase in the disease burden and progression during the waiting period to the point where patients are not eligible for CAR-T cell treatment , 10. Eve-T cells . TherefoIt should also be noted that, the expanded clinical application of FasTCAR platform needs to be further evaluated. For GC007F cells there are more studies needed to determine dose based on current findings. Larger studies are needed to further evaluate the efficacy including PFS and OS. The risk benefit needs to be further characterized including evaluation of preventive strategies to manage CRS. Lastly also duration of response needs to be further evaluated in studies to determine follow in strategies in case of MRD relapse.In summary, our results demonstrate that compared with C-CAR-T cells, GC007F cells produced with the novel FasTCAR platform have a superior expansion capacity and a younger phenotype and are less exhausted. Data generated show preliminary efficacy results in patients with R/R B-ALL that are promising and need further evaluation. Moreover, this first-in-human clinical study, conducted in patients with R/R B-ALL in China, showed that CD19-directed GC007F cell therapy were effective in eliminating CD19 positive B-ALL with a manageable toxicity. The safety, efficacy, and potential long-term clinical benefits of GC007F cell therapy warrant further clinical evaluation.Supplementary information


Botrytis cinerea (B. cinerea). However, its specific regulatory mechanism remains to be further investigated. In non-heading Chinese cabbage  ssp. Chinensis), our previous study showed that BcWRKY33A is induced not only by salt stress, but also by B. cinerea infection. Here, we noticed that BcWRKY33A is expressed in trichomes and confer plant defense resistance. Disease symptoms and qRT-PCR analyses revealed that BcWRKY33A-overexpressing and -silencing lines were less and more severely impaired, respectively, than wild type upon B. cinerea treatment. Meanwhile, the transcripts’ abundance of indolic glucosinolates’ (IGSs) biosynthetic genes is consistent with plants’ B. cinerea tolerance. Identification and expression pattern analysis of BcMYB51s showed that BcMYB51-3 has a similar trend to BcWRKY33A upon B. cinerea infection. Moreover, BcWRKY33A directly binds to the BcMYB51-3 promoter, which was jointly confirmed by Y1H, dual-LUC, and EMSA assays. The importance of MYB51, the homolog of BcMYB51-3, in the BcWRKY33A-mediated B. cinerea resistance was also verified using the TRV-based VIGS system. Overall, our data concludes that BcWRKY33A directly activates the expression of BcMYB51-3 and downstream IGSs’ biosynthetic genes, thereby improving the B. cinerea tolerance of NHCC plants.The transcription factor WRKY33 is a vital regulator of the biological process of the necrotrophic fungus  PIWRKY65 from Paeonia lactiflora performed a positive role in regulating the jasmonic acid (JA) and salicylic acid (SA) signaling pathways in response to the infection of Alternaria tenuissima . To. ToB. cin plants . Meanwhied lines . In the  qRT-PCR a, as exp3A lines b. The fu cinerea c,d.B. cinerea, indicating the positive defense ability of WRKY33 and BcWRKY33A , with totally different expressions [BcMYB51-3 upon B. cinerea infection, whether BcMYB51-1 has redundant or unique functions with BcMYB51-3 upon B. cinerea infection requires further verification.Additionally, among three BcMYB51s, phylogenetic analysis showed that BcMYB51-3 shared a close relationship with BcMYB51-1 compared with BcMYB51-2 c; the exressions . AlthougB. cinerea spreads widely in plants, causing severe crop losses. Recent studies have shown that CCCH-type protein C3H14 enhances plant tolerance to B. cinerea depending on WRKY33 signaling [B. cinerea, the detailed molecular mechanism is not clear [B. cinerea via directly binding and activating the expression of BcMYB51-3, and this result was jointly confirmed by Y1H, LUC, and EMSA assay  was impaired, with the reduced transcriptional abundance of some IGSs’ biosynthetic genes, indicating that MYB51 is required in this process  in an artificial climate chamber at 22/18 °C, respectively. The BcWRKY33A-overexpressing Arabidopsis lines harbor recombinant construct 35S:BcWRKY33A-GFP, and the BcWRKY33A-silencing NHCC lines harbor recombinant construct pTY-BcWRKY33A. Methods of plant transformation were performed as described in our previous study [us study .http://tbir.njau.edu.cn/NhCCDbHubs/index.jsp, accessed on 20 May 2021) [http://brassicadb.cn/#/, accessed on 20 May 2021) [The sequence information of BcMYB51s was obtained from the Non-heading Chinese Cabbage Database (ay 2021) , and MYBay 2021) . Gene stay 2021) , the mulay 2021) , and theBcMYB51-1, BcMYB51-2, and BcMYB51-3 genes without termination codon were cloned and inserted into the NdeI-KpnI-cut pRI101 vector to obtain recombinant plasmids  background were produced using a TRV-based VIGS system with slight modifications [PDS and MYB51 cDNA fragments was amplified using primers: PDS-F (5′-TTCTGCGGCGAATTTGCCTTATCAAAACG-3′), PDS-R (5′-AGAAACTCTTAACCGTGCCATCGTCATTGAG-3′), MYB51-F (5′-AGCTCGTGGACTACCAGGAA-3′), and MYB51-R (5′-TTGACGTTCATAGACCGGCG-3′). The resulting PCR products were inserted into the EcoRI-XbaI-cut TRV2 to obtain recombinant constructs (TRV-PDS and TRV-MYB51). After transformation using the Agrobacterium tumefaciens strain GV3101, pTRV1 and pTRV2 constructs were co-injected into fifteen-day-old 35S:BcWRKY33A (# 4) seedlings with needleless 1 mL syringes. After infiltrating the entire leaves of the seedlings, they were kept in dark conditions overnight.The NHCC silence lines were generated using the TYMV-based VIGS system . pTY andications . In brieBcMYB51s were amplified from the gDNA of NHCC ‘suzhouqing’ and the products were inserted into the pAbAi vector. After confirming the appropriate inhibition concentration of aureobasidin A (AbA), the interaction of pAbAi-pBcMYB51s and cBcWRKY33A or nBcWRKY33A was determined using the lithium acetate method  in Y1H Gold strains [In yeast one-hybrid assay, the insertion of the full-length BcWRKY33A protein into the pGADT7 vector may cause yeast cell death for unknown reasons. To avoid its toxic effects on yeast, we truncated the BcWRKY33A according to its amino acid sequence and motifs to nBcWRKY33A  and cBcWRKY33A (159-476 aa), the starting codon ATG was added to the 5′ end of cBcWRKY33A to ensure its normal translation , and theBcMYB51-3 was inserted into the PstI-BamHI-cut pGreenII-0800-LUC vector as the reporter vector using a cloning kit . 35S:BcWRKY33A-GFP and 35S:GFP (control) were used as effector vectors, and relative primers are shown in GV3101 (pSoup), the mixture of effector and reporter (1:1) was injected into leaves of tobacco. D-luciferin was sprayed onto the leaves and a plant imaging system  was used to record the LUC imaging. The LUC/REN was detected and measured using by a dual-luciferase reporter assay kit  [The 2000-bp promoter of , China) .EMSA was applied using a chemiluminescent EMSA kit  according to the instruction. The recombinant proteins of BcWRKY33A were purified as referred to in our previous study . The DNA® qPCR SYBR Green Master Mix (Low Rox Plus)  on the QuantStudio®5 system and analyzed using the 2−ΔΔCT method, normalized to ELF4A (AT1G80000) and BcGAPDH (BraC09g068080.1) for Arabidopsis and NHCC. All primers used in qRT-PCR are shown in Total RNA was mainly extracted from leaves of plants using a TRIzaol reagent, and cDNA was synthesized using an Evo M-MLV Mix Kit with gDNA Clean for qPCR (Accurate Biotechnology (Hunan) Co., Ltd., Changsha, China). The gene relative expressions were detected using Hieff


Iris laevigata is ideal for gardening and landscaping in northeast China because of its beautiful flowers and strong cold resistance. However, the short length of flowering time  greatly limits its applications. Molecular breeding and engineering hold high potential for producing I. laevigata of desirable flowering properties. A prerequisite is to identify and characterize key flowering control genes, the identity of which remains largely unknown in I. laevigata due to the lack of genome information. To fill this knowledge gap, we used sequencing data of the I. laevigata transcriptome to identify MADS-box gene-encoding transcription factors that have been shown to play key roles in developmental processes, including flowering. Our data revealed 41 putative MADS-box genes, which consisted of 8 type I  and 33 type II members . We then selected IlSEP3 and IlSVP for functional studies and found that both are localized to the nucleus and that they interact physically in vitro. Ectopic expression of IlSEP3 in Arabidopsis resulted in early flowering (32 days) compared to that of control plants (36 days), which could be mediated by modulating the expression of FT, SOC1, AP1, SVP, SPL3, VRN1, and GA20OX. By contrast, plants overexpressing IlSVP were phenotypically similar to that of wild type. Our functional validation of IlSEP3 was consistent with the notion that SEP3 promotes flowering in multiple plant species and indicated that IlSEP3 regulates flowering in I. laevigata. Taken together, this work provided a systematic identification of MADS-box genes in I. laevigata and demonstrated that the flowering time of I. laevigata can be genetically controlled by altering the expression of key MADS-box genes. Iris laevigata (family of Iridaceae) is an herbaceous flowering plant species growing in the temperate regions of the Northern Hemisphere [I. laevigata of high ornamental values. In addition, I. laevigata is strongly tolerant of cold [I. laevigata is an ideal gardening plant in the northeast region of China with high commercial value. Nonetheless, the potential of I. laevigata in gardening and landscaping is limited due to a short length of flowering time, typically only 2 days for an individual flower and 20–25 days for the population. Because the flower is the most important ornamental part of I. laevigata, it is critical for breeders to identify key factors controlling flowering time and use genetic tools to improve flowering traits [misphere . The lar of cold . Thus, Ig traits .FLOWERING LOCUS T (FT), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), APETALA1 (AP1), and FLOWERING LOCUS C (FLC). Much of detailed molecular works has been performed in the model plant Arabidopsis [I. laevigata, very few studies have been reported on the flowering control. Most studies used plants in the same genus, focusing on the characterization of the reproductive growth and modulation of flowering time via changing agronomic parameters, such as photoperiod and temperature, or using plant growth regulators [I. laevigata remain largely unknown.Flowering is a critical developmental process featuring the transition from vegetative to reproductive growth in plants . Moleculbidopsis . In I. lgulators ,12,13,14Arabidopsis, more than 100 genes involved in the flowering pathways have been characterized [agamous has long been recognized as a key player in stamen and carpel development [Arabidopsis agamous gene encodes a transcription factor that is highly similar to homeotic proteins in yeast , snapdragon , and humans . Collectively, these genes were named the MADS-box gene family using the first letter of the four founding gene members [C, and MIKC*, have been recognized based on variances in the intervening domain [In the model plant elopment . Sequenc members . MADS-bo members . Two subg domain .Arabidopsis [Arabidopsis, the rice genome consists of a smaller MADS-box gene family with 75 members that are also categorized into five groups . Since then, MADS-box genes have been documented in various plant species including bread wheat [Given the essential role of MADS-box transcription factors in flowering, extensive work has been performed in the identification of the encoding genes and delineation of gene functions ,24,25,26bidopsis . MADS-bobidopsis . Comparead wheat , willow ad wheat , bamboo ad wheat , watermeI. laevigata are lagging far behind compared to those of other plant species. Consequently, little is known on the molecular mechanisms behind the flowering control in I. laevigata. In addition, the identity and function of MADS-box genes in I. laevigata have not been reported. To overcome the unavailability of genome data, our group recently performed RNA-sequencing using the flower tissue of I. laevigata [I. laevigata [I. laevigata and characterize selected genes in the context of flowering control. Here, we report the identification of 41 putative MADS-box genes and phylogenetic/structural analysis of candidate genes. We selected two genes for further functional studies and found that both IlSEP3 (SEPALLATA3) and IlSVP (SHORT VEGETATIVE PHASE) encode proteins that are localized into the nucleus. Interestingly, the two proteins showed physical interactions. Overexpressing IlSEP3, but not IlSVP, promoted flowering in Arabidopsis possibly by regulating the expression of genes in multiple pathways.Due to the lack of genome information, molecular studies in aevigata . The valaevigata . Using aI. laevigata. Among the 8 type I genes, 5 and 3 belong to the Mα and Mβ subfamily, respectively. Type II MADS-box genes consist of 2 MIKC* members and 31 MIKCC members, which can be further divided into the following 7 subtypes: SEP-like (n = 7), AGL6-like (n = 1), SQUA-like (n = 5), AG-like (n = 1), TM3-like (n = 5), DEF/GLO-like (n = 11), and STMADS11-like (n = 1).A total of 41 non-redundant MADS-box genes were identified from our full-length transcriptomics database . PhylogeIlSEP3 and IlSVP . Accordingly, the predicted tertiary structures also showed the dominance of α-helix  and the circadian clock control including FKF1, ELF3, PRR7, TOC1, ZTL, DOF5.5, and COP1  and PCR screening  compared to the controls (mean of 36.08 d). However, the difference was not statistically significant (p > 0.05). Thus, plants overexpressing IlSVP were not used for further experiments in this study.Phenotypical analysis of three IlSEP3 promotes early flowering, the mRNA abundance of various endogenous flowering time genes was determined in the transgenic plants . However, our data is comparable to 39 MADS-box genes in the Louisiana Irises (Iris fulva), 25 of which belonged to the MIKCC type [Iris atropurpurea, an endemic species in Israel [C genes identified in this study, 28 were found in the I. atropurpurea transcriptome. Thus, these data suggested similar gene numbers and subtypes among closely related Iris species.In this study, we identified 41 MADS-box genes in KCC type . Notablyn Israel . CompareC subgroup members are the most extensively studied MADS-box genes. Based on phylogenetic analysis, 12 subgroups have been found in Arabidopsis [FLC-like, AGL15-like, GGM13-like, AGL17-like, and AGL12-like, were not identified in I. laevigata. This is consistent with a previous report, where FLC-like, GGM13-like, TM8-like, AGL15-like, AGL17-like, and AGL12-like were not identified in the Louisiana Irises [Iris is supportive of the notion that FLC-like is specific to dicots such as Arabidopsis [AGL15-like, GGM13-like, AGL17-like, and AGL12-like showed high mRNA expression levels in the root [I. laevigata.Currently, the MIKCbidopsis . Nonethea Irises . Lackingbidopsis ,28,30. BC branch. Both IlSEP3 (belonging to the SEP-like sub-branch) and IlSVP (belonging to the STMADS11-like sub-branch) showed high sequence similarities to their corresponding homologs in other plant species. The physical interactions between IlSEP3 and IlSVP indicated that they may function as a complex in vivo regulating flowering. This challenges the conventional view that SVP and SEP3 function in the early stage of flowering initiation and floral organ formation, respectively [For functional studies, we selected two members in the MIKCectively . Insteadectively . Experimectively ,32,33,34IlSEP3 and IlSVP, which showed that IlSEP3 promotes flowering. This is consistent with previous studies in which an earlier flower phenotype was observed in transgenic Arabidopsis overexpressing SEP3 from different plant species including lavender [Ziziphus jujuba Mill. [Isatis indigotica) [IiSEP3-2 and IiSEP3-3 using virus-induced gene silencing (VIGS) resulted in delayed flowering in woad [SPE3s in Arabidopsis often resulted in curly and smaller leaves compared to WT. Thus, our results and data from others strongly support that SPE3 is a positive player in flowering.Our overexpression experiments provided a direct assessment of molecular functions of lavender , Ziziphuba Mill. , lily [3ba Mill. , and woaigotica) . Convers in woad . In addiIlSEP3 led to perturbed expression patterns of key flowering genes including FT, SOC1, AP1, VRN1, AG20OX, as well CO (downregulation). Previous studies also showed that overexpressing of these upregulated genes  promotes flowering [IlSEP3 can modulate various flowering pathways, including the gibberellic acid-dependent pathway and the vernalization pathway in promoting early flowering in transgenic plants. It is also noteworthy that overexpressing IlSEP3 resulted in a higher expression of SVP. A similar repressuring effect on SVP has been found in Arabidopsis plants overexpressing either the SEP3-2 or SEP3-3 splicing variant of woad [SVP was upregulated before flowering and downregulated after flowering in Arabidopsis plants overexpressing ZjSEP3 [IlSEP3-overexpression plants.Mechanistically, we found that overexpressing lowering ,40,41. T of woad . More ing ZjSEP3 . It is pIlSVP. Although SVP is often considered a repressor of flowering, the literature is divided on its molecular function. For example, ectopic expression of kiwifruit SVP3 (Actinidia spp.) in A. deliciosa showed no impact on flower time but led to prolonged flowers [TaVrn1 (encoding another MADS-box transcription factor) [SVP from sweet cherry resulted in a delay in flowering and the production of flowers with curly sepals in Arabidopsis [SVPs among different plant species remains unclear. Nonetheless, SVP may represent a gene characterized with functional variations as SVPs from the same species can exhibit distinct functions. For instance, the four SVPs in kiwifruit showed gene-specific expression patterns and functions during vegetative growth, flowering, and bud dormancy [SPVs have also been observed in Arabidopsis, in which SVP and SGL24 can function in concert with each other during one developmental stage  but perform opposite roles for another process  [IlSVP in the corresponding Arabidopsis mutant to see whether it can rescue the svp mutant.By contrast, we found no impact on flowering time by overexpressing  flowers . TaVrt2, factor) . In addibidopsis . Thus, idormancy . The duatiation) ,46. In tI. laevigata (unpublished from our group) was used to identify the MADS-box gene family. The open reading frame (ORF) of the obtained sequences was predicted by the TBtools . Domain analysis was performed using the online Batch CD-Search tool . Sequences harboring the MADS-box domain were kept and subjected to a second-round ORF analysis  to ensure the integrity of the transcript.An annotated transcriptomics dataset of I. laevigata and Arabidopsis. MADS-box proteins of Arabidopsis were downloaded from Phytozome . Sequence alignment was performed using Clustal W, and the neighbor-joining (NJ) method was used for tree construction with a bootstrap of 1000 times. Annotation of the phylogenetic tree was performed using iTOL .Phylogenetic analysis was performed using MEGA X with MADS-box sequences from I. laevigata plants grown in the nursery of Northeast Forestry University (May 2020) using a kit (KANGWEI). Agarose gel electrophoresis was used to check the quality of the RNA, and DNA Eraser was used to remove genomic DNA. Next, 1 µg of total RNA was used for reverse transcription (TaKaRa). Amplification of target genes was performed using PCR with the following primers: 5′-TATTCAATGGTGAGGGGGAGAGTGGAGC-3′ and 5′-TTATGTGCAGGGATGGCTTCCGTGAGAC-3′ for IlSEP3; and 5′-TCTTTCTCCTCTGTTGCTGTGT-3′ and 5′-GAAGCTCTACTGCATCATCGTG-3′ for IlSVP. The obtained gene fragments were ligated into the pEASY-Blunt Zero Cloning Vector (TRANSS) and transformed into E. coli. Positive clones were selected for sequencing (QINGKE). All procedures were performed per instructions of respective kits.Total RNA was extracted from the leaf and flower of https://web.expasy.org/protparam/, accessed on 1 January 2022). The hydrophobicity/hydrophilicity scale was assessed using ProtScale . Prediction of the secondary and tertiary structures was performed using SOPMA and SWISS-MODEL, respectively. Potential phosphorylation sites were predicted by the NetPhos software. Subcellular localization was predicted using Wolf Psort.Physical and chemical parameters were predicted using the ProtParam tool  and 5′-GGATCCGTCCGGAAGCCATCCCTGCACATAACCA-3′ (BamHI). Primers for IlSEP3 into GADT7 were 5′-ATATGGCCATGGAGGCCAGTGAATTCATTCAATGGTGAGGGGGAGAGTGGAGC-3′ and 5′-ATCTGCAGCTCGAGCTCGATGGATCCGTCCGGAAGCCATCCCTGCACATAACCA-3′ . Primers for IlSVP into pGBKT7 were 5′-ATATGGCCATGGAGGCCAGTGAATTCATGGCGAGGGAGAAGATACAG-3′  and 5′-ATCTGCAGCTCGAGCTCGATGGATCCCTGCATCATCGTGCCCTTC-3′. Primers for IlSVP into pGADT7 were 5′-CCATGGAGGCCAGTGAATTCATGGCGAGGGAGAAGATACAG-3′ and 5′-AGCTCGAGCTCGATGGATCCCTGCATCATCGTGCCCTTC-3′. pGBKT7-IlSEP3 was obtained by subcloning IlSEP3 into the pre-linearized pGBKT7 vector (NdeI and BamHI). pGADT7-IlSEP3 was obtained by subcloning IlSEP3 into GADT7 via (EcoRI and BamHI) homologous recombination using the ClonExpress II One Step Cloning Kit. In the same way, pGADT7-IlSVP (EcoRI and BamHI) and pGBKT7-IlSVP(EcoRI and BamHI) were obtained.To construct vectors for Y2H, the coding sequences of IlSEP or pGBKT7-IlSVP into Y2HGold and growing the cells on SD/-Trp. For the autoactivation test, cells transformed with pGBKT7-IlSEP or pGBKT7-IlSVP were grown on SD/-Trp/X-α-gal/AbA. For co-transformation, eight groups were assessed: the test group (pGBKT7-IlSEP3 and pGADT7-IlSVP), (pGBKT7-IlSVP and pGADT7-IlSEP3), (pGBKT7-IlSEP3 and empty), (pGBKT7-IlSVP and empty), (empty and pGADT7-IlSVP), (empty and pGADT7-IlSEP3), positive control (pGADT7-T and pGBKT7-53), and negative control (pGADT7-T and pGBKT7-Lam). Transformed cells were diluted and then grown on either SD/-Trp/-Leu or SD/-Trp/-Leu/-Ade/-His/X-α-Gal/ABA at 28 °C for 48 h.The Y2HGold yeast was used as the bait strain. Transformation into the competent cells was performed as described before . ToxicitIlSEP3-GFP and pCAMBIA1300-Pro35S: IlSVP--GFP vectors were constructed by homologous recombination. Briefly, the full-length coding region was amplified using the following primers with homology arms (underlined): 5′-TTGATACATATGCCCGTCGACTATTCAATGGTGAGGGGGAGAGTGGAGC-3′ and 5′-CCCTTGCTCACCATGGATCCGTCCGGAAGCCATCCCTGCACATAACCA-3′ for IlSEP3; and 5′-TTGATACATATGCCCGTCGACTCTTTCTCCTCTGTTGCTGTGT-3′ and 5′-CCCTTGCTCACCATGGATCCGAAGCTCTGCATCATCGTGCCC-3′ for IlSVP. These constructs, along with the empty vector , were then delivered into Agrobacterium tumefaciens GV3101 by the freeze-and-thaw method [For gene overexpression, pCAMBIA1300-Pro35S: w method . PositivIlSEP3-GFP and IlSVP-GFP proteins in Nicotiana benthamiana leaves according to previously established methods [IlSEP3-GFP or pCAMBIA1300-Pro35S: IlSVP--GFP were cultured. Bacterial cells were collected by centrifuge and resuspended in 10 mM MaCl2 to OD600 of 1.5, followed by the addition of acetosyringone to a final concentration of 200 µM. After activation at dark for 3 h, the bacterial solution was injected into the downside of the leaf using a syringe. Fusion proteins in the epidermal cells of the transformed plants were determined under microscopy.Subcellular localization was performed by transient expression of the  methods . BrieflyA. thaliana (ecotype Col-0) was performed using the floral dipping method [IlSEP3 and IlSVP were determined by qRT-PCR using the seeding leaf of the transgenic plants. Actin2 (AT3G18780) was used as a control. Relative gene expression was determined using the 2−ΔΔCT method. Three lines of a high level of expression were selected.Transformation of g method . Seeds oPhenotypical analyses were performed as described previously ,52,53. TCO, GA20OX, VRN1, SPL3, FCA, SVP, FT, SOC1, AP1, and FLC) was determined by RT-PCR. Two-week-old seedlings were used. The selection of these genes and the time for gene expression analysis were based on previous studies [AtACT2 control (2−ΔΔCT method) was shown.The expression of 10 well-known genes in flowering control  would be beneficial because of a general lacking flowering plants in wetland during that time window of the year.It would be intriguing to generate transgenic