Progressive functional deterioration in the cochlea is associated with age-related hearing loss (ARHL). However, the cellular and molecular basis underlying cochlear aging remains largely unknown. Here, we established a dynamic single-cell transcriptomic landscape of mouse cochlear aging, in which we characterized aging-associated transcriptomic changes in 27 different cochlear cell types across five different time points. Overall, our analysis pinpoints loss of proteostasis and elevated apoptosis as the hallmark features of cochlear aging, highlights unexpected age-related transcriptional fluctuations in intermediate cells localized in the stria vascularis (SV) and demonstrates that upregulation of endoplasmic reticulum (ER) chaperon protein HSP90AA1 mitigates ER stress-induced damages associated with aging. Our work suggests that targeting unfolded protein response pathways may help alleviate aging-related SV atrophy and hence delay the progression of ARHL. Age-related hearing loss (ARHL), also known as presbycusis, is a progressive and irreversible hearing impairment caused by cochlear aging. According to epidemiologic studies, the risk of developing ARHL increases rapidly after the age of 40, with a third of people over 65 estimated to be suffering from disabling hearing loss . Owing t+ gradient, vital for sound sensation and audition  approaches have identified transcriptional signatures associated with aging and related diseases within multiple organs and heterogeneous tissues . HoweverIn this study, we established a comprehensive single-cell transcriptomic atlas of mouse cochlear aging at high-temporal resolution. In the SV, we found that ICs showed pronounced transcriptional alterations during aging, among which elevation of unfolded protein response (UPR) and apoptosis were the most profound. Notably, we discovered that the chaperon HSP90AA1 was elevated during aging, and the activation of endogenous HSP90AA1 alleviated ER stress-induced damages in SV cells, suggesting a compensatory mechanism that may help to prevent SV from aging-associated atrophy. Collectively, our study constitutes a rich resource for identification of cell type-specific molecular alterations along the time course of cochlear aging, and as such, will enable the discovery of cochlear aging-associated biomarkers, and help identify cellular and molecular programs that can be targeted therapeutically in presbycusis.To uncover the cellular and molecular mechanisms of cochlear aging, we used C57BL/6J mice, a widely used rodent model of ARHL . To estaPou4f3+, Pcp4+), Deiter cell and pillar cell , Nudt4+ pillar cell (Nudt4+), and inner phalangeal cell/inner border cell  , satellite glial cell , Schwann cell , chondrocyte   , marginal cell , basal cell , and capillary endothelial cell     (Nelson S100a6+) . Cells ilc26a2+) , and ost Runx2+)    and S3A.nd Vwf+)  and S3A nd Vwf+) . Cells ilc26a7+)  and S3A lc26a7+) , as welllc26a7+)  and S3A.lc26a7+) . For exaSyne1 and Dnajc5b, and the latter expressing Slc26a5 and Kcnq4 (Chchd10+), type IB , type IC (Pou4f1+), type II (Th+), and an oligo1+ SGN subtype  analysis. We found that the CV increased during aging, as manifested by elevated CV values at 2-, 5-, 12-, and 15-month-old compared with that of 1-month-old, and peaked at 5-month-old . When we cochlea . AltogetAs samples were segregated transcriptionally by age , we nextTceb2 and Wbp5; downregulated: Elob and Cebpd) and translation   of PDEGs, we observed a panel of PDEGs with high frequency at the early stage (2 M vs. 1 M), while a larger number of PDEGs emerged with low frequency at later stage (15 M vs. 1 M) . This pad Rack1) and S5B. d Rack1) . The dowd Rack1) . The oveFurthermore, functional enrichment analysis and transcriptional network analysis of PDEGs from four pairwise comparisons uncovered similar yet asynchronous characteristics during cochlear aging . For exaCdkn1a, Lmna, Gadd45b, and Gadd45g  . We, theHsp90aa1, Calr, and Hspa5), (ii) apoptotic signaling pathway , (iii) positive regulation of adaptive immune response , and (iv) response to oxidative stress  , extracellular matrix organization , rhythmic process , and cation transmembrane transport  (We next sought to resolve steadily upregulated and downregulated DDEGs (module 6 or module 1) in different cell types throughout the entire temporal window, as such insight can inform mechanistic contributions to sustained and unidirectional effects on cochlear aging . Based ond Gpx4)  and S8B, Atp1a1)  and S8B.H2-K1, B2m, H2-D1, Isg15, Bst2, Hsp90aa1, Hsp90ab1, Bst2, Ifi27l2a, Mt2, S100a8, and the top 10 shared downregulated genes were Rbm3, Dct, Sparc, Dbp, Coch, Col1a2, Atp1b1, Ccnd2, Ptgds, and Car14  and Stat3 , Irf7 (Interferon regulatory factor 7), whereas others were involved in the UPR, such as Xbp1 (X-Box binding protein 1), Atf4 (Activating transcription factor 4), and Ddit3 (DNA damage inducible transcript 3), and apoptosis, including Atf3 (activating transcription factor 3) (Dbp (D-Box-binding PAR BZIP transcription factor), Nr1d1 (nuclear receptor subfamily 1 group D member 1), and Nr1d2 (nuclear receptor subfamily 1 group D member 2) are involved in circadian rhythm regulation  . As for gulation . Among ut manner , suggest+) and the proportion of TUNEL-positive cells markedly increased in SV tissues from 5- and 15-month-old mouse cochleae relative to those in 1-month-old mice , ranked as the top one, was one of the most frequently occurring upregulated genes overlapped by DDEGs and annotated genes in Aging Atlas database as mentioned earlier (in situ hybridization (RNA–ISH) and Western blot assays also demonstrated the age-dependent elevation of HSP90AA1 levels   by RT-qPCR   , and foucin (TM) , which wnockdown . Conversnockdown   . In concHsp90aa1. In functional experiments, activation of HSP90AA1 can alleviate ER stress-induced damages. In summary, our work provides a rich resource for mining cellular and molecular mechanisms of cochlear aging at high temporal and single-cell resolution, and for advancing development of diagnostic and therapeutic interventional strategies against ARHL  are in urgent need  Biotechnology Co. Ltd. and kept in a quiet environment with enough water and food under a standard 12:12-h light-dark (LD) cycles with 22°C and 55% humidity.SV cells (SV–k1) were purchased from Bluefbio in China. Cell culture was carried out as reported previously . In brieThe ABR threshold is determined as described previously . BrieflyDPOAE responses of 2f1-f2 were measured using the Real-time Signal Processing System II from Tucker-Davis Technologies as described previously . Output Endocochlear potential determination was performed as described previously . BrieflyAfter sacrifice by cervical vertebrae dislocation, the temporal bones were dissociated from mouse inner ear, a hole on the apical cochleae was made and tissue was fixed in 4% paraformaldehyde  in PBS for about 24 h at 4°C, followed by three washes with PBS and decalcification with 10% EDTA for 72 h at 4°C to completely soften the cochlea. For paraffin-embedded sections, the cochleae were dehydrated by gradient alcohol and then embedded in paraffin, and cut into 5 μm sections. For the frozen sections, the completely decalcified cochleae were dehydrated with 15% sucrose solution and 30% sucrose solution and were embedded in optimal cutting temperature (OCT) compound , snap-frozen in liquid nitrogen and then stored at −80°C. Frozen samples were cut into 8-μm sections using a CM 1850 cryostat (Leica Microsystems Nussloch).H&E staining was performed as previously described . Slides Frozen sections were washed in PBS for 5 min and then fixed by 4% paraformaldehyde  in PBS for 25 min at room temperature (RT). Paraffin-embedded sections were deparaffinized in xylene and rehydrated through gradient alcohol . After rinsing in distilled water, antigen retrieval was performed by microwaving the slides in 10 mmol/L sodium citrate buffer (pH 6.0) three times for 5 min each. Upon cooling down to RT, the sections were rinsed in PBS for three times. Then frozen sections or paraffin-embedded sections were permeabilized and blocked for 1 h at RT in PBS with 0.4% Triton™ X-100  and 10% donkey serum in PBS, respectively. Sections were then incubated with the appropriate primary antibodies , Sigma, T2200 Rabbit anti-myosin 7a, Proteus Bioscience, 25-6790; Rabbit anti-CALR, Abcam, ab92516; Rabbit anti-MRP8, Abcam, ab180735; Rabbit anti-S100A9, Abcam, ab92507) in 10% donkey serum overnight at 4°C, followed by incubating with secondary antibodies and Hoechst 33342  for 1 h at RT. After additional several washes, the sections were mounted with VECTASHIELD Antifade Mounting Medium , and images were obtained with a confocal laser-scanning microscope .Aggresome staining was performed following the manufacturer’s protocol . Frozen sections were fixed by 4% paraformaldehyde  in PBS for 15 min followed by permeabilization with 0.3% Triton™ X-100  for 7 min, incubated with the Aggresome dye  for 3 min and then destained in 1% acetic acid for 30 min. The sections were then blocked with 10% donkey serum in PBS for 1 h after washing thoroughly with PBS and followed by incubation with rabbit anti-Kcnj10 antibody  overnight at 4°C. The sections were then counterstained with Hoechst 33342  and fluorescence-labeled secondary antibody  for 1 h at RT after washing with PBS for 3 times (10 min each). Image was obtained by Confocal laser-scanning system . The aggresome intensity was quantified by Image J software and normalized to that of 1 month old mice.TUNEL  staining was conducted on paraffin sections utilizing the Kit  following the manufacturer’s protocol. Then, the slides were counterstained with Hoechst 33342  for visualization of nucleus. Finally, the slides were mounted with VECTASHIELD Antifade Mounting Medium  and the percentages of TUNEL-positive cells were quantified by Image J software. The number of TUNEL-positive cells was normalized to that of 1 month old mice.Hsp90aa1 fragment from mouse cDNA and subsequent labeling of the RNA probe were as follows: Forward: gaggaaacccagacccaaga, Reverse: gatcccccagctgaggactc. DIG RNA Labeling Mix (Roche Diagnostics) were used to transcribe DIG-labeled RNA probes by T7 and T3 RNA polymerases. Then, RNA–ISH was performed following a previously described method . Briefly, the freshly collected cells were stained with Annexin V-EGFP Apoptosis Detection Kit and then analyzed by BD LSRFortesa flow cytometer.The analysis of cellular aggresome intensity was performed following the manufacturer’s instructions. The freshly collected cells were stained by PROTEOSTAT® Aggresome detection kit  and then analyzed by BD LSRFortesa flow cytometer .Hsp90aa1, guide RNA targeting Hsp90aa1 and two non-targeting controls (NTCs) was cloned into LentiCRISPRv2 vector (Addgene #52961). For activation of endogenous expression of HSP90AA1, guide RNA targeting the transcriptional start site (TSS) locus of Hsp90aa1 and two NTCs were constructed into lentiSAM v2 vector (Addgene #75112). To produce lentivirus particles, HEK293T cells were co-transfected with transfer plasmid lentiviral sgRNA plasmids or lentiMPH v2 (Addgene #89308), along with packaging plasmids psPAX2[AU: Please note that the extra opening single quotes have been removed.] (Addgene #12260) and pMD2.G (Addgene #12259) by Lipofectamine 3000 transfection reagent . The supernatants containing viral particles were collected 48 and 72 h later and were ultra-centrifuged at 19,400 ×g for 2.5 h to obtain virus particles. To knockdown the expression of Hsp90aa1, SV cells were transduced with lentiviral vector LentiCRISPRv2 for 48 h in the presence of 10 μg/mL polybrene following selection with puromycin for 5 days. To transcriptionally activate the endogenous expression of HSP90AA1, SV cells were co-transduced with the produced lentiviral vectors LentiSAM v2 and LentiMPH v2 for 48 h in the presence of 10 μg/mL polybrene before selection with blasticidin and hygromycin for 5 days. To verify the successful knockdown or activation of HSP90AA1, the selected cells were collected for Western blot to test the protein levels of HSP90AA1.The CRISPR/Cas9-mediated gene knockdown and activation were performed as previously described . For knog for 30 min to extract the supernatant for protein concentration quantification. Then the protein concentration of each sample was measured by BCA kit following manufacturer’s instruction. Each sample was electrophoresed by SDS–PAGE and then electrotransferred to PVDF membrane (Millipore) which was then blocked with 5% skimmed milk (powder from BBI Life Sciences) and incubated with the primary antibodies  overnight at 4°C, and then incubated with the secondary antibodies conjugated with horseradish peroxidase (HRP). At last, the visualization and data processing were performed by a ChemiDoc XRS system (Bio-Rad).Cells were lysed with SDS lysis buffer [containing 4% SDS and 100 mmol/L Tris-HCl (pH = 6.8)] and heated at 105°C for 10 min. Mouse cochleae were grinded and then lysed with 200 μL SDS buffer for 24 h at 4°C followed by heating at 105°C for 10 min and then centrifuged at 21,100 ×Gapdh expression, calculated using the ∆∆Cq method. The primers used for RT–qPCR are as follows: Xbp1-Forward, GACAGAGAGTCAAACTAACGTGG; Xbp1-Reverse, GTCCAGCAGGCAAGAAGGT; Calr-Forward, TGGCTGCTCCCAATAATGTCT; Calr-Reverse, GAGGGTAGTGACCAAAAGATGG; Hsp90b1-Forward, TCGTCAGAGCTGATGATGAAGT; Hsp90b1-Reverse, GCGTTTAACCCATCCAACTGAAT; Ddit3-Forward, CTGGAAGCCTGGTATGAGGAT; Ddit3-Reverse, CAGGGTCAAGAGTAGTGAAGGT; Gapdh-Forward, TGGATTTGGACGCATTGGTC; Gapdh-Reverse, TTTGCACTGGTACGTGTTGAT.RNA was extracted according to the manufacturer’s instructions after cells were lysed by TRIzol™ Reagent  and the concentration of RNA was measured after dissolving in about 20 μL DEPC water (Sangon Biotech). 2 μg RNA was subjected to reverse transcription according to the instructions of the GoScript™ reverse transcription system (Promega) to obtain cDNA. RT-qPCR was then performed with the qPCR Mix (TOYOBO) on a CFX384 RT-PCR system (Bio-Rad). The relative mRNA expression level of each gene was normalized to For RNA-seq, 1.5 μg total RNA was provided to the Novogene Bioinformatics Technology Co. Ltd. for subsequent quality control, library construction and high-throughput sequencing. Briefly, Next Ultra RNA Library Prep Kit for Illumina (NEB) was used for sequencing library construction and the resultant libraries were sequenced on an Illumina paired-end sequencing platform by 150-bp read length.l-Cysteine , 0.5 mmol/L EDTA, 15 mmol/L HEPES , and 10 K units/mL DNase I  at 37°C for 30 min, pipetted 10 times every 10 min. 50 μL 10% fetal bovine serum  was added to terminate the digestion followed by pipetting 100 times in order to obtain single cell suspension. After a centrifugation with 1500 rpm at 4°C for 5 min, the cell pellet was resuspended and filtered with a 40-μm strainer  and washed with 1 mL of L15 medium. Filtered cell suspension was collected directly into a 1.5 mL EP tube and Hoechst , Propidium Iodide were added, followed by FACS (BD Influx) to remove the debris and dead cells. The obtained single cells were centrifuged with 2000 rpm at 4°C for 5 min and resuspended in 0.04% bovine serum albumin  in PBS for scRNA-seq.The samples for scRNA-seq included 1-, 2-, 5-, 12-, and 15-month-old mice and each age group contains 5 males and 5 females. The cochleae of mice of the same age and sex were pooled together for scRNA-seq. Briefly, the cochleae were taken out and placed in chilled 500 μL Leibovitz’s L-15 medium  quickly after mice were sacrificed by cervical dislocation. Afterward, temporal bones of mice were isolated, and the overlying bone was extracted, leaving the lateral wall, the modiolus with the spiral ganglion, the spiral limbus, the inner sulcus, the organ of Corti and the outer sulcus remaining. Microdissected tissues from each mouse were pooled and transferred into 500 μL digestion solution I containing Leibovitz’s L-15 medium , 200 unit/mL collagenase IV  and 10 K unit/mL deoxyribonuclease I  and incubated at 37°C for 30 min, pipetted once every 10 min. Thirty minutes later, digestion solution I was changed to digestion solution II containing 1× EBSS , 20 unit/mL papain , 1 mmol/L Single cells were captured by droplet-based microfluidic technology and the construction of single cell transcriptional libraries was performed by the Chromium 10× Single-Cell Instrument (10× Genomics) and 10× Genomics Chromium Single Cell 3ʹ GEM Library and Gel Bead Kit v3. In brief, cells were loaded in each channel with a target output of 5000 cells per sample with appropriate cell concentration measured by Moxi GO II (Orflo Technologies). All the reactions were performed in the Bio-Rad C1000 Touch Thermal cycler with 96 Deep-Well Reaction Module in which 12 cycles were used for cDNA amplification and sample identification. Amplified cDNAs and final libraries were then evaluated on a Fragment Analyzer (AATI) using a High Sensitivity NGS Analysis Kit . The average fragment length of the 10× cDNA libraries was assessed with the AATI, and quantified by qPCR using the Kapa Quantification kit. All the libraries were diluted and pooled together for each run of NovaSeq sequencing. All the libraries were sequenced on the NovaSeq 6000 Sequencing System (Illumina).cellranger count function, which was aligned to the mouse reference genome (mm10) and generate the gene expression matrix. The filtered gene expression matrix was used for downstream analysis.Cell Ranger single-cell software suite (version 3.1.0) (10× Genomics) with default parameters was used to align and quantify the dataset. The quantification of the sample-specific FASTQ file was evaluated by the −6”. The “find.pK” function was used to calculate the optimal pK value. After quality control, 45,972 cells remained and were used for downstream bioinformatic analyses. In order to better eliminate the false positive of biological heterogeneity caused by technical factors such as sequencing depth in scRNA-seq data, we normalized and scaled the data based on R package SCTransform (version 0.3.2)  were used to define cell identity. Dimensionality reduction was performed with “RunUMAP” function. Marker genes for each cluster were determined with the Wilcoxon rank-sum test by “FindAllMarkers” function. Only those with “avg_logFC” > 0.5 and “p_val_adj” < 0.05 were considered as marker genes. Cell types were identified based on the expression of classic marker genes. Marker genes for each cell type are shown in Downstream analysis for scRNA-seq data was implemented by the single-cell toolkit Seurat (version 3.2.0)  in R ve to identn 0.3.2)  to reveaThe algorithm called Ratio of Global Unshifted Entropy (ROGUE) provided by Zhang et al.  was occuFindVariableGenes’’ function of Seurat was used to identify highly variable genes (HVGs). The top 10% variable genes  were selected for downstream analysis. Next, the absolute value of the cell-paired-distance dc,x was calculated between each HVGs expression in all cells of the young individuals and the old individuals in each cell type c:Analysis of age-relevant CV was used to observe the aging effects on different cell types . The “FiFinally, the arithmetic mean of dc,x , and the standard deviation of dc,x as  were calculated. Accordingly, the aging-related transcriptional variation of each cell type is defined by the following formula:FindMarkers’’ in Seurat to identify aging-associated PDEGs between 1-month-old and each consecutive time point. The log (fold change) (LogFC) and adjusted P-value of each PDEG were calculated by the non-parametric two-sided Wilcoxon rank-sum test and only those with |“avg_logFC”| > 0.25 and “p_val_adj” < 0.05 were considered to be aging-associated PDEGs. We also ran similar analyses using 2-month-old mice as reference and got PDEGs between neighboring ages. The PDEGs are listed in We used the function of ‘‘plot_pseudotime_heatmap” function in R package Monocle2  in each cell type. Only those genes with |“avg_logFC”| > 0.25 and “p_val_adj” < 0.05 are considered age-related DEGs. Second, for each cell type, we used the above genes to construct an expression matrix , and then sort all cells by age scale . Next, we used the “genSmoothCurves” function to fit smooth spline curves for the gene expression matrix dynamics along age time in a gene-wise manner and return the corresponding response matrix. Finally, the “ward. D2” method was used to cluster the matrix by row hierarchical clustering through “hclust” function, and the clustering results were assigned to 8 groups. The R package pheatmap (version 1.0.12) was used to visualize the age-dependent genes expression pattern. The DDEGs are listed in To identify age-dependent DDEGs, we borrowed the method of the “Monocle2  and custP < 0.01) were displayed.GO analysis of DEGs was performed by Metascape  and visut-tests and Benjamini-Hochberg multiple testing p.adjust were used for downstream analysis.To comprehensively assess the expression levels of individual gene in the pathways, we also adopted Gene set variation analysis (GSVA) , a quantThe transcriptional regulatory network analysis was performed with the GENIE3 (version 1.6.0)  and RcisAUCell_buildRankings” function, with default parameters. The canonical pathway database was downloaded from the MsigDB database, KEGG database and GO database. These gene sets were used to score each cell. Area-under-the-curve (AUC) values were calculated  based on gene expression rankings. Genes in each gene set are listed in To score individual cells for pathway activities, we used the R package AUCell (version 1.8.0) . We usedHsp90aa1 sgRNA were collected for RNA-seq analysis using an Illumina sequencing platform. RNA sequencing libraries were prepared as previously reported (Mouse SV cells were transduced with the sg-NTC (Control), sg-reported . BrieflyP-value (p.adjust) < 0.05 and |log2 (fold change)| > 0.58. The DEGs are listed in Trim Galore (version 0.4.5) software was used for automate adapter trimming and quality control, and Hisat2 (version 2.0.4)  with deft-test. For gene set score analysis, statistical analysis was performed using two-sided Wilcoxon rank-sum tests. P-values < 0.05 are considered statistically significant.Data are presented as the mean ± SEMs. The statistical analyses were performed using GraphPad Prism (v8) with unpaired two tailed Student’s https://doi.org/10.1093/procel/pwac058.The online version contains supplementary material available at pwac058_suppl_Supplementary_FiguresClick here for additional data file.pwac058_suppl_Supplementary_Table_S1Click here for additional data file.pwac058_suppl_Supplementary_Table_S2Click here for additional data file.pwac058_suppl_Supplementary_Table_S3Click here for additional data file.pwac058_suppl_Supplementary_Table_S4Click here for additional data file.pwac058_suppl_Supplementary_Table_S5Click here for additional data file.


Escherichia coli (STEC) is a foodborne pathogen that causes gastrointestinal infections, ranging from acute diarrhea and dysentery to life-threatening diseases such as Hemolytic Uremic Syndrome. Currently, a vaccine to prevent STEC infection is an unmet medical need.Shiga toxin-producing We developed a chimeric protein-based vaccine targeting seven virulence factors of STEC, including the Stx2B subunit, Tir, Intimin, EspA, Cah, OmpT, and AggA proteins. Immunization of mice with this vaccine candidate elicited significant humoral and cellular immune responses against STEC. High levels of specific IgG antibodies were found in the serum and feces of immunized mice. However, specific IgA antibodies were not detected in either serum or feces. Furthermore, a significantly higher percentage of antigen-specific CD4+ T cells producing IFN-γ, IL-4, and IL-17 was observed in the spleens of immunized mice. Notably, the immunized mice showed decreased shedding of STEC O157:H7 and STEC O91:H21 strains and were protected against weight loss during experimental infection. Additionally, infection with the STEC O91:H21 strain resulted in kidney damage in control unimmunized mice; however, the extent of damage was slightly lower in immunized mice. Our findings suggest that IgG antibodies induced by this vaccine candidate may have a role in inhibiting bacterial adhesion and complement-mediated killing.This study provides evidence that IgG responses are involved in the host defense against STEC. However, our results do not rule out that other classes of antibodies also participate in the protection against this pathogen. Additional work is needed to improve the protection conferred by our vaccine candidate and to elucidate the relevant immune responses that lead to complete protection against this pathogen. Escherichia coli (STEC) are a group of foodborne pathogens that cause diarrhea and dysentery. In some cases, these infections can lead to more severe and potentially deadly complications, such as Hemolytic Uremic Syndrome (HUS), mainly in children under 5 years old and the elderly . During infection, this toxin can spread systemically and cause damage to the kidneys and central nervous system, leading to HUS .E. coli (EHEC), harbor the pathogenicity island (PAI) called Locus of Enterocyte Effacement (LEE)  and the murine pathogen Citrobacter rodentium, thus these bacteria known as “A/E pathogens.”Adhesion and colonization of the intestine are key steps in STEC infection. A group of STEC strains, formerly called enterohemorrhagic nt (LEE) . These snt (LEE) . One of bacteria . Additiobacteria , 8. Adhebacteria –11. The Studying the pathogenesis of STEC infection in human volunteers is not possible due to the risk developing HUS and dying. Ruminants do not develop diseases due to colonization by this bacterium. Although laboratory animals such as rabbits and mice have been used as models of STEC infection, none of them fully reproduces the symptoms and diseases observed in humans , 13. HowCurrently, there is no licensed vaccine against STEC for humans, and vaccines licensed for use in cattle reduce, but do not eliminate, the colonization and spread of these bacteria , 16. FurSeveral promising vaccine candidates for STEC have been developed based on proteins encoded in the LEE PAI . ExampleThe biotechnological revolution, particularly the advancements in gene synthesis, has opened new doors in the rational design of more efficient and cost-effective vaccines –34. VaccIn this study, we developed two chimeric proteins containing antigenic domains from seven virulence factors of STEC, including Stx2B, Tir, Intimin, AggA, EspA, Cah, and OmpT proteins. These chimeric proteins were combined and formulated with the AddaVax™ adjuvant . Subsequently, we evaluated the potential of these chimeras as vaccine candidates against STEC using the streptomycin-treated mouse model. Overall, our results show that this vaccine formulation elicited high levels of specific IgG antibodies and provided partial protection against STEC, including the LEE-positive O157:H7 and the LEE-negative O91:H21 strains. These results highlight the potential of chimeric proteins for the development of new vaccines against bacterial pathogens.r) mutants of STEC O157:H7 86-24 and STEC O91:H21 V07-4-4 strains, which were obtained from previous studies  server (The design and ) server . These t) server  with a t) server  with a t) server . For thiThe predicted three-dimensional structure of Chimera 3 (Chi3) and Chimera 4 (Chi4) was constructed using RaptorX  and PhyrE. coli expression and cloned into the pET52b(+) vector  with an N-terminal 10xHis-tag. The ClearColi® BL21(DE3) strain  was then transformed with recombinant plasmids containing synthetic genes. To purify recombinant proteins, transformant E. coli strains were grown in LB-Miller Medium containing ampicillin (100 µg/ml) at 37°C. When the culture reached an optical density ~1.2 at 600 nm, it was supplemented with 1 mM IPTG for 4 h. Subsequently, bacteria were harvested by centrifugation and resuspended with lysis buffer  followed by sonication. The pellet obtained after centrifugation was dissolved using 8M urea. Denatured chimeric proteins were further purified by using a Ni-column  in an AKTA FPLC system . The purified chimeric proteins were refolded and sterilized using a 0.22 μm filter before being stored in aliquots at -20°C until use. The concentration was determined by Bradford protein assay  with BSA as standard. Standard SDS-PAGE and Western blot confirmation determined the protein purity and molecular weight. In addition, the reactivity of Chimeric proteins to IgG and IgA present in human and cattle sera was assessed by ELISA assay, as previously described (Synthetic genes coding for Chi3 and Chi4 proteins were ordered from GenScript Biotech (USA). The nucleotide sequences were optimized for escribed , 29. HumIn general, vaccines containing multiple antigens have shown to enhance protective efficacy compared to those containing fewer antigens , 36, 37.ad libitum and maintained on a 12-h light cycle. Mice were monitored on a daily basis using a score system to assess the perception of pain, distress, and discomfort. The scoring sheet employed was a modified version of the supervision protocol proposed by Morton and Griffiths, 1985  (Protocol 19250-ODO-UCH). Female BALB/c mice (5−6 weeks old) were housed in a pathogen-free and biosafety level 2 facility with water and food hs, 1985 . BrieflyTwo types of immunization studies were performed, one active and one passive Figure 1For passive immunization experiments, a pool of sera was obtained from five actively immunized mice (Chi3 + Chi4 + AddaVax™) two weeks after the last booster immunization. As a control, another set of sera was obtained from five control mice (PBS + AddaVax™). IgG was then purified with the Melon™ Gel IgG Spin Purification kit  and the concentration of purified IgG was determined using the ELISA Flex kit – Mouse IgG . Then, ten female BALB/c mice (10−11 weeks old) were randomly distributed into two experimental groups (n=5 for each group). One group of mice was passively immunized, through the tail vein, with 200 µg of anti-Chi3/Chi4 IgG and two days later with a similar dose. The other group of mice served as control and received 200 µg of control IgG (2SO4 (50 µl/well). The absorbance of the solution in each well was determined at 450 nm using a Synergy HT microplate reader . The results were reported as the reciprocal of the highest titer, giving an optical density (OD) reading of at least the mean ±2 standard deviations compared to the baseline sera or fecal supernatants. Each sample was determined twice in duplicate, and results were shown as the mean reciprocal endpoint titer.Flat-Bottom 96-well ELISA plates  were incubated with a mixture of 1 µg of each chimeric protein diluted in 100 µl of Phosphate-Buffered Saline  overnight at 4°C. Then, they were incubated with 300 µl/well of blocking solution  for 1 h at 37°C. The plates were washed 3 times with 300 µl/well of PBS containing 0.05% Tween 20 (T-PBS). Mice sera or fecal supernatants were serially diluted in blocking solution (100 µl/well) and incubated for 1h at 37°C. After 4 washes with T-PBS (400 µl/well), secondary antibody-HRP conjugate anti-mouse IgG (Cat #610-1302), anti-mouse IgG1(Cat #610-103-040), anti-mouse IgG2a (Cat #610-103-041), anti-mouse IgG2b (Cat #610-103-042), anti-mouse IgG3 (Cat #610-103-042), or anti-mouse IgA (Cat #610-4306), all from Rockland Immunochemicals (USA), were diluted 1:2000 in blocking solution (100 µl/well) and incubated for 60 min at room temperature (RT). After 4 washes with Tris-buffered saline (TBS)  containing 0.05% Tween 20, TMB  substrate was incubated for 10 min at RT. The reaction was stopped with 2N Hin vitro with heat-killed STEC O157:H7 (2 μg/well). After 72 hours, cells were collected and washed with FACS stain buffer . Cells were incubated with purified rat anti-mouse CD16/32 antibody  to block non-specific binding to FcɣR. Subsequently, the staining was carried out with the following specific anti-mouse monoclonal antibodies: TCR β-FITC , IL4-PE Cyanine 7 , CD4-APC Cyanine 7  ; IFNg-Alexa Fluor 700 , IL17A-PE, CF594 , . For intracellular antigen staining, the cells were permeabilized with Foxp3/Transcription Factor Staining Buffer . For detection of cytokines expressed in CD4+ T cells, ex vivo stimulation of splenocytes was performed with phorbol 12-myristate 13-acetate   and Ionomycin (4 µg/mL)  for four hours at 37°C in the presence of Monensin (4 µg/mL) , and Brefeldine A (4 µg/mL) . To analyze the percentage of cytokines expressed in CD4+ T cells, we adjusted the gate according to the fluorescence minus one (FMO) control. The flow cytometry assay was performed in an 18-color flow cytometer (BD LSR FORTESSA X-20). The analysis from acquired cells was carried out in the FlowJo V10 software Spleens from immunized or control mice were collected two weeks after the last immunization. Then, cell suspensions were obtained by using a 40 µm cell strainer  and cold RPMI  supplemented with 10% FBS and 1% penicillin-streptomycin), after removing erythrocytes by Red Blood Cell (RBC) Lysis Buffer . Splenocytes were cultured in V-Shaped-Bottom 96-well plates  (approximately 4 X 105 cells/well) at 37°C, 5% CO2 in RPMI supplemented with 10% FBS and 1% penicillin-streptomycin. Then, splenocytes were stimulated ® Ultra 15 ml tubes  and subsequently ultracentrifuged at 235,000 x g for 2 h at 4°C. The pellet (OMVs) was resuspended in 1 ml of 20 mM TRIS-HCl (pH 8.0) and stored at -80°C until use.The OMVs were isolated as previously described , with moTEM visualization and immunogold labeling were performed as previously described , with moTwo different infection protocols were carried out using the streptomycin-treated mouse model .ad libitum water containing streptomycin (5 g/l) for two days prior to infection. In addition, the stool was confirmed to be free of streptomycin-resistant E. coli at the time of infection. Bacterial strains were grown overnight in low-glucose DMEM medium containing 50 µg/ml streptomycin at 37°C with shaking. Before inoculation, food and water were restricted overnight (12 h). The next morning, mice were inoculated orally with 50 µl of bacterial suspension containing 1×109 CFU of STEC O157:H21 86-24 strain or 1×107 CFU of STEC O91:H21 V07-4-4 strain, which are non-lethal doses, according to previous studies . The weight of the mice was recorded every three days.Within the second challenge study, the passively immunized mice were inoculated orally following the second infection protocol , embedded in paraffin wax, and sectioned at 5 μm. The sections were then stained with hematoxylin and eosin (H/E). Pathological evaluation of the H/E-stained tissue sections was carried out by a pathologist blinded to the experimental design.5 cells/well) at 37°C, 5% CO2 in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin. The bacterial strains (STEC O157:H7 or STEC O91:H21) were grown overnight in low glucose DMEM at 37°C with agitation. The next day, an aliquot was diluted 1:50 in the same medium and incubated at 37°C for 3-4 h with agitation. The serum from five mice immunized with Chi3 + Chi4 + AddaVax™ was pooled and inactivated at 56°C for 45 min. Sera from five non-immunized mice (PBS + AddaVax™) were pooled, inactivated, and used as a control. Next, an inoculum of ~106 CFU of the bacteria was incubated in DMEM medium or DMEM medium containing inactivated hyperimmune or control serum at a 1:5 dilution for 30 min at 37°C with gentle agitation. After that, the bacteria were used to infect Caco-2 cells for 2 h at 37°C with 5% CO2. Five washes with PBS removed non-adherent bacteria, and adherent bacteria were recovered by lysis with 0.1% Triton X-100, serially diluted in PBS, and plated on MacConkey agar plates for enumeration. The result was expressed as the percentage of adherent bacteria in relation to the number of bacteria added, normalized with the percentage of adherent bacteria that were incubated in DMEM medium (without serum). The assay was performed twice in duplicate.Caco-2 cells were cultured in 24-well plates (approximately 4 X 106 CFU of bacteria (STEC O157:H7 or STEC O91:H21) was incubated with sera from immunized mice or control sera at a dilution of 1:5, either active or inactivated, and inactivated sera supplemented with exogenous mouse complement . The incubation was for 1 h at 37°C with slight agitation. Surviving bacteria were determined by serial dilutions and plating on MacConkey agar plates. The serum bactericidal activity was calculated using the equation: /bacterial CFU in control sera X 100. The assay was performed twice in duplicate.Serum bactericidal assays were performed as described , with moAll statistical analysis was done using GraphPad Prism software (v9.0) . For all statistical tests, a P value of <0.05 was considered significant. Statistical differences in humoral and cellular responses between groups were determined by Mann-Whitney test. Statistical differences in bacterial colonization and body weighs between groups were determined by two-way ANOVA test, followed by Sidak’s multiple comparison test. Statistical differences between groups in the histological analysis were determined by two-tailed Student’s t test. Statistical differences between groups in the adhesion assay experiments were determined by Mann-Whitney test. Statistical differences between groups in the Serum bactericidal activity assay were determined by Kruskal-Wallis test.For the rational design of a chimeric protein-based vaccine against STEC, antigenic proteins previously evaluated as immunogens in a mouse model were prioritized. Among these proteins are the B subunit of the Shiga toxin 2 (Stx2B), Tir, Intimin, AggA, EspA, Cah and OmpT. in silico using several immunobioinformatics tools, as described in material and methods section. As a result, several epitopes were predicted, and some of them were located consecutively or partially overlapping, suggesting the existence of antigenic domains  strain and subsequently purified by column chromatography. In addition, analysis of these proteins by SDS-PAGE indicated that they are stable and have the predicted molecular weight . Subsequently, mice were immunized following the protocol described in Notably, after three doses of the vaccine formulation, mice generated significantly higher levels of IgG anti-Chi3/Chi4 antibodies in both serum and feces compared to control mice  of STEC O157:H7 Figure 4In marked contrast to the IgG antibody response, the levels of anti-Chi3/Chi4 IgA antibodies in serum and feces  were almost undetectable, and no differences were observed between groups , where they bind to virulent bacteria expressing LEE, leading to their elimination by neutrophils that migrate through the epithelium. In contrast, the “avirulent” C. rodentium population is not opsonized by IgG but is eventually excluded by the microbiota.The murine pathogen i et al. . In thisA or IgM . More reA or IgM  describeRegarding STEC, it has been reported that vaccine candidates based on proteins encoded in LEE  and on Stx2B induce specific IgG antibodies that reduce the shedding of this pathogen in mice , 27, 77.In passive immunized mice, protection against colonization by STEC O157:H7 was not observed Figure 7E. coli, such as the O91:H21 strain V07-4-4, can cause kidney damage and, in some cases, even lead to death  (Protocol 19250-ODO-UCH), Universidad de Chile.In vivo experiments, DM, RG-B, PP, DS, CV, LG, and AO. In vitro experiments, DM, RG-B, PP, and LG. Histological analysis, HM. Data interpretation and writing the manuscript, DM and LC. Review and edition, LC and RV All authors contributed to the article and approved the submitted version.Conceptualization and experimental design, DM, RV, and LC.


Macrophages (MΦs) are the most abundant leukocytes in mammalian ovaries that have heterogeneity and plasticity. A body of evidence has indicated that these cells are important in maintaining ovarian homeostasis and they play critical roles in ovarian physiological events, such as folliculogenesis, ovulation, corpus luteum formation and regression. As females age, ovarian tissue microenvironment is typified by chronic inflammation with exacerbated ovarian fibrosis. In response to specific danger signals within aged ovaries, macrophages polarize into different M1 or M2 phenotypes, and specialize in unique functions to participate in the ovarian aging process. In this review, we will focus on the physiologic roles of MΦs in normal ovarian functions. Furthermore, we will discuss the roles of MΦs in the process of ovarian senescence, as well as the novel techniques applied in this field. It produces oocytes and multiple reproductive hormones including estrogen, progesterone and androgens . Unlike Macrophages (MΦs) are a central population of leukocytes in the innate immune system, with high levels of heterogeneity and plasticity in various tissues –11. They2MΦs constitute a vital component of innate immune system, and play important roles during infections and inflammation. They are often distributed in multiple tissues/organs of the body , 18. Pre2.1MΦs display strong heterogeneity and plasticity in their phenotypes and functions when exposed to various tissue microenvironments . In resp2.2Typically, activated MΦs express a variety of receptors, including co-stimulatory and antigen presenting molecules , chemotactic/activating cytokine receptors, pattern recognition receptors, and opsonic receptors . MΦs perWith MΦs polarization into M1 and M2 phenotypes, they exhibit enormous functional heterogeneity , 20. Spe3MΦs are the most abundant immune cells in mammalian ovaries. The number and distribution of these cells change during ovarian cycles , 29. Acc3.1+ M1 MΦs increases significantly around developing follicles, while the proportion of CD206+ M2 MΦs does not. Moreover, depletion of CD11c+ M1 MΦs using diphtheria toxin injection in mice leads to follicular impairment, vasculature impairment and ovarian hemorrhage, whereas depletion of CD206+ M2 MΦs does not  secreted by pituitary gland initiates ovulation, which involves preovulatory follicles rupture at the apex and extrude cumulus cell-oocyte complex . Animal Following ovulation, the remains of ovarian follicles undergo tissue remodeling, involving luteinization of follicular theca cells and granulosa cells, and vascularization, to develop a corpus luteum (CL) . The CL 4As a key reproductive organ in females, the ovary, however, ages early in life , 3, 39. 4.1Mounting evidence suggests that ovarian aging in mammals is associated with a sterile chronic inflammation in ovaries, which adversely affects ovarian function and oocyte quality , 52, 53.Recently, it has been proposed that MΦs are responsible for age-associated inflammation within the ovary , 14, 39.4.2In addition to chronic inflammation, stromal fibrosis within ovaries is another hallmark of mammalian ovarian senescence , 57. Ova4.3IL-6, TNF-α, IL-17, iNOS, ASC and NLRP3. By contrast, M2 phenotype did not show significant changes  technology to transcriptomic analysis of ovaries, follicles as well as MΦs subpopulations , 67. Div ovaries –74. Henc5Ovarian MΦs play pivotal roles in normal ovarian functions and ovarian aging. During reproductive senescence, danger signals within aged ovaries induce MΦs polarization into different M1/M2 phenotypes. Perturbation of balance of M1/M2 phenotypes in aged ovaries dictates chronic inflammatory milieu concurrent with stromal fibrosis, leading to follicular loss and ovarian dysfunction. To regulate the balance between M1 and M2 subsets might be a promising therapeutic strategy for women with advanced reproductive age. Future studies are still needed to further unravel the roles of MΦs in ovarian aging and develop a new approach to ameliorate ovarian decay.MT: Writing – original draft. MZ: Writing – review & editing. YS: Supervision, Writing – review & editing.


Phoenix dactylifera L.) fruits contain high concentrations of phenolic compounds, particularly flavonoids and other micronutrients, which impact human health due to their potent antioxidant, anti-inflammatory, and anticancer characteristics. In the present study, the effect of ethyl acetate, hydroethanol, hydromethanol, and aqueous extract from three date palm varieties  on phytochemical profiles and antioxidant and anticancer activities was investigated. Fruit extracts were screened for their antioxidant activity using the DPPH· method. Phenolic constituents were quantified and identified using HPLC-DAD. Extracts  were assessed for cytotoxicity on nine human cancer cell lines, i.e., MG-63, HCT116, MCF7, MDA-MB-231, HEPG2, HUH7, A549, H460, and HFB4, using the sulphorhodamine-B (SRB) assay. Results showed that the ethyl acetate extract of the Sukkari fruits has the greatest antioxidant potential with an IC50 value of 132.4 ± 0.3 μg·mL−1, while the aqueous extract of Ajwa date fruits exhibited the lowest antioxidant effect with an IC50 value of 867.1 ± 0.3 μg·mL−1. The extracts exhibited potent to moderate anticancer activities against the investigated cancer cell line in a source-dependent manner. Methanol extract of Siwi fruits exhibited the most potent anticancer activity (IC50 = 99 ± 1.6 µg·mL−1), followed by the same extract of Sukkari fruits with an IC50 value of 119 ± 3.5 µg·mL−1 against the cell line of human breast cancer (MDA-MB-231). Additionally, principal component analysis (PCA) was investigated to determine the relationship among the investigated traits and treatments. Our findings reveal that date palm fruit-derived extracts are excellent sources of biologically active constituents and substantiate their potential use in new anticancer strategies from natural resources.Date palm ( Date fruit is distinguished by its high content of simple monosaccharides, e.g., fructose and glucose 65–85%) ,2,3,4. R–85% ,2,3tivities ,8,9. In tivities .p-coumaric acid, and flavonoid glycosides, i.e., apigenin, quercetin, and luteolin [Nowadays, antioxidants derived from natural sources are being used for a wide range of purposes because they are ecologically safe, culturally more acceptable, and less hazardous than synthetic ones ,12. Moreluteolin ,14.These bioactive compounds must be extracted, identified, and quantified to develop functional foods and nutraceuticals. The extraction technique is an essential step in this process . HoweverAccording to the WHO, one of the main causes of death all over the world after cardiovascular diseases is cancer . Among cβ-glucans [β-glucans [β-carotene, which is also found in date fruits but in relatively small amounts [Diet and lifestyle play a significant role in cancer prevention and treatment . Date pa-glucans . Further-glucans . Moreove-glucans . Further-glucans . Dates c-glucans . Additio-glucans  and E in-glucans , vitamin amounts ,27,28. D amounts . Moreove amounts . It is w amounts .One of the extraordinary dates is the Ajwa date, which exists only in Madina, Saudi Arabia, with its high nutritional and medical value ,13,14. AThe highest content of proteins and nutrition exists in the variety of Sukkari . FurtherDespite several studies on the date palm varieties as safe and edible natural resources, to date, there is no data regarding the phytochemical profiles of the diversity of chemical constituents and the antioxidant and anticancer activities of the effects of ethyl acetate extract of three varieties of date palm . So, the present study was conducted to assess the effects of ethyl acetate, hydroethanol, and hydromethanol, as well as aqueous fruit extracts of three date palm varieties , on phytochemical profiles, antioxidants, and anticancer activities, which are expected to substantiate their potential use in new anticancer strategies from safe and natural resources.All materials used for the experimental work, including extractions and the analytical protocols, have been purchased from various suppliers, i.e., gallic acid, rutin, DPPH·, Folin–Ciocalteu reagent, and aluminum chloride anhydrous were acquired from Sigma Aldrich . Ethyl acetate, methanol, and ethanol were from Advent Chembio Pvt. Ltd.  and have been distilled before use. Sodium carbonate was from Fluka . Nineteen pure phenolic compounds  for quantification were acquired from Sigma-Aldrich .Ajwa and Sukkari date palm fruits were obtained from a date factory in Al-Madinah al-Munawwarah City, Saudi Arabia. Siwi date fruits were obtained from the date factory in Fayoum City, Egypt. The samples were randomly selected from a semi-dry batch without consideration for size, shape, color, or appearance. Seeds were removed from their fruits, air-dried at room temperature, then powdered by a laboratory blender , and stored in airtight bottles at −80 °C  until use.2O, 70%), hydromethanol , and water (H2O) in conical flasks. Using the ultrasound-assisted solvent extraction (UASE) technique, the extraction was carried out by placing the conical flasks in a Probe Sonicator homogenizer  for 30 min [A known amount of each sample was weighed (50 g/250 mL) and extracted individually with ethyl acetate (EtOAc), hydroethanol  was determined according to A.O.A.C. . MoisturThe determination of proteins in terms of nitrogen was performed using the micro Kjeldahl method . The nitrogen value was converted to protein by multiplying by a factor of 6.25. The ash values were obtained by heating date fruit pulp (5 g) on the Bunsen burner until the formation of fumes became obvious, at which point the sample was moved into the furnace at 550 °C and remained in it until a gray-colored sample was formed . After cThe Anthrone method  was used2O2 for 30 min after heating at 25 °C. Next, after cooling, 30% of the 1 mL of H2O2 aliquot was mixed and filtered. Using 20 mL of distilled water, the filtrate was diluted [Powdered samples of the dates were subjected to acid digestion, and 0.5 g of powdered samples was digested with 3.5 mL each of conc. sulfuric acid and 30% H diluted . Then th2O) and 5 mL of 6 M hydrochloric acid (HCl), then heated for 24 h at 100 °C, then filtrated, and the obtained filtrate was used for the analysis. HPLC analysis was conducted using an Agilent 1260 system , a 1260 binary pump equipped with a 1260 diode array fluorescence detector, and an Eclipse Plus C18 column , using (A) sodium phosphate dibasic and sodium borate buffer, pH 8.2 (A) and (B) ACN: MeOH: H2O 45:45:10, as the mobile phase. Using a gradient series of 98% A and 2% B (0 min); 98% A and 2% B (0.84 min); 43% A and 57% B (33.4 min); 0% A and 100% B 382 (33.5 min); 0% A and 100% B (39.3 min); 98% A and 2% B (39.4 min); 98% A and 2% B 383 (40.0 min) for the elution, column temperature was preserved at 40 °C. The DAD was observed at 338 nm (bandwidth 10 nm). The fluorescence detector was adjusted as follows: from 0 to 27 min at 340/450 nm (excitation/emission) and from 27 to 35 min at 266/306 (excitation/emission).The amino acid makeup of the analyzed date fruits was determined using the technique outlined by Jajic et al. . One gra50) was calculated by the analysis of the linear regression of the dose-response curve, plotting inhibition percentage versus the concentration of the extract (10–320 µg·mL−1). All measurements were carried out in triplicate. Control experiments  were executed similarly and simultaneously as the dates palm extract assays.The DPPH· radical-scavenging activity (DPPH· RSA) of all the extracts was calculated by the 2,2-diphenyl-1-picrylhydrazyl (DPPH·) free radical . The conThe amount of total phenolic in the dates palm extracts was measured by the Folin–Ciocalteu reagent using the procedure of Yu et al. . The totv/v) (solvent B) as eluent, and a 0.8 mL/min flow rate for the whole run (70 min). The resulting peaks were detected simultaneously at 280 nm for the benzoic acid derivatives and 320 nm for the cinnamic acid derivatives. Using a 0.45 µm Acrodisc syringe filter , all samples were filtered before injection according to Kim et al. [HPLC analysis was performed for the identification of phenolic constituents by HPLC-DAD analysis on an Agilent 1100 series liquid chromatography pump equipped with an autosampler and a diode-array detector, using an Eclipse XDB-C18  with a C18 guard column , using acetonitrile (solvent A) and 2% acetic acid in water  assay [3 cells/well in 96-wells. They were allowed to connect for 24 h before extract incubation, following the treatment of the cells with concentrations of 62.5, 125, 250, and 500 µg·mL−1 for MG-63, HCT116, MCF7, MDA-MB-231, HEPG2, HuH7, A549, H460, and HFB4 cells. A triplicate for each concentration was used, and incubation was extended for 48 h. Using DMSO as a control vehicle (1% v/v). After 48 h, cells were treated with 20% trichloroacetic acid to be fixed and stained with 0.4% SRB dye. Each well’s optical density (O.D.) was determined at 570 nm using an ELISA microplate reader . The mean survival fraction at each extract concentration was determined: O.D. of the treated cells divided by the control cells O.D.Human osteosarcoma cell line (MG-63), human colon cancer cell line (HCT116), human breast cancer cell lines (MCF7 and MDA-MB-231), human hepatocellular cancer cell lines (HEPG2 and HuH7), human lung cancer cell lines (A549 and H460), and human normal melanocyte cell line (HFB4) were used for the screening and were obtained from the American Type Culture Collection  and were maintained at the Pharmacology Unit Cancer, Biology Department, National Cancer Institute (NCI), Cairo University, Cairo, Egypt, in DMEM containing 10% fetal bovine serum, 0.2% NaHCOB) assay . FleetinUsing the statistical software program Genstat versions: 14.2 , data were examined using the analysis of variance (ANOVA) approach. Utilizing the Duncan multiple range test (DMRT) with a significance level of 0.05, the variance between means was compared. The principal component analysis (PCA) was carried out utilizing the R version 4.2.3 statistical software package factoextra .The average chemical composition of the three studied date varieties is presented in Dates are well known for their high sugar content; the total sugar level of the samples ranged from 75.2 ± 1.75 to 80.2 ± 2.02%, with a significant difference between Ajwa and Siwi/Sukkari. The Ajwa date showed the lowest total carbohydrate content. The reducing sugars represented most of the sugars found in the studied date varieties, where the percentage of reducing sugars ranged between 71.5 ± 1.25 and 75.6 ± 1.05, and the Sukkari date recorded the highest percentage, while the lowest percentage was in the Ajwa variety. Our findings are similar to those described by Perveen and Bokahri  and grea−1 DW) followed by phosphorus (109.9 ± 2.07–231.3 ± 6.25 mg 100 g−1 DW), magnesium (91.59 ± 3.52–151.1 ± 2.32 mg 100 g−1 DW), and calcium (55.73 ± 1.87–128.7 ± 3.22 mg 100 g−1 DW). The results support earlier research that showed dates had a high potassium concentration [−1 DW), followed by zinc, while copper was recorded as the lowest concentration in all samples. The results showed that the Ajwa date was superior to Siwi and Sukkari in their content of macro- and micro-elements, with highly significant differences except for iron, where Sukkari and Siwi dates recorded a greater amount than the Ajwa dates. Siwi variety recorded higher iron content with 6.81 ± 1.69 mg/100 g−1 of DW.ntration . RegardiThe obtained results agreed with those described by Siddeeg et al. and Trabzuni et al. for the Sukkari date ,54, by EAlthough date fruits are not a strong source of protein, date protein contains most of the essential amino acids needed for the human metabolic system, notably cell growth and regeneration . The resThe non-polar amino acid proline was present in the highest amounts in the Siwi date and was the most prevalent amino acid in all studied date types. Additionally, the results revealed that all investigated dates contained high concentrations of aspartic acid, leucine, phenylalanine, and glutamine. Histidine recorded the lowest concentration among the identified amino acids in all date varieties. Ali et al., Hamad et al., and Khaled et al. obtained highly similar results for Ajwa and Sukkari dates grown in Saudi Arabia ,59,60. IIt is observable that the Sukkari date had higher concentrations of aspartic acid and tyrosine than the other varieties, while it had the lowest concentration of leucine compared to the Siwi and Ajwa dates. Ajwa date was reported to have ample proteinogenic and non-proteinogenic amino acids . The non−1 dried extract) had the highest TPC level, followed by Ajwa date EtOAc extract (23.97 ± 0.29 mg GAE g−1 dried extract) and Siwi date EtOAc extract (16.22 ± 0.30 mg GAE·g−1 dried extract), while the TPC of the EtOH/H2O 70%, MeOH/H2O 80%, and H2O in the Ajwa date were the highest  than the Sukkari and Siwi date in all extracts except the EtOAc extract.It is well known that phenolic and flavonoid substances have a direct impact on biological processes in plants, including antioxidant , antibac−1 dried extract in EtOAc to 1.17 ± 0.26 mg RE·g−1 dried extract in H2O in the case of Sukkari, almost as much as the differences in phenolic contents. As displayed in −1 dried extract) and Siwi date EtOAc extract (13.06 ± 0.30 mg RE·g−1 dried extract), whereas the order of TFC in the distinct other extracts of the three varieties of dates  followed the sequence EtOH/H2O 70% extracts > MeOH/H2O 80% extracts > H2O extracts, with the TFC ranging from 10.28 ± 0.29 to 1.17 ± 0.26.The TFC ranged from 17.40 ± 0.26 mg RE·g2O extract has much greater TPCs than the alcoholic extract, particularly in Ajwa dates, contrary to our result. In comparison to the studies of Al-Farsi et al., Mohamed et al., and Salah et al., the amounts of phenolics and flavonoids were greater in this study [According to Saleh et al. , the H2Ois study ,63,64. Tis study . Furtheris study . Therefois study .The antioxidant effect of the three date varieties was measured using DPPH·. A significant variance among the date varieties was recorded regarding the type of solvent applied for extraction and the amounts and quality of phenolic and flavonoid components in each extract. 50 value of 132.4 ± 0.3 μg mL−1, while the aqueous extract of Ajwa date fruits showed the lowest antioxidant effect with an IC50 value of 867.1 ± 0.3 μg mL−1. The EtOAc extract of all date varieties has the highest antioxidant activity of all the extracts of the three date varieties, as shown in 2O extract exhibited the lowest level of antioxidant activity among all the extracts of the three date varieties  (Our results showed that the ethyl acetate extract of Sukkari fruits has the highest antioxidant activity with an ICSukkari) . AlthougBased on the above analysis of the antioxidant effect, the TPC and TFC of the examined extracts of different date types and the EtOAc extract of the examined date fruits were analyzed using HPLC-DAD analysis, revealing the presence of different phenolic and flavonoid compounds that were identified through the comparison with the available standards according to UV data and the retention times .−1), syringic acid (27.815 µg·g−1), and sinapic acid (22.860 µg·g−1). At the same time, the major phenolic compounds detected in Sukkari extract were p-hydroxybenzoic acid (81.34 µg·g−1), catechin (63.30 µg·g−1), chlorogenic acid (50.35 µg·g−1), ferulic acid (122.8 µg·g−1), sinapic acid (51.73 µg·g−1), and kaempferol (20.47 µg·g−1). Therefore, considering that major compounds have a concentration greater than 20 µg·g−1, we found that the Sukkari cultivar had six major compounds whereas the Ajwa cultivar had three, and according to this consideration, there were no compounds with a higher concentration in the Siwi cultivar. However, since there is a scarcity of research on phenolic and flavonoid constituents in ethyl acetate extract from date fruits, we could not compare it with earlier work.The major phenolic constituents of Ajwa date included p-hydroxybenzoic acid (59.127 µg·gThe phenolic (phenolic acids and flavonoids) compounds in the ethyl acetate extract of Sukkari dates are present in concentrations significantly higher than their counterparts in Ajwa and Siwi dates. In addition, there are also detected compounds in the Sukkari date with high concentrations devoid of Ajwa and Siwi cultivar extracts. The following are the phenolic and flavonoid compounds detected in Sukkari extract alone, without the other extracts: (a) Kaempferol, which is effective in scavenging radicals and combating cancer ,67, (b) 2O 70%, and MeOH/H2O 80% extracts at concentrations ranging from 62.5 to 500 μg·mL−1 after exposure to the tested date varieties for 48 h. was assessed in human cancer and normal cells using the sulphorhodamine-B (SRB) assay. Human osteosarcoma cell line (MG-63), human colon cancer cell line (HCT116), human breast cancer cell lines (MCF7 and MDA-MB-231), human hepatocellular cancer cell lines (HEPG2 and HuH7), human lung cancer cell lines (A549 and H460), and human normal melanocyte cell line (HFB4) were used for the screening.The cytotoxic activity of EtOAc, EtOH/H50; µg·mL−1) all examined extracts against the tested cancer cell lines is shown in 2O 80% extract of Siwi fruits exhibited the most potent anticancer activity (IC50= 99 ± 1.6 µg·mL−1), followed by the same extract of Sukkari fruits with an IC50 value of 119 ± 3.5 µg·mL−1 against the cell line of human breast cancer (MDA-MB-231). As a result, Siwi dates have a greater impact than expected. This effect on this type of cancer cell in particular may be due to components other than phenolic compounds, i.e., mono and sesqui-terpenes that are not detected herein in the Siwi date, that might be responsible for this effect [Cytotoxicity of  in the Siwi date variety. They identified 20 monoterpenes and 5 sesquiterpenes, beside quercetin, and 1 phenolic acid, namely coumaric acid, in the non-polar solvents by GC-MS/ESIMS, which showed positive radical scavenging activity and moderate antibacterial activities against Gram (+) and Gram (−) bacteria. The four extracts displayed varying cytotoxic activities against HeLa and HEK293T cancer cell lines, with higher ethyl acetate and MeOH/H2O 70% extracts and slightly less for both n-butanol and chloroform extracts. These results are consistent with our findings and interpretations.To the best of our knowledge, our study is the second report that has dealt with the Siwi variety biologically and phytochemically. The Siwi variety was first examined by El-Sayed et al. , who rep50 values less than 125 μg·mL−1 could be a promising candidate for further advancement as a cancer therapeutic agent, whereas samples with IC50 values between 125 and 5000 μg·mL−1 were regarded to have moderate potential for future development into a cancer therapeutic agent [Several studies proposed that samples with ICic agent ,81,82,83Natural substances have a long history of being used as cancer prevention agents. It has chemo-preventive properties. Several studies have shown that numerous plant-based substances, including taxol, vincristine, and vinblastine, have potent anticancer activities with little or no adverse effects . Ajwa daPhoenix dactylifera L. includes phenolic acids, carotene, polyphenols, anthocyanins, and tannins, among other phytochemicals. Another investigation on the phenolic content of Ajwa dates reported the existence of three significant phenols in pharmacology: catechin, caffeic acid, and rutin [nd rutin ,85,86,87nd rutin .4 in rat hepatocytes.El et al.  reportedParanthaman  revealedPrincipal component analysis (PCA) was employed to evaluate the association among the investigated traits and treatments, as shown in 2O extract) in all varieties to hydroalcoholic and non-polar solvent (EtOAc extract) (2O), T8 (Siwi × H2O), and T12 (Sukkari × H2O)) were much more spread out, implying dissimilarity. Dim 1 divided the treatment levels into two groups: the cancer cell lines and TPC and TFC were situated on the positive side, but those of moderate and lower values were located on the negative side (2O 70%), T3 (Ajwa × MeOH/H2O 80%), T6 (Siwi × EtOH/H2O 70%), T7 (Siwi × MeOH/H2O 80%), T10 (Sukkari × EtOH/H2O 70%), and T11 (Sukkari × MeOH/H2O 80%) or TPC and TFC (T1 (Ajwa × EtOAc), T5 (Siwi × EtOAc) and T9 (Sukkari × EtOAc)). Cancer cell lines and their attributes were associated with TPC and TFC in the EtOAc extracts of three varieties of date tested (T1 (Ajwa), T5 (Siwi), and T9 (Sukkari)) in Dim 1, whereas extraction yields and IC50 were associated with polar extraction. The adjacent vectors of traits reflect a strong positive association, while vectors with larger angles prove a weak association, and opposite vectors (at 180 °C) reveal a negative relationship. A strong positive association was observed among various solvents  and all their attributes, while there was a negative association with extraction yields and IC50 values. As expected from the findings previously discussed, different solvents and related traits were positively correlated and opposite those of extraction yields and IC50 values. Moreover, a significant difference was detected between ethyl acetate extracts and other extracts tested. Hence, the PCA biplot supported the aforementioned presented findings.The first two PCAs (Dim 1 and Dim 2) accounted for 85.70% of the variability. Dim 1 explained 63.6% of the variation and was related to decreasing the polarity gradient from polar solvent (Hextract) . The incive side . Dim 2 e50 values. This outcome hints that the biologically active components of the date fruits Ajwa, Siwi, and Sukkari may be effective natural anticancer medicines. Unlike current anticancer drugs, these findings open up the possibilities of using date palm fruit-derived extracts as a new anticancer strategy from safe and edible natural resources.The work aimed at studying the nutritional value, antioxidant, and anticancer activity effects of ethyl acetate, hydroethanol, hydromethanol, and aqueous fruit extracts of three date palm varieties , as well as the phytochemical profiles of their ethyl acetate extract only. The ethyl acetate extract of Sukkari fruits displayed the maximum antioxidant activity. Furthermore, all the examined extracts exhibited strong to moderate anticancer activities against the examined cancer cell lines in a source-dependent manner. Moreover, the PCA biplot reflects a strong positive association among various solvents  and all their attributes, while there was a negative association with extraction yields and IC


T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to a persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3, and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct, with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation. T cells become activated in the cellular interaction with antigen-presenting cells (APC). Recognition of a peptide/MHC complex on the APC surface by the T cell receptor (TCR) mediates immunological specificity and provides a strong stimulatory signal. This signal is tuned by the engagement of multiple costimulatory and coinhibitory receptors engaging ligands on the APC. An important challenge is to determine unique and overlapping functions among these coregulatory receptors. T cells express five key inhibitory receptors, CTLA-4, PD-1, LAG3, TIM3, and TIGIT ,3. Whiletrans-Golgi network, and TIM3 accumulates at the cellular interface with APCs [Vesicular localization of individual inhibitory receptors has already been characterized, that of CTLA-4 most extensively ,7. Briefith APCs ,24. HoweBALB/c CL4 TCR transgenic mice (RRID:IMSR_JAX:005307)  were breThe complete medium consisted of RPMI-1640 plus L-glutamine  supplemented with 10% FBS , 50 µM β-Mercaptoethanol (Gibco), and PenStrep (Gibco) at 100 U/mL penicillin and 100 µg/mL streptomycin. Renca cells (RRID:CVCL_2174) were maintained in complete medium. Renca cells are negative for CD86 (unpublished) and CEACAM-1  but exprAntibodies used are described in the order: antigen, fluorophore if applicable, clone, supplier, dilution, RRID:For Western Blotting:SNX1, polyclonal, Abcam, 1:1000, RRID:AB_296721SNX2, 13, BD Biosciences, 1:1000, RRID:AB_398834SNX5, polyclonal, Proteintech, 1:1000, RRID:AB_2192708SNX6, D-5, Santa Cruz Biotechnology, 1:1000, RRID:AB_10842310SNX27, polyclonal, Proteintech, 1:1000, RRID:AB_10888628AP-1 γ adaptin, polyclonal, Proteintech, 1:1000, RRID:AB_2058209AP-2 α adaptin, polyclonal, Proteintech, 1:1000, RRID:AB_2056316VPS35, polyclonal, Abcam, 1:1000, RRID:AB_10696107GLUT1, EPR3915, Abcam, 1:1000, RRID:AB_10903230Polycystin-2, polyclonal, Santa Cruz Biotechnology, 1:1000, RRID:AB_2163376Anti-mouse IgG, Alexa Fluor 680, Invitrogen, 1:20,000, RRID:AB_2633278Anti-rabbit IgG, Alexa Fluor 800, Invitrogen, 1:20,000, RRID:AB_2633284For immunostaining and T cell priming:EAA-1, c45b10, Cell Signaling Technology, 1:200, RRID:AB_2096811Anti-rabbit IgG, Alexa Fluor 568, Invitrogen, 1:400, RRID:AB_143157CD3ε, 145-2C11, Biolegend, 5 µg/mL, RRID:AB_11149115CD28, 37.51, BioLegend, 1 µg/mL, RRID:AB_11147170CD3ε, FITC, 145-2C11, Biolegend, 1:50, RRID:AB_312671CD4, PerCP/Cy5.5, RM4-5, BioLegend, 1:50, RRID:AB_893326CD45, APC, 30-F11, BioLegend, 1:50, RRID:AB_312976CD8α, APC/Fire750, 53-6.7, BioLegend, 1:50, RRID:AB_2572113CTLA-4, PE, UC10-4B9, BioLegend, 1:50, RRID:AB_313254PD-1, BV605, 29F.1A12, BioLegend, 1:50, RRID:AB_2562616LAG3, APC, C9B7W, BioLegend, 1:50, RRID:AB_10639935TIM3, PE/Cy7, RMT3-23, BioLegend, 1:50, RRID:AB_2571933TIGIT, BV421, 1G9, BioLegend, 1:50, RRID:AB_2687311For the retroviral expression of all receptors, stimulatory and inhibitory, we used the same previously described plasmid backbone where expression of a receptor with a C-terminally fused eGFP is driven by the Mouse Moloney Leukemia Virus long terminal repeat ,30,31.+ T cells, red blood cells were removed from dissociated spleens using ACK lysis buffer (Gibco), and splenocytes were plated at a density of 5 × 106 cells per well in 1 mL of complete medium in a 24-well plate. KdHA peptide (IYSTVASSL) was added to a final concentration of 1 μg/mL, and cells were incubated overnight. Primed cultures were stringently washed five times in PBS to remove unbound peptides and then retrovirally transduced if desired (detailed below). Subsequently, cells were re-plated at 4 × 106 cells per well in 2 mL IL-2 medium in a 24-well plate. Thus, further CTL expansion was independent of TCR engagement. CTLs were sorted and imaged on day four. For 5C.C7 CD4+ T cells, dissociated lymph node cells were plated at a density of 5 × 106 cells per well, in 1 mL of complete medium, in a 24-well plate. MCC peptide (ANERADLIAYLKQATK) was added to a final concentration of 3 µM. T cells were transduced after overnight culture (detailed below). Following transduction, cells were re-plated at 4 × 106 cells per well in 2 mL IL-2 medium in a 24-well plate. T cells were sorted and imaged on day four or five.Procedures used for the isolation and stimulation of TCR transgenic T cells have been extensively detailed elsewhere ,29,30,316 cells per well. Thus, CTLs were expanded with continuous TCR engagement. At 48 h post T cell activation, cells were taken out from the coated plate and stained.For the flow cytometry experiments to quantify endogenous inhibitory receptor cell surface expression, a 24-well cell culture plate was coated with 5 μg/mL αCD3 and 1 μg/mL αCD28 in phosphate-buffered saline (PBS) at 4 °C overnight. Red blood cells were removed from dissociated BALB/c spleens using ACK lysis buffer (Gibco), and splenocytes were plated onto a 100 mm tissue culture dish for 2 h at 37 °C. Non-adherent cells were taken out and seeded onto the αCD3/αCD28-coated plate at 5 × 10g, at 32 °C. Following centrifugation, the supernatant was replaced with an IL-2 medium.Procedures and plasmids used for retroviral transduction of TCR transgenic T cells have been extensively detailed elsewhere ,30,31. B2 and 0.5 mM MgCl2) plus DRAQ7. A BD Influx cell sorter (BD Bioscience) was used to isolate GFP-positive T cells [6 Renca cells were pulsed with KdHA peptide at a final concentration of 2 µg/mL for 1 h. These pulsed Renca cells will be referred to as Renca+HA cells. Glass-bottomed 384-well optical imaging plates (Brooks Life Science Systems) were used for all imaging experiments. Imaging was done at 37 °C using a Perkin Elmer UltraVIEW ERS 6FE confocal system attached to a Leica DM I6000 inverted epifluorescence microscope and a Yokogawa CSU22 spinning disk. A 40× oil-immersion lens (NA = 1.25) was used for all imaging experiments unless otherwise stated. Prior to imaging, 50 µL of imaging buffer was added to the well, followed by 5 µL of T cells. T cells were allowed to settle for several minutes. Once a monolayer of T cells had formed, 4 µL of Renca+HA cells were carefully added to the top of the well. Images were acquired for 15 min. Every 20 s, a z-stack of 21 GFP images (1 µm z-spacing) was acquired as well as a single, mid-plane differential interference contrast (DIC) reference image. After imaging, GFP files were saved as individual z-stacks for each time point, and both DIC and GFP files were exported as TIFF files for subsequent analysis.Approximately 72 h after retroviral transduction, T cell cultures were resuspended in ‘imaging buffer’  of the maximum intensity along that profile. The membrane-to-interior ratio of fluorescence intensity for each refined cell slice was calculated by splitting the cells in two based on distance from the cell surface. For this, the width of the membrane region was estimated by fitting orthogonal Gaussian profiles around the refined cell surfaces. To estimate membrane width for each cell, the smallest 5/20 (inhibitory receptors/TCRζ) Gaussian sigma values were averaged and multiplied by 2/3 . The fluorescence intensity within the membrane and interior regions was measured and extrapolated to three dimensions, assuming isotropy.CL4 CTL retrovirally transduced to express inhibitory receptor GFP fusion proteins were imaged on an Olympus IXplore SpinSR imaging system with a 60× oil immersion lens (NA = 1.50) at 37 °C. A z-stack of 65 GFP images (0.25 µm z-spacing) was acquired, as well as a single, mid-plane differential interference contrast (DIC) reference image. The association of inhibitory receptor-GFP fluorescence with the cell edge was measured using the ModularImageAnalysis ) 37% HCl in 100% ethanol) at room temperature for 15 min and baked at 60 °C for up to 30 min. Then, 50 μL of coating solution  + 2.5 μg/mL ICAM1-Fc  was incubated overnight at 4 °C. Alternatively, the glass bottom of a 384-well MatriPlate was coated with 1 mg/mL biotin-BSA , followed by 200 µg/mL NeutrAvidin (ThermoFisher) and then 10 µg/mL biotin hamster anti-mouse CD3εantibody  and kept in the imaging buffer  FBS). No differences in TIRF imaging between the two coating methods were observed, and data were therefore pooled. Live TIRF images were acquired using a Leica AM TIRF microscopy multicolor system attached to a Leica DMI 6000 inverted epifluorescence microscope equipped with 488 nm and 561 nm laser lines. A Leica HCX PL APO 100× oil immersion lens (NA = 1.47) was used. Images were acquired at frame rate every 333 ms. For dual color experiments, the laser was switched between frames resulting in image acquisition in each color every 735 ms. The onset of T cell activation was determined as the first frame in a series of rapidly spreading TIRF signals.To image vesicular insertion into the plasma membrane, T cells were activated by a flat glass surface coated with αCD3ε mAb and soluble ICAM-1. The glass bottom of a 384-well MatriPlate  was washed with 50 μL 1% acid alcohol  was drawn across the widest region of a cell and divided into ten segments. The mean fluorescence intensity of each segment was measured and normalized to the segment with the highest fluorescent intensity.To determine the frequency of vesicular insertion, a machine learning-based image analysis algorithm was developed to automatically identify vesicular insertion events at the plasma membrane. The training data of more than 100 annotated events were chosen to show the rapid appearance of a fluorescence signal followed by a slower dispersion as a first category or the sustained maintenance of this signal as a second category. These changes in fluorescence intensity and dynamics are most consistent with the release of vesicular content and, less certain, the formation of a receptor cluster, respectively. Alternate explanations can’t be excluded. The algorithm contained four sequential steps. (i) Blur detection: During time-lapse TIRF imaging, the particle signal might become blurred due to focus drift. To resolve this issue, we developed a support vector machine (SVM) classifier [https://www.mathworks.com/matlabcentral/fileexchange/27314-focus-measure’ (accessed on 1 September 2022) as the feature set. The training data was generated by human annotation of time series images. The parameters in the SVM were optimized using 10-fold cross-validation. (ii) Particle detection: The general idea of particle detection is to identify particles using a two-sample t-test for local maximum versus local minimum. First, the region of interest (ROI) for particle detection for all time points was determined by 90% of Otsu’s thresholding [t-test. The sample for the candidate point was the pixel values of the five brightest pixels p-value of t-test. If the p-value is below a predetermined threshold (0.001 in this case), the point is considered a valid particle; otherwise, it is classified as a noise point. This process is repeated for all candidate points and all frames. (iii) Point tracking. After point detection, we used the simpletracker library ) to track all the particles over time. We used the nearest neighbor algorithm in the linkage with a linkage threshold of p-value of each particle over the local background via a two-sample t-test. The training data was generated by human annotation of time series images. The SVM classifier was trained on the annotated data with 10-fold cross-validation. The events were predicted for each particle at each time point. The output file of this analysis was a list of events with onset time, duration, coordinates within the entire imaging field, and classification. As only a small fraction of the identified events consisted of rapid fluorescence appearance followed by sustained maintenance, all events were included in the analysis.assifier  to identsholding  on the asholding . After tFor colocalization analysis in dual color TIRF experiments, colocalization over time was measured using a workflow compiled in the ModularImageAnalysis . Any remaining binary regions that had become merged were split apart using a distance-based watershed transform [3 to 500 μm3 accepted size range were discarded. Cells were tracked through time [ransform . Individransform , and anyugh time , and anyAPEX proteomics: 2 × 106 CL4 T cells transduced and FACS sorted to express CTLA-4-GFP, LAG3-GFP, or TIM3-GFP were incubated with 500 μM biotin-phenol  in IL-2 medium at 37 °C for 30 min. Cells were collected and incubated with 1 mM H2O2 (Sigma-Aldrich) for exactly 30 s and spun for 30 s at 311 g. It should be noted that biotinylation can, in principle, occur both in the vesicular lumen and at the cell surface. However, as the majority of the cellular pool of the inhibitory receptors is intracellular and as extracellular labeling requires the use of the enzymatically more active unmodified horseradish peroxidase [v/v) NP40, 1% (v/v) HALTTM protease and phosphatase inhibitor cocktail (ThermoFisher), pH = 7.5) for 20 min. To the 20 μL lysis buffer, prewashed streptavidin beads  were added per sample and rotated for 2 h. The beads were washed twice with wash buffer  and then wash buffer without NP40 and processed for proteomic analysis. Biotinylated proteins were digested into peptides and labeled with TMT 6-plex tandem mass tags. Using liquid chromatography–tandem mass spectrometry, peptide sequence information was obtained by an MS1 scan, and then the reporter group of the TMT 6-plex labels was cleaved to detect their specific m/z by MS/MS whose intensity represents the relative amount of the peptide from the corresponding samples [roxidase , the con samples . Mass sp samples . The sea samples , which sAPEX electron microscopy: 3 × 106 Renca cells were pulsed with 2 μg/mL KdHA peptide for 1 h. At least 1 × 106 APEX2-CTLA-4/LAG-3/TIM-3- GFP overexpressing CL4 T cells were sorted by FACS. Both cell types were suspended in 100 μL 10% FBS/PBS/1 mM CaCl2/0.5 mM MgCl2, mixed, and spun 30 s at 311 g. The supernatant was removed, and the cell pellet was incubated at 37 °C for 5 min. The cell pellet was fixed in 2.5% glutaraldehyde/3 mM CaCl2/0.1 M sodium cacodylate, pH 7.4 for 5 min at room temperature, then 1 h on ice. The cell pellet was washed with 0.1 M sodium cacodylate, pH7.4 (wash buffer), blocked in 30 mM glycine in wash buffer, then washed again in wash buffer, each step for 10 min. The wash buffer was replaced with a DAB + 0.03% H2O2 solution for 5 min. Cells were washed 10 min in wash buffer, followed by 30 min incubation with 1% osmium tetroxide. The cell pellet was washed with distilled water, fixed in 3% uranyl acetate for 20 min, washed with distilled water, and dehydrated through 70%, 80%, 90%, and 96%, and 3 times 100% ethanol, 10 min each. The pellet was infiltrated in 100% EPON resin, rotating overnight at room temperature, then embedded in fresh 100% EPON resin, polymerized in 12–24 h at 65 °C. Samples were cut into 20 μm sections and imaged by transmission electron microscopy.Indirect immunostaining for EAA1: 20,000 Renca cells in 50 µL complete medium were seeded in a 384-well plate (Corning) and incubated overnight at 37 °C. Renca cells were incubated with 1 µg/µL of HA peptide for 1 h. Then, 100,000 CL4 T cells transduced with GFP-tagged co-inhibitory receptors  were added and left for 10 min at 37 °C. Cells were fixed in 50 µL 4% PFA for 20 min at 4 °C. Each well was washed with 50 µL of PBS. The reaction was quenched with 50 µL of 50 mM NH4Cl for 10 min at 4 °C, and wells were washed three times with 50 µL PBS. Cells were permeabilized using 50 µL of 0.02% Triton X-100 in PBS for 20 min at 4 °C. Cells were washed with 50 µL of PBS and incubated with 50 µL of blocking solution (1% BSA in PBS) for 30 min at room temperature. Cells were stained with 50 µL anti-EEA1 mAb  at a dilution of 1:200 in 1% BSA in PBS with Fc Block at the same dilution overnight at 4 °C. Cells were washed three times with 50 µL of PBS. The wells were incubated with 50 µL of secondary antibody  diluted 1:400 in 1% BSA in PBS with Fc Block at 1:200 for 1 h at room temperature. Wells were washed three times and left in 50 µL of PBS at room temperature. Nuclei were stained with Hoechst 33258 (Invitrogen) diluted 1:5000 in PBS for 10 min at room temperature.Lysotracker/Mitotracker staining: 20,000 Renca cells in 50 µL complete medium were seeded in a 384-well plate (Corning) and incubated overnight at 37 °C. Renca cells were incubated with 1 µg/µL of HA peptide for 1 h. Then, 300,000 CL4 T cells transduced with GFP-tagged co-inhibitory receptors  were suspended in IL-2 medium at 37 °C containing either 65 nM LysoTracker® Deep Red (ThermoFisher Scientific) or 25 nM MitoTracker™ Red CMXRos for 1 h or 30 min, respectively. Following incubation, cells were resuspended in a 37 °C IL-2 medium. Then, 100,000 CL4 T cells were added and left for 10 min at 37 °C. Cells were fixed and stained with Hoechst 33258 as described in the EEA1 staining protocol.Image acquisition: All images were collected with a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DMi8 inverted epifluorescence microscope. An HC PL APO CS2 40× oil immersion objective (NA = 1.3) was used. GFP constructs were excited with a 488 nm line of the white light laser, Hoechst-stained nuclei with the 406 nm line of the diode laser, and LysoTracker/EEA1/MitoTracker with the 561 nm line of the white light laser. The pinhole was set to 1 Airy unit (AU). A zoom factor of 6 was used to acquire all the images.Colocalization analysis: All images were analyzed using the Fiji-ImageJ [i-ImageJ ,34 softw3 in volume were removed [Sets of images without Hoechst stain were analyzed using an image analysis workflow created using the MIA plugin  [Sets of images with Hoechst stain were analyzed using an image analysis workflow created using the MIA plugin  . Adjacenntation) . To calcntation) . Red andImmunostaining to determine the fraction of an inhibitory receptor on the cell surface: 3 × 106 day 3 BALB/c T cells were stained for surface receptors in 1% BSA/PBS for 30 min at room temperature. The surface-stained cells were washed twice with PBS. One third of the cells was directly analyzed by flow cytometry; two thirds of the cells were fixed with 750 μL fixation buffer  for 30 min, washed twice with 1× permeabilization buffer (10% (v/v) in ddH2O, BioLegend, #421002), stained again in 1× permeabilization buffer for 30 min at room temperature and analyzed by flow cytometry.g for 10 min at 4 °C. Two percent of the cell lysate supernatant was kept as an input sample, and the rest of the supernatant was incubated with the equilibrated GFP-trap beads for 1 h at 4 °C on a rotator. GFP-trap beads were washed three times with IP lysis buffer and once with IP lysis buffer without NP-40. Proteins were eluted from the beads and denatured in 2× sample buffer (4% SDS in lysis buffer) at 95 °C for 10 min. Proteins were detected by Western blotting using a LI-COR Odyssey scanner.Based on the pEGFP.C1 vector (Clontech), plasmids for the CMV promoter-driven expression of fusion proteins of GFP with either only the inhibitory receptor cytoplasmic domain or the joint transmembrane plus cytoplasmic domain were generated. HEK293T cells were grown to 90% confluence in 15 cm culture dishes and transfected with 15 μg DNA of GFP-inhibitory receptor cytoplasmic domain or transmembrane plus cytoplasmic domain constructs using 1 μL of a 10 mM polyethyleneimine stock solution in 5 mL Opti-MEM I reduced Serum Medium . GFP-trap beads  were aliquoted to 1.5 μL per sample and equilibrated by three washes in IP lysis buffer , pH = 7.4). HEK2393T cells were washed twice with ice-cold PBS, lysed by prechilled IP lysis buffer on ice, and centrifuged at 13,000× + 5C.C7 T cell receptor (TCR) transgenic T cells [p < 0.01) smaller fraction of the total fluorescence was located at the cell edge as opposed to the cell interior (p < 0.01) more clustered than that of any other receptors except for CD2 (p < 0.01) more clustered than that of the other three inhibitory receptors, PD-1, TIGIT, and TIM3  smaller fraction of the total fluorescence of all five inhibitory receptors as compared to that of the TCR was located at the cell edge as opposed to the cell interior (p < 0.0001) clustered than that of the other four inhibitory receptors and that of the TCR (p < 0.05) than that of the TCR, PD-1, TIGIT, and TIM3 . We use the interaction of CTL from CL4 TCR transgenic mice with Renca renal carcinoma cells as a model of tumor cell cytolysis . We haverst step A. Similainterior B. Using  the TCR C. The diand TIM3 B. The saand TIM3 .p < 0.01) less than the 59 ± 2% of the TCR, a stimulatory receptor that nevertheless also continuously cycles through vesicular compartments [To provide an estimate of which fraction of the inhibitory receptors is located at the plasma membrane versus the cell interior, we acquired a smaller image data set of live CL4 CTL expressing TCRζ or the five inhibitory receptors right at the resolution limit of confocal microscopy A. Even tartments ,61 , the MTOC reorients within two minutes from behind the nucleus to the cellular interface ,64. To dAs inhibitory receptors are localized in vesicles, one should be able to detect the insertion of such vesicles into the T cell plasma membrane upon T cell activation. To test this suggestion, we imaged the activation of CL4 T cells on glass coverslips coated with the stimulatory anti-TCR antibody 145-2C11 with or without the extracellular domain of the costimulatory ligand ICAM-1 using total internal reflection (TIRF) microscopy. CL4 T cells spread on the cover slips as an indication of activation A, and evTo determine whether the divergent insertion of vesicles carrying different inhibitory receptors resulted in distinct inhibitory receptor distributions across the T cell coverslip interface, we performed a line scan analysis of the TRIF data where we measured the fluorescence intensity in ten equally sized segments of a line of a width of 4.5 µm across the entire interface. The CTLA-4 distribution was focused consistently more on the center of the interface A–D. WhilIn summary, across all imaging assays, the five inhibitory receptors displayed distributions consistent with a predominant vesicular localization. This contrasted with the stronger enrichment of the TCR, the costimulatory receptors CD28 and CD2, and CD6 at the cell edge. When analyzed in conjunction with all imaging assays, distinct localization profiles emerged for all inhibitory receptors G,H. CTLATo corroborate distinct subcellular localization of inhibitory receptors in molecular terms, we used APEX2 proteomics ,66, a biOur extracellular fusion of inhibitory receptors to APEX2 allows for the labelling of vesicles harboring the receptors in electron micrographs . We coulp < 0.05) higher than that of the other three receptors investigated (p < 0.05) more strongly associated with endosomes than LAG3 and TIM3, reaching an average Pearson’s correlation coefficient of 0.45 ± 0.1. While a further investigation of the time dependence of vesicular associations is beyond the scope of this manuscript, the data suggest that vesicular associations vary over time and that such variation differs between the inhibitory receptors. We also investigated the association of inhibitory receptors with mitochondria, largely as a negative control but also as the APEX proteomics found enrichment of mitochondrial proteins such as the phosphate carrier Slc25a3 in the lumen of vesicles carrying LAG3. Colocalization with mitochondria was minimal, as expected (To further characterize vesicles harboring inhibitory receptors, we incubated CL4 CTL expressing GFP fusion proteins of the inhibitory receptors for 10 min with Renca target cells incubated with a high concentration, 2 µg/mL of HA agonist peptide. We then stained the cell couples with an antibody against EEA1 to label early endosomes and with Lysotracker and Mitotracker to label lysosomes and mitochondria, respectively. We automatically segmented the cytoplasm of individual T cells  and detestigated A,B. As sstigated C,D, suggstigated . At thisexpected E,F. OverThe cytoplasmic domains of the inhibitory receptors contain multiple sorting motifs conserved between mice and humans that may mediate the selective binding of key regulators of receptor trafficking A. To detTo strengthen the association with vesicular structures, we generated fusion proteins of GFP followed by the transmembrane and cytoplasmic domains of the inhibitory receptors and repeated the pull-down experiments. The distribution of these fusion proteins appeared more punctuate F, consis3 receptors [Here, we have shown that the majority of the cellular pool of five inhibitory receptors resides in the cell interior  in contreceptors , or funceceptors , can shoOur data are only the start of a comprehensive investigation of the subcellular localization of inhibitory receptors. Future work will have to characterize in detail how such distributions change during T cell activation as a function of time. The dynamic data E and theWhich processes limit plasma membrane localization of inhibitory receptors? A likely means to limit plasma membrane expression is constitutive endocytosis. Consistent with this suggestion, the endosomal marker transferrin receptor was highly enriched in the vicinity proteomes of all three inhibitory receptors studied, CTLA-4, LAG3, and TIM3 B. MoreovHow can distinct vesicular distributions of the five inhibitory receptors be maintained? Conceptually, distinct vesicular distributions can be achieved through receptor-specific recruitment into defined subsets of vesicles or through differential association with the same set of vesicular structures. Given the highly interconnected, dynamic, and molecularly complex nature of the vesicular system, these two options may simply be two ends of a continuous spectrum. Even though marker proteins such as EEA-1 or LAMP-1 are commonly used to distinguish different vesicle types, there are extensive contacts between the different vesicle types and vesicles with microtubules as principal mediators of vesicular trafficking ,75,76. IIt seems unlikely that distinct vesicle subtypes underpin the differential localization of the five inhibitory receptors in T cells. Sorting motifs in the inhibitory receptor cytoplasmic domains are overlapping A. PulldoUnderstanding the distinct distributions of the five inhibitory receptors has translational potential. Even without detailed mechanistic insight, it allows for unique roles of individual inhibitory receptors, even in T cells that express all five of them. Such unique roles are emerging. For example, in our work, blocking TIM3 but not PD-1 can reactivate suppressed CTL ,28. Furt