Journal: Cell reports

Article Title: Integration of T helper and BCR signals governs enhanced plasma cell differentiation of memory B cells by regulation of CD45 phosphatase activity

doi: 10.1016/j.celrep.2021.109525

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: APC-Vio770 Recombinant human anti B220(clone REA755) , Miltenyi Biotec , Cat# 130–110-849; RRID:AB_2658286.

Techniques: Negative Control, Recombinant, Purification, Staining, Gene Expression, Lysis, Immunoprecipitation, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Cell Isolation, Software, Microscopy

Journal: Cell Reports Medicine

Article Title: Vaccine-induced ICOS + CD38 + circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways

doi: 10.1016/j.xcrm.2021.100262

Figure Lengend Snippet:

Article Snippet: Anti-human Survivin (REA459) , Miltenyi , Cat # 130-106-741; RRID: AB_2653606.

Techniques: Recombinant, Staining, Gene Expression, Software

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Hepatitis B (Engerix-B) vaccination and experimental design. (Top) Timeline of vaccination and blood collection. (Bottom) Memory CD4 T cells were magnetically enriched and FACS-sorted from two time points (day 0 and day 60) for TCRβ repertoire sequencing. Peptide matrix pools were used to map CD4 T cell epitopes of the vaccine from peripheral blood mononuclear cells (PBMCs) collected at day 60 and to select single peptides. After 7 days of in vitro expansion, single peptide-specific and master peptide pool-specific CFSE low CD4 T cells from PBMCs collected at day 60 were FACS-sorted in two technical replicates for TCRβ repertoire sequencing. PBMCs collected at days 0, 60, 180, and 365 were stimulated with the master peptide pool (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry. ( B ) Vaccinee cohort can be classified into three groups as determined by anti-hepatitis B surface (anti-HBs) titer over four times points. Early-converters seroconverted at day 60, late-converters seroconverted at day 180 or day 365, and non–converters did not have an anti-HBs titer higher than 10 IU/ml at any of the time points.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Sequencing, In Vitro, Expressing, Flow Cytometry

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Scatter plot of the DNA-based TCRβ reads for each vaccinee at each time point. ( B ) Scatter plot of number of unique TCRβ amino acid sequences for each vaccinee at each time point, where the shape denotes the response as based on antibody titer. ( C ) Overview of unique TCRβ amino acid sequences in the memory CD4 T cell repertoire of each vaccinee. The bottom blue bar denotes those TCR sequences that were found at both time points. The green and red bars denote the number of unique TCR sequences at each time point. The total bar height thus represents the total number of unique memory CD4 T cell clonotypes sequences for a specific vaccinee. ( D ) Frequency of unique HBsAg-specific TCRβ sequences out of total sequenced TCRβ sequences between two time points for all vaccinees colored and faceted by group. ( E ) Change in frequency (clone read count/total counts) of those HBsAg-specific CD4 T cells present at both time points. The (ns) mark denotes a non-significant paired Wilcoxon signed-rank test (p-value = 0.7577). ( F ) Receiver operating characteristic (ROC) curve using R hbs to differentiate between age-matched early-converters and late-converters in a leave-one-out cross-validation at day 0. Age-matching was accomplished retaining only samples in the age range 40–55. A Wilcoxon test was used to confirm that there was no difference in age distributions between early- and late-converters (p-value = 0.60, mean EC = 44.5 years, mean LC 45.1 years). Diagonal line denotes a random classifier. Reported is the area under the curve (AUC) and its 95% confidence interval.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Biomarker Discovery

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Comparison of the memory CD4 TCRβ repertoire diversity, as shown by breadth (number of unique TCRs) and entropy (Shannon equitability index) between day 0 and day 60. Indices are available in . ( B ) Frequency of unique vaccine-specific TCRβ sequences out of total sequenced TCRβ sequences between two time points for all vaccinees colored by group. Frequencies are available in . ( C ) Sequenced CD4 + TCR memory repertoire of vaccinee H35 at day 60. Each TCR clonotype is represented by a node. TCRs are connected by an edge if their Hamming distance is one. Only clusters with at least three TCRs are shown. TCR clonotypes in red are the vaccine-specific TCRβ sequences that were not present prior to vaccination. ( D ) Frequency of vaccine-specific TCRβ sequences within memory CD4 T cell repertoire normalized by number of HBsAg-specific TCRβ sequences found for each vaccinee at time point 60. Frequencies are available in . Figure 2—source data 1. Breadth and entropy of T cell receptor β (TCRβ) repertoire. Breadth (number of unique TCRs) and entropy (Shannon equitability index) of the memory CD4 TCRβ repertoire at two time points, day 0 and day 60. Figure 2—source data 2. Frequency of unique hepatitis B surface antigen (HBsAg)-specific T cell receptor β (TCRβ) sequences. Frequency of unique HBsAg-specific TCRβ sequences out of unique total TCRβ sequences in the memory CD4 T cell repertoire at two time points, day 0 and day 60. Figure 2—source data 3. Frequency of normalized hepatitis B surface antigen (HBsAg)-specific T cell receptor β (TCRβ) sequences. Frequency of vaccine-specific TCRβ sequences within memory CD4 T cell repertoire normalized by number of HBsAg-specific TCRβ sequences found for each vaccinee at day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Comparison

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Shown is the frequency of CFSE low CD4 T cells out of total CD4 T cells for all vaccinees, vaccinees per group and for each vaccinee. Peripheral blood mononuclear cells from day 60 were labeled with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with a pool of peptides spanning hepatitis B (HB) surface antigen (HBsAg) (peptide pool) and single peptides selected based on epitope mapping of the entire antigen (single peptide). After day 7 of in vitro expansion, cells were stained with antibodies to surface markers (CD3, CD4, and CD8) that enable gating on viable CD4 T cells. CFSE intensity was used to identify and sort CFSE low cells for T cell receptor (TCR) repertoire analysis of antigen-specific CD4 T cells.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Labeling, In Vitro, Staining

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Overview of the detected HBsAg epitope-specific TCRβ sequences. Each bar corresponds to unique TCRβ sequences found against a single 15mer HBsAg peptide, with 11 amino acid overlap to each subsequent peptide. Bars in blue denote those epitopes for which 10 or more volunteers had a strong T cell reaction. Motif logos on top of bars denote a sampling of the most common TCRβ amino acid sequence motifs for those epitopes. ( B ) Scatter plot with the frequency of predicted HBsAg epitope-specific and bystander TCRβ sequences at day 60. These make up respectively the numerator and denominator of the HBsAg-predictive ratio, R hbs . Predictions done as a leave-one-out cross-validation. Each circle represents a vaccinee with the color denoting the response group (blue: early-converter, yellow: late-converter, red: non-converter). ( C ) HBsAg-predictive ratio, R hbs , when calculated on the memory CD4 TCRβ repertoires at day 60. ( D ) HBsAg-predictive ratio, R hbs , when calculated on the memory CD4 TCRβ repertoires at day 0. ( E ) Receiver operating characteristic (ROC) curve using R hbs to differentiate between early-converters and late-converters in a leave-one-out cross-validation at day 0. Reported is the area under the curve (AUC) and its 95% confidence interval. Data for B, C, D, and E are available in . ( F ) ROC curve using R hbs to differentiate between age-matched early-converters and late-converters in a leave-one-out cross-validation at day 0. Age-matching was accomplished retaining only samples in the age range 40–55. A Wilcoxon test was used to confirm that there was no difference in age distributions between early- and late-converters (p-value = 0.60, mean EC = 44.5 years, mean LC 45.1 years). Diagonal line denotes a random classifier. Reported is the area under the curve (AUC) and its 95% confidence interval. Figure 3—source data 1. Hepatitis B surface antigen (HBsAg)-predictive ratio ( R hb ) data. Frequency of predicted HBsAg epitope-specific and bystander T cell receptor β (TCRβ) sequences; and HBsAg-predictive ratio, R hbs , calculated on the memory CD4 TCRβ repertoires at two time points, day 0 and day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Sampling, Sequencing, Biomarker Discovery

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: ( A ) Gating strategy started by a lymphocyte gate, followed by gating on viable CD3 + CD8 − T cells. Doublets were excluded using doublet discrimination (area against the height of forward scatter pulse) before gating on CD4 + T cells. Next, CD45RA, CXCR5, CD25, and CD127 were used to identify main subsets of CD4 T cells using Boolean gates as specified in the accompanying table. ( B ) Shown an example of gating for CD154 (CD40L) and CD137 (4-1BB) for cells left unstimulated (left) and cells stimulated with a master peptide pool (right) for an early-converter vaccinee at day 60.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques:

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of a pool of peptides of hepatitis B surface antigen (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry. ( A ) CD40L + 4-1BB − and CD40L − 4-1BB + CD4 T cells from day 60 were gated on and then overlaid in a contour plots of CD25 versus CD127 to assess T COV and T REG phenotype. ( B ) Summary plot of median fluorescence intensity (MFI) of CD25 and CD127 for all vaccinees. Wilcoxon signed-rank with paired analysis; statistical significance was indicated with ****p ≤ 0.0001.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of the master peptide pool (hepatitis B surface antigen [HBsAg]) and assessed for converse expression of 4-1BB and CD40L by flow cytometry on days 0, 60, 180, and 365. Shown is number of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells after subtraction of responses in negative control. ( A ) Aggregate analysis from vaccinees (including early-, late-, and non-converters) showing a peak of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60 (day 60 after first dose of the vaccine and day 30 after second dose), declining thereafter. Shown are numbers of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells. ( B ) Correlation between the difference in antibody titer between day 365 and day 0 and vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60. ( C ) Aggregate analysis from early- and late-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell in early-converters and lack thereof in late-converters. ( D ) Aggregate analysis from early- and late-converter vaccinees showing no significant differences in vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 0. Data for A, B, C, and D are available in . ( E ) Receiver operating characteristic (ROC) curves for R hbs from day 0 data in a leave-one-out cross-validation compared to the frequency of vaccine-specific CD40L + 4-1BB − memory CD4 T cell out of 10 6 memory CD4 T cells for each vaccinee at time points 60 (area under the curve [AUC] = 0.84), 180 (AUC = 0.56), and 365 (AUC = 0.57). ( F ) Receiver operating characteristic (ROC) curves for R hbs from day 0 data in a leave-one-out cross-validation compared to the frequency of vaccine-specific CD40L − 4-1BB + memory CD4 T cell out of 10 6 memory CD4 T cells for each vaccinee at time points 60 (AUC = 0.62), 180 (AUC = 0.56), and 365 (AUC = 0.52). Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001 rs , Spearman’s correlation coefficient, −1≤ rs ≤ 1; rs and p-value by Spearman’s correlation test. Figure 4—source data 1. Ex vivo T cell assay and serological data. Numbers of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell out of 10 6 memory CD4 T cells and antibody titers at the four time points, days 0, 60, 180, and 365.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Negative Control, Biomarker Discovery, Ex Vivo

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Correlation between the difference in antibody titer between day 365 and day 0 and vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell at day 60 colored by vaccinee group and labeled with vaccinee ID. rs , Spearman’s correlation coefficient, −1≤ rs ≤ 1; rs and p-value by Spearman’s correlation test.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Labeling

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees were stimulated with 2 μg/ml of a pool of peptides of hepatitis B surface antigen (HBsAg) and assessed for converse expression of 4-1BB and CD40L by flow cytometry on days 0, 60, 180, and 365. ( A ) Aggregate analysis from early-, late-, and non-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + memory CD4 T cell in early-converters and lack thereof in late and non-converters. Shown are numbers of vaccine-specific memory CD4 T cells out of 10 6 memory CD4 T cells after subtraction of responses in negative control (see Materials and methods for details). ( B ) Aggregate analysis from early-, late-, and non-converter vaccinees showing a significant induction of vaccine-specific CD40L + 4-1BB − and CD40L − 4-1BB + CD4 T cell in early-converters and lack thereof in late and non-converters. Shown are numbers of vaccine-specific CD4 T cells out of 10 6 CD4 T cells after subtraction of responses in negative control (see Materials and methods for details).

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Flow Cytometry, Negative Control

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Peripheral blood mononuclear cells (PBMCs) from vaccinees at day 0 (prior to vaccination) were phenotyped for expression of markers of T REG . ( A ) Aggregate analysis of 4-1BB + CD45RA − T REG within CD45RA − T REG CD4 T cells in early- and late- and non-converter vaccinees before vaccination. ( B ) Aggregate analysis of the median fluorescence intensity of 4-1BB in T H , cT FH , T REG , and cT FR cells before vaccination. ( C ) Aggregate analysis of the median fluorescence intensity of 4-1BB (left panel) and CD25 (right panel) in CD45RA − T REG and CD45RA + T REG cells before vaccination. Data for A, B, and C are available in . ( D ) Frequency of T REG , CD45RA − T REG , and CD45RA + T REG cells within total CD4 T cells in early-, late-, and non-converter vaccinees before vaccination. ( E ) Composition of T REG compartment as determined by expression of 4-1BB and CD45RA in early-, late-, and non-converter vaccinees before vaccination. Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. Figure 5—source data 1. Frequency of 4-1BB + CD45RA − T REG cells and median fluorescence intensity data. Frequency of 4-1BB + CD45RA − T REG within CD45RA− T REG CD4 T cells, and median fluorescence intensity of 4-1BB in T H , cT FH , T REG , and cT FR cells, and of 4-1BB and CD25 in CD45RA − T REG and CD45RA + T REG cells before vaccination.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Expressing, Fluorescence

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet: Aggregate analysis of the frequency of 4-1BB + CD45RA − T REG within CD45RA − T REG CD4 T cells in early-, late-, and non-converter vaccinees at days 0, 60, 180, and 365. Statistical significance was indicated with ns p > 0.05, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques:

Journal: eLife

Article Title: Preexisting memory CD4 T cells in naïve individuals confer robust immunity upon hepatitis B vaccination

doi: 10.7554/eLife.68388

Figure Lengend Snippet:

Article Snippet: Antibody , CD4-APC (REA623) (recombinant antibodies, REAfinity) , Miltenyi Biotec , Cat# 130-113-222 , FACS (1/50 per test).

Techniques: Recombinant, Sequencing, Software, Staining, Virus

Journal: iScience

Article Title: IL-23 tunes inflammatory functions of human mucosal-associated invariant T cells

doi: 10.1016/j.isci.2025.111898

Figure Lengend Snippet:

Article Snippet: CD8 PerCP-Vio700 (Clone REA734) , Miltenyi Biotec , Cat#130-110-682; RRID: AB_2659249.

Techniques: Control, Recombinant, CRISPR, Negative Control, Software

Journal: Cell reports

Article Title: Activation of GPR44 decreases severity of myeloid leukemia via specific targeting of leukemia initiating stem cells

doi: 10.1016/j.celrep.2023.112794

Figure Lengend Snippet:

Article Snippet: Mouse anti-mouse CD45.1 (FITC) , Miltenyi Biotec , Cat# 130–124-211; RRID: AB_2857674.

Techniques: Virus, Recombinant, Enzyme-linked Immunosorbent Assay, Binding Assay, Staining, Modification, Saline, Concentration Assay, Over Expression, Protein Extraction, Membrane, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Protease Inhibitor, CCK-8 Assay, Reverse Transcription, Selection, Plasmid Preparation, Knock-Out, Software, Real-time Polymerase Chain Reaction, Flow Cytometry

Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Analysis of Plaur-miR1-3p and Plaur-miR1-5p expression in Neuro2a cells and mouse posterior cortex. (A) qPCR of Plaur-miR1-3p and Plaur-miR1-5p expression in Neuro2a cells. The data are expressed as mean ± SEM ( n = 3), normalized to Snord95 expression as a reference gene. (B) qPCR of Plaur expression in Neuro2a cells. The data are expressed as mean ± SEM ( n = 3), normalized to Actb (encodes β-actin) expression as a reference gene. For (A,B) , the columns are: Neuro2a WT—control Neuro2a cells; Neuro2a-KO-uPAR—uPAR-deficient Neuro2a cells; Neuro2a Plaur-miR1—Neuro2a cells with ectopic Plaur-pre-miR1 expression; Neuro2a-KO-uPAR Plaur-miR1—uPAR-deficient Neuro2a cells with ectopic Plaur-pre-miR1 expression. The data were analyzed by using analysis of variance followed by Dunnett’s multiple comparisons test using GraphPad Prism software. (C) qPCR of Plaur-miR1-5p and Plaur-miR1-3p in the posterior cortex 0 and 3 h after endogenous Plaur induction. The data are expressed as mean ± SEM ( n = 4). The data were analyzed by using a one-sample t -test with GraphPad Prism software. Statistical significance in (A–C) is indicated by bars and asterisks as follows: * p < 0.05; ** p < 0.01; *** p < 0.001. (D) Sequence results of 19 TA vector clones containing Plaur-miR1-5p sequences generated in (C) . Four of 19 (20%) clones were 22–24 bp in length and could correspond to mature miRNA. In addition, 8 of 19 (40%) clones were 22–31 bp in length and could correspond to other RNA fragments isolated from posterior cortex small RNA fraction . (E) Clones 1, 9, 11, and 16 show the Plaur-miR1-5p sequence, demonstrated as alignment with Plaur-pre-miR1. The asterisks in (D,E) indicate that the aligned sequences match at that position.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Expressing, Control, Software, Sequencing, Plasmid Preparation, Clone Assay, Generated, Isolation

Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Plaur-miR1 (Plaur-miR1-3p and Plaur-miR1-5p) expression altered the expression of the predicted target genes Mef2d and Snrnp200 in Neuro2a cells. qPCR analysis of (A) Mef2d and (B) Snrnp200 in Neuro2a WT, Neuro2a KO uPAR, Neuro2a WT, and Neuro2a KO uPAR cells after pBl-U6-Plaur-pre-miR1 expression. The data presented as mean ± SEM ( n = 3), normalized to Actb (encodes β-actin) expression as a reference gene. The data were analyzed by using analysis of variance followed by Dunnett’s multiple comparisons test with GraphPad Prism software. Statistical significance is indicated by bars and asterisks as follows: ** p < 0.01; *** p < 0.001.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Expressing, Software

Journal: Frontiers in Molecular Neuroscience

Article Title: Identification of a Novel Small RNA Encoded in the Mouse Urokinase Receptor uPAR Gene ( Plaur ) and Its Molecular Target Mef2d

doi: 10.3389/fnmol.2022.865858

Figure Lengend Snippet: Results of the luciferase reporter assay for the confirmation of specificity of Plaur-miR1-5p effect on its predicted targets. Neuro2a cells were co-transfected with pBl-U6-Plaur-pre-miR1 and pGL3 vector coding 3-UTR or CDS sequences. Empty vectors pBl-U6 and pGL3 were used as controls. Data were normalized by luciferase activity in Neuro2a cells co-transfected with pBl-U6-Plaur-pre-miR1 and pGL3 vectors. The data are presented as mean ± SEM ( n = 3) and compared using two-way ANOVA followed by Šídák’s multiple comparisons test with GraphPad Prism software. Statistical significance is indicated by bars and asterisks as follows: * p < 0.05.

Article Snippet: Mouse Neuro2a neuroblastoma cells (ATCC ® CCL-131TM University Boulevard Manassas, VA, United States) not exceeding 20 passages were cultured in complete medium—Dulbecco’s Modified Eagle’s Medium (DMEM) (#21969035), 10% fetal bovine serum (FBS, Gibco, #10270-106, United Kingdom), 1 × Minimum Essential Medium (MEM) Non-Essential Amino Acids Solution (#11140050) and 1 × antibiotic-antimycotic solution (#15240062; all from Gibco, Life Technologies, Bleiswijk, Netherlands)—at 37 ° C in an atmosphere with 5% CO 2 .

Techniques: Luciferase, Reporter Assay, Transfection, Plasmid Preparation, Activity Assay, Software

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A , B ) Plasmids expressing human YAP5SA (2 mg/kg) plus RFP control (2 mg/kg) or mEPDR1 (2 mg/kg), together with plasmids expressing PB transposase (0.8 mg/kg), were delivered into mice by hydrodynamic injection ( n = 6 male mice per group). Liver tumors were analyzed 110 days after injection. Photographs show livers ( A ) and tumor numbers ( B ) were determined. Data were presented as the mean ± SEM. ( C ) Dimensionality reduction and visualization based on T-distributed stochastic neighbor embedding (t-SNE) analysis of a subset of mouse liver immunocytes from the indicated group in ( A ). ( D ) Statistical difference analysis for immune cell subsets was obtained from dimensionality reduction analysis in panel ( C ). n = 5 male mice per group and data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of the immune co-suppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in liver CD8 + T cells from the indicated group in ( A ). n = 6 male mice per group and data were presented as the mean ± SD. ( F ) Schema of coculture of human CD8 + T cells with HepG2 cells expressing Flag-EV or Flag-EPDR1. ( G ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. n = 3 independent experiments and the data were presented as the mean ± SD. ( H – J ) Hepa 1-6 cells stably expressing Flag-EV or Flag-mEPDR1 were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group), and α-CD8 (4 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block CD8 + T cells and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( H ), growth curves ( I ), and relative tumor burdens ( J ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM. Data information: Statistical significance was determined by two-way ANOVA ( D , I ), one-way ANOVA ( J ), and two-tailed unpaired Student’s t -test ( B , E , G ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Expressing, Control, Injection, Flow Cytometry, Stable Transfection, Blocking Assay, Two Tailed Test

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A ) Western blotting analysis of the protein level of Myc-tag mEPDR1 in liver samples from the YAP5SA-induced mouse model. Calnexin served as a loading control. ( B ) t-SNE plot showing the expression level marker genes for distinct subpopulations of lymphocytes. ( C ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1 + TIM-3 + ) cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( D ) Schema of coculture of mouse CD8 + T cells with Hepa 1-6 cells expressing Flag-EV or Flag-mEPDR1. ( E ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) and immune effector molecules (IFNγ, GzmB) positive cells in CD8 + T cells after coculture with the indicated tumor cells. Data were presented as the mean ± SD of three independent experiments ( n = 3). ( F ) Flowrate analysis of the amount of CD8 + T cells in blood from the mice with the indicated manipulation. ( G – I ) Hepa 1–6 cells stably expressing NTC or shEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. The figure depicts xenografts ( A ), growth curves ( B ), and tumor weights ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( H ), and mean ± SD ( I ), respectively. ( J ) Flow cytometric analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( G ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometric analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( G ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-tailed unpaired Student’s t -test ( C , E ), two-way ANOVA ( H ), and one-way ANOVA ( I – K ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Western Blot, Control, Expressing, Marker, Flow Cytometry, Stable Transfection, Injection, Two Tailed Test

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were infected with shNTC or shPD-L1, and subsequently subcutaneously injected into C57BL/6J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1-6 cells stably expressing Flag-EV, Flag-mTRIM21, Flag-mEPDR1, or Flag-mTRIM21 plus Flag-mEPDR1 both were injected subcutaneously into C57BL/6 J mice ( n = 6 male mice per group). Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of membrane-bound PD-L1 in the indicated group in ( A ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). ( K ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD ( n = 6). Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – K ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Stable Transfection, Expressing, Infection, Injection, Flow Cytometry, Membrane

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A – C ) Hepa 1-6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group), and α-PD-L1 (6 mg/kg) neutralizing antibody was injected intraperitoneally four times (twice a week starting at 10 days after inoculation) to block PD-L1 and IgG2b was used as control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( A ), growth curves ( B ), and tumor weight ( C ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( B ) and mean ± SD ( C ), respectively. ( D ) Flow cytometry analysis of ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( A ). Data were presented as the mean ± SD. ( E ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, Granzyme B) positive cells in tumor CD8 + T cells from the indicated groups in ( A ). Data were presented as the mean ± SD. ( F – H ) Hepa 1–6 cells stably expressing Flag-EV and Flag-mEPDR1 were injected subcutaneously into C57BL/6J mice ( n = 6 male mice per group). BAY11-7082 was used to inhibit the NF-Κb pathway, and the vehicle was used as a control. Tumor size was measured starting at 10 days after inoculation. Photographs show xenografts ( F ), growth curves ( G ), and final tumor weight ( H ) determined at the end of the experiment (day 25). Data were presented as the mean ± SEM ( G ), and mean ± SD ( H ), respectively. ( I ) Flow cytometry analysis of the ratio of immunosuppressive molecules (PD-1, TIM-3) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. ( J ) Flow cytometry analysis of the ratio of immune effector molecules (IFNγ, GzmB) positive cells in tumor CD8 + T cells from the indicated group in ( F ). Data were presented as the mean ± SD. Data information: Statistical significance was determined by two-way ANOVA ( B , G ) and one-way ANOVA ( C – E , H – J ). .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Stable Transfection, Expressing, Injection, Blocking Assay, Control, Flow Cytometry

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: ( A ) Representative immunohistochemistry images of EPDR1, PD-L1, p65, and CD8 staining in HCC specimens; scale bars, 50 μm. ( B ) Correlation analysis of EPDR1 and PD-L1 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( C ) Correlation analysis of EPDR1 and nucleus located p65 positive signal in HCC specimens, P values and R were calculated by two-tailed Person’s correlation analysis. ( D ) Summary: In the presence of EPDR1, intracellular EPDR1 directly binds to TRIM21 in competition with IKBKB, thereby disrupting the ubiquitylation and degradation of IKBKB, leading to enhanced NF-κB signaling and downstream PD-L1 expression, which exacerbates the exhaustion of CD8 + T cells and contributes to tumor progression. .

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Immunohistochemistry, Staining, Two Tailed Test, Expressing

Journal: The EMBO Journal

Article Title: EPDR1 promotes PD-L1 expression and tumor immune evasion by inhibiting TRIM21-dependent ubiquitylation of IkappaB kinase-β

doi: 10.1038/s44318-024-00201-6

Figure Lengend Snippet: Reagents and tools table

Article Snippet: InVivoMAb anti-mouse CD8 , BioXcell , BE0061.

Techniques: Recombinant, Purification, Sequencing, Control, Modification, Cell Stimulation, Ligation, Cloning, cDNA Synthesis, SYBR Green Assay, Software