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: 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: 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: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Antigenic stimulation of human splenic mononuclear cells mimics the T CD4 + response of GC reaction (A) Splenic mononuclear cells (splenocytes) were stimulated with CytoStim and cultured for 3 days in the presence of cytokines (IL-7, IL-12, activin) (A). CXCR5 and PD-1 expressions among CD4+ T cells were assessed. Representative flow plots showing CXCR5 and PD-1 expression on CD4 + T cells from ex vivo splenocytes and splenocytes cultured for 3, 5, and 10 days (left) and the percentage of Tfh among CD4+ T cells (right). (B) Representative flow plots showing IL-21 production by Tfh (left) and the percentage of IL-21-positive cells among Tfh (right). (C) Representative histogram showing ICOS expression among Tfh (left) and relative expression of ICOS among Tfh (right). (D) Gating strategy allowing the identification of PD-1 neg Tfh, non-GC Tfh, and GC Tfh among total CXCR5 + PD-1 + cells ex vivo or after 3 days of stimulation of splenocytes or PBMCs in polarizing cytokines. (E) Percentage of total CXCR5 + PD-1 + cells including PD-1 neg Tfh, non-GC Tfh, and GC Tfh among CD4 + T cells ex vivo or after 3 days of culture using splenocytes or PBMCs (n = 7–14). (F and G) Mean fluorescence intensity of PD-1 (F) and CXCR5 (G) expression on ex vivo GC Tfh and GC Tfh D3 splenic cells. (H) Representative flow plots showing IL-21 and IFNγ production by non-GC Tfh D3 and GC Tfh D3 cells 3 days after splenocyte stimulation. (I) Percentage of IL-21- and/or IFNγ-positive cells among non-GC Tfh D3 cells and GC Tfh D3 . (J) Gating strategy for analysis of Bcl6 expression in CD4 + T cells and histograms showing Bcl6 mean fluorescence intensity for GC Tfh D3 , non-GC Tfh D3 , and CXCR5 − PD-1 - CD4 + T cell subsets (n = 14). Each symbol represents an individual donor. A Wilcoxon matched pairs test was performed; ∗, p < 0.05; ∗∗, p < 0.005.

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Cell Culture, Expressing, Ex Vivo, Fluorescence

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet: Distinct CD4 + T cell subsets contribute to the generation of Tfh with heterogeneous functional profiles (A) Mean fluorescence intensity of the CXCR5 marker expressed by CXCR5 + PD-1 + cells derived from (1) naive, (2) MemPD-1 neg , (3) MemPD-1 neg and Tfh. (B) Frequency of IL-21- and/or IFNγ-positive cells among CXCR5 + PD-1 + cells at day 3. (C – E) Representative flow plots showing CXCR3, ICOS, and CD40L expression by CXCR5 + PD-1 + cells (left panel) and frequency of CXCR3-, ICOS-, and CD40L-positive cells among CXCR5 + PD-1 + cells at day 3 (right panel). (F) Ex vivo cells or their respective Tfh D3 counterparts obtained after 3 days of splenocyte culture were co-cultured with autologous B cells for 7 days. (G) Box plots represent the frequency of CD27 + CD38 + cells among CD19 + cells, the concentration of total immunoglobulins and the absolute number of live B cells after co-culture. (H) Quantification of IgG1, IgG4, and IgA in the co-culture supernatants. Each symbol (A–H) represents an individual donor. (A–H) A Wilcoxon matched pairs test was performed, ∗, p < 0.05; ∗∗, p < 0.005; ∗∗∗, p < 0.001.

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Functional Assay, Fluorescence, Marker, Derivative Assay, Expressing, Ex Vivo, Cell Culture, Concentration Assay, Co-Culture Assay

Journal: iScience

Article Title: Naive and memory CD4 + T cell subsets can contribute to the generation of human Tfh cells

doi: 10.1016/j.isci.2021.103566

Figure Lengend Snippet:

Article Snippet: FITC anti-human IFNγ , Miltenyi Biotec , Cat# 130-113-497; RRID:AB_2733587.

Techniques: Cell Analysis, Purification, Virus, Recombinant, Blocking Assay, Antibody Labeling, Transfection, Software

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Identification of a novel homozygous IFNAR2 variant leading to IFNAR2 deficiency. (A) IFNAR2 protein sequence with known deleterious variants marked. (B) Pedigrees of affected individuals originating from Greenland (P1), Canada (P2 and P3), and Alaska (P4 and P5). Double lines refer to consanguinity. (C) Principal component analysis of 4,630 Greenlandic individuals combined with three super populations from the 1000 Genomes Project. (D) Local ancestry haplotypes of admixed and p.Ser53Pro IFNAR2 heterozygous Greenlandic individuals colored according to ancestry. Each heterozygous individual is represented with two haplotypes, one for the p.Ser53Pro haplotype (top) and one for the WT haplotype (bottom). The fraction of Inuit ancestry of the WT and p.Ser53Pro haplotype is estimated at the location of the variant. (E) IFNAR2 cell surface expression analyzed by flow cytometry in PBMCs and distinct cell subsets of healthy controls (C1–C5) and patients (P1 and P2). Representative of a single experiment in P1 and P2 PBMC. (F) Immunoblotting showing IFNAR2 expression in whole-cell lysates of PBMCs from P1 compared to three healthy controls (C1–C3). GAPDH was used as loading control. Not repeated due to limited patient material. (G) RT-qPCR of whole-cell RNA lysates of PBMCs evaluating IFNAR2 mRNA levels relative to TBP for P1 compared to three healthy controls (pooled, mean ± SD of n = 3 biological replicates; **, P < 0.01, Welch’s t test). Representative of a single experiment in P1 PBMC. Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Variant Assay, Sequencing, Expressing, Flow Cytometry, Western Blot, Control, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Clinical information and variant modelling. (A) Chest radiograph from P3 demonstrating pneumonitis and ARDS associated with influenza A infection. (B) Summary of the outputs of in silico prediction software. (C) Conservation of the p.Ser53Pro IFNAR2 residue across the indicated species. (D) Graphical models of the binary complex between human IFNα2 and IFNAR2 WT and p.Ser53Pro proteins (PDB accession no. 3S9D ) were created using PyMOL software version 2.0.7. (E) Displayed are all rare exonic variants (gnomAD MAF < 0.01) in genes related to primary immunodeficiency identified by WGS in P1. All genotype quality scores were 99. CADD, Combined Annotation Dependent Depletion.

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Variant Assay, Infection, In Silico, Software, Residue

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Defective signaling and ISG induction downstream of IFNAR2 p.Ser53Pro. (A) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNβ 100 IU/ml for 5, 15, or 30 min. Whole-cell lysates were harvested for Western blotting for the visualization of pSTAT1, STAT1, and GAPDH, which was used as the loading control. Not repeated due to limited patient material. (B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were treated with IFNβ 100 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and the visualization of IFNAR2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (C) Primary IFNAR2 −/− dermal fibroblasts reconstituted with lentiviruses expressing GFP, WT, or p.Ser53Pro IFNAR2 were treated with IFNα2b or IFNγ 1,000 IU/ml for 30 min. Whole-cell lysates were harvested for immunoblotting and visualization of pJAK1, JAK1, pSTAT2, STAT2, STAT1, pSTAT1, and GAPDH as the loading control. One representative immunoblot of n = 3 independent experiments is shown. (D) PBMCs from P1 and three healthy controls (C1–C3) were treated with IFNβ 100 IU/ml for 6 h. Total RNA was purified for RT-qPCR of IFIT1 and IRF7 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001, ns, non-significant; two-way ANOVA with Šidák’s test for multiple comparisons). (E) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the mother of P1 (Het) were treated with IFNβ 100 IU/ml for 6–24 h. Total RNA was purified for RT-qPCR of MX1 and IFIT1 relative to TBP (mean ± SD of n = 3 independent experiments; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Dunnett’s test for multiple comparisons). (F) Primary dermal fibroblasts from P1 or a healthy control (C1) were treated with IFNα2b 1,000 IU/ml overnight. Whole-cell lysates were prepared for immunoblotting and visualization of MX1, USP18, RSAD2, ISG15, and α-tubulin (ATUB) as the loading control. One representative immunoblot of n = 3 independent experiments is shown. Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Western Blot, Control, Expressing, Purification, Quantitative RT-PCR

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: In vitro functional modelling of the IFNAR2 p.Ser53Pro mutant. (A) EBV-transformed B cells from P2 and a healthy control (C1) were treated with IFNα2b 1,000 IU/ml for 30 min. Whole cell lysates were harvested for immunoblotting and visualization of STAT1, pSTAT1 and GAPDH as loading control. Representative of n = 2 repeat experiments. (B) Graphical summary of experimental strategy. IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. (C) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control and protein lysates prepared for immunoblotting for IFNAR2, GFP, and GAPDH loading control. Representative of n = 3 independent experiments. (D) IFNAR2-deficient dermal fibroblasts were reconstituted with WT or mutant IFNAR2 lentiviral constructs or an empty vector control. After overnight stimulation with IFNα2b or IFNγ (1,000 IU/ml) protein lysates were prepared for immunoblotting for the ISG products MX1, USP18, RSAD2 and ISG15 alongside GAPDH loading control. Representative of n = 3 independent experiments. (E) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2 , were pre-treated with IFNα2b or IFNγ (1,000 IU/ml) overnight, prior to infection with HSV1 (17 + strain, MOI 0.01). At 48 h after infection, lysates were prepared for immunoblotting for HSV gD, alongside MX1, RSAD2, ISG15 and GAPDH as loading control. Representative of n = 3 independent experiments. (F) Primary IFNAR2-deficient fibroblasts, stably reconstituted with empty vector, WT IFNAR2 or p.Ser53Pro IFNAR2 , were pre-treated with IFNα2b or IFNγ (1,000 IU/ml) overnight, before infection with a cytopathic dose of EMCV. At 24 h after infection, cell viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent experiments, one-way ANOVA with Šidák’s multiple comparisons test; ****, P < 0.0001; ns, non-significant). Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: In Vitro, Functional Assay, Mutagenesis, Transformation Assay, Control, Western Blot, Construct, Plasmid Preparation, Stable Transfection, Infection, Imaging, Viability Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Impaired viral control in patient fibroblasts bearing homozygous IFNAR2 p.Ser53Pro. (A and B) SV40-immortalized dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were pretreated with IFNβ 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 0.001. (A) At 24, 48, and 72 hpi, supernatants were sampled and titrated for TCID50 (geometric mean ± SD of n = 3 independent repeats; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001, two-way ANOVA with Tukey’s test for multiple comparisons). (B) At 72 hpi, the cells were lysed for immunoblotting of whole-cell lysates for the HSV1 protein VP5 and GAPDH as the loading control. One representative immunoblot of n = 3 independent repeat experiments is shown. (C) SV40-immortalized dermal fibroblasts from P1 and a healthy control were pretreated with IFNβ 100 IU/ml for 24 h before infection with cell-free VZV at an MOI of 1. At 48 hpi, total RNA was harvested for RT-qPCR, evaluating the levels of VZV immediate early ORF63 and late ORF40 transcripts, respectively, relative to GAPDH (mean ± SD of n = 3 independent repeats; *, P < 0.05; ***, P < 0.001, two-way ANOVA with Šidák’s test for multiple comparisons). (D) SV40-immortalized dermal fibroblasts from P1 and one healthy control were pretreated with IFNβ 100 IU/ml for 24 h before infection with MeV (Edmonston strain, MOI = 0.00083). At 96 hpi, supernatants were harvested and titrated for TCID50 (geometric mean ± SD of n = 3 independent repeats; ****, P < 0.0001, two-way ANOVA with Tukey’s test for multiple comparisons). (E and F) Primary dermal fibroblasts from P1, a healthy control (C1), the heterozygous mother of P1 (Het) and a known IFNAR2-deficient patient ( IFNAR2 −/− ) were pretreated with IFNα2b 1,000 IU/ml for 16 h before infection with MuV at an MOI 0.1 (MuV, Enders strain). At 72 hpi, (E) viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent repeats; **, P < 0.01; ****, P < 0.0001, two-way ANOVA with Dunnett’s test for multiple comparisons) and (F) whole cell lysates were prepared for immunoblotting and visualization of MuV nucleoprotein (NP) and GAPDH as loading control. One representative immunoblot of n = 3 independent repeat experiments is shown. (G) Primary dermal fibroblasts from P1 and a healthy control (C1) were pretreated with IFNα2b 1,000 IU/ml for 16 h before infection with a cytopathic dose of EMCV. At 24 hpi, cell viability was assessed in an imaging based live cell viability assay (mean ± SD of n = 3 independent repeats; ****, P < 0.0001, two-way ANOVA with Dunnett’s test for multiple comparisons). Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Control, Infection, Western Blot, Quantitative RT-PCR, Imaging, Viability Assay

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Defective processing of p.Ser53Pro IFNAR2 through the secretory pathway. (A) Primary dermal fibroblasts from P1, healthy controls (C1–3), and the heterozygous mother of P1 (Het) were lysed for immunoblotting of whole cell lysates for IFNAR2 protein and α-tubulin (ATUB) as loading control. Densitometry analysis of IFNAR2 expression relative to loading control (mean ± SD of n = 3 independent experiments; *, P < 0.05; **, P < 0.01; ****, P < 0.0001, Welch’s one-way ANOVA with Dunnett’s test for multiple comparisons). (B) Total RNA from primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) was purified for RT-qPCR evaluating IFNAR2 mRNA levels relative to TBP (mean ± SD of n = 3 independent experiments; *, P < 0.05; **, P < 0.01, one-way ANOVA with Tukey’s test for multiple comparisons). (C) Model of change to IFNAR2 protein entropy from the p.Ser53Pro substitution, prepared using DynaMUT. Red color indicates a gain of flexibility, blue a gain of stability. (D) HEK 293 FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection, the cells were treated with CHX at the indicated concentrations or DMSO vehicle control, for a further 24 h, before whole-cell lysates were prepared for immunoblotting for IFNAR2, and α-tubulin (ATUB) as loading control. Expression of IFNAR2 protein relative to loading control was assessed by densitometry analysis and expressed as a proportion of the DMSO-treated control. Displayed is a representative immunoblot of two independent experiments. (E) Left: HEK293FT cells were transfected with lentiviral expression constructs expressing IFNAR2 WT or IFNAR2 p.Ser53Pro or empty vector. Whole cell lysates were prepared and treated with PNGase F before immunoblotting for IFNAR2 and GAPDH as loading control. Displayed is a representative immunoblot of n = 3 independent experiments. (E) Right: Primary dermal fibroblasts of a healthy control were stably transduced with a lentiviral vector encoding IFNAR2 WT or IFNAR2 p.Ser53Pro. Whole cell protein lysates were harvested and treated with O-glycosidase and neuraminidase before immunoblotting for IFNAR2 and GAPDH as loading control. Displayed is a representative immunoblot of n = 2 independent experiments. (F) HEK293FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection cells were fixed, immunostained, and imaged by confocal microscopy for expression of the Golgi marker RCAS1 (left) or the ER marker calnexin (right) alongside IFNAR2. Scale bars represent 10 μm. Shown are the results of correlation analysis of IFNAR2 and the relevant organelle marker in individual cells (mean ± SD of n = 9 WT and n = 12 S53P cells analyzed; ***, P < 0.001, t test). Representative of n = 2 repeat experiments. (G) HEK293FT cells were transfected with HA-tagged expression constructs encoding WT or p.Ser53Pro IFNAR2 . 24 h after transfection cell surface expression of IFNAR2 was assessed by flow cytometry. Representative of n = 2 repeat experiments. Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Western Blot, Control, Expressing, Purification, Quantitative RT-PCR, Transfection, Construct, Plasmid Preparation, Stable Transfection, Transduction, Confocal Microscopy, Marker, Flow Cytometry

Journal: The Journal of Experimental Medicine

Article Title: Life-threatening viral disease in a novel form of autosomal recessive IFNAR2 deficiency in the Arctic

doi: 10.1084/jem.20212427

Figure Lengend Snippet: Reconstitution of patient fibroblasts with WT IFNAR2 restores IFNAR2 signaling, ISG induction and viral control. (A and B) Primary dermal fibroblasts from P1, two healthy controls (C1 and C2), and the heterozygous mother of P1 (Het) were transduced with lentiviral vectors encoding EGFP , IFNAR2 WT, or IFNAR2 p.Ser53Pro. 2 d after transduction, cells were pretreated with IFNβ 100 IU/ml for either (A) 30 min before being lysed and harvested for whole cell lysates for immunoblotting or (B) 6 h before being lysed and harvested for total RNA for RT-qPCR of MX1 and IFIT1 relative to GAPDH (mean ± SD of n = 3 independent repeats; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons). For A, one representative immunoblot of three independent experiments is shown. (C and D) SV40-immortalized dermal fibroblasts from P1 and a healthy control were transduced with lentiviral vectors encoding EGFP or IFNAR2 WT. 2 d after transduction, cells were pretreated with IFNβ 100 IU/ml for 24 h before infection with HSV1 (KOS strain) at an MOI of 1. At 24 h p.i., supernatants were harvested and titrated for (C) TCID50 (geometric mean ± SD of n = 3 independent replicates; ****, P < 0.0001; ns, non-significant; two-way ANOVA with Tukey’s test for multiple comparisons) and the cells were lysed for Western blotting for the HSV1 protein VP5, IFNAR2, and GAPDH as loading control (D). Source data are available for this figure: .

Article Snippet: Immunophenotyping of PBMCs from P1 and healthy controls was performed by flow cytometry using the following monoclonal mouse anti-human antibodies: IFNAR2 PE (clone REA124, REAfinity; Miltenyi Biotec 130-099-555) in a panel with CD14 FITC (BD, 555397), CD3 PerCP-Cy5.5 (BD, 624060), CD19 PE-Cy7 (BD, 341113), CD4 APC (BD, 345771), CD8 APC-Cy7 (BD, 348813), and CD45 V500 (BD, 560777).

Techniques: Control, Transduction, Western Blot, Quantitative RT-PCR, Infection

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: A-i, Sorting strategy (CD45−CD31−) identifying CD34+CD56− or CD34−; A-ii, CD34+CD90− and CD34+CD90+ cells; A-iii, CD56+CD82+ cells; A-iv, CD90+ cells; B, Work-flow; C-E, adipogenic, fibrogenic and myogenic differentiation of CD34+CD90−CD56− (C), CD34+CD90+CD56− (D) and CD56+CD82+CD34− (E) populations by IHC; F, % mononuclear cells in human skeletal muscle (n=17, study 2, non-T2DM); G, PDGFRα in FAPs (scalebar 50 μm); H, COL1A1 expressing CD90+CD56− (FAPs), CD56+ (MuSCs) and CD90−CD56− (Unidentified) cells (n=6, study 2, non-T2DM) after 3–9 days in vitro; I, Clonal experiment; J, Colony formation (%) of single sorted FAPs and MuSCs (n=12, study 2, non-T2DM); K, Limiting dilution assay on FAPs and MuSCs (n=3, study 2, non-T2DM), Solid line is non-linear fit; L, MyHC+/Desmin+ myotubes from single sorted MuSCs; M, Perilipin1+/Collagen1+ cells from single sorted FAPs; N, Biological processes in FAPs (n=3, study 2, non-T2DM); O, % of EdU+ MuSCs and FAPs (n=10–16, study 2, non-T2DM); P, EdU+ MuSCs (i) and FAPs (ii) 96h post sort. Cells obtained from of mm. Rectus Abdominus/Gastrocnemius. *p<0.05, **p<0.01 and ***p<0.001.

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Expressing, In Vitro, Limiting Dilution Assay

Journal: Cell metabolism

Article Title: Human skeletal muscle CD90 + fibro-adipogenic progenitors are associated with muscle degeneration in type 2 diabetic patients

doi: 10.1016/j.cmet.2021.10.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The cells were then resuspended in wash buffer and incubated in MACS human FcR blocking solution (20μl/sample, cat no . 130–059-901, Miltenyi Biotec) and primary antibodies against CD45-FITC (12μl/sample, Cat no . 130–114-567, Clone 5B1, Miltenyi Biotec), CD31-FITC (4μl/sample, Cat no . 130–110-668, Clone REA730, Miltenyi Biotec), CD90-PE (3.2μl/sample, Clone 5E10, eBioscience, Thermo Fisher Scientific), CD56-BV421 (1:100, Cat no . 562752, BD Bioscience, San Jose, CA, USA), CD82-PE-vio770 (Cat no . 130–101-302 10μl/sample, Miltenyi Biotec) and CD34-APC (20μl/sample, clone 581, BD Bioscience) in darkness at 4°C for 30 min. Propidium iodide (PI, 10μl/sample, cat 556463, BD Bioscience) immediately before sorting to exclude non-viable cells.

Techniques: Recombinant, Blocking Assay, Staining, Imaging, Sample Prep, Software, Flow Cytometry, RNA Sequencing, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: MTL-CEBPA and pembrolizumab combination treatment is associated with changes across tumor types, consistent with immune inflammation and activation (A) Fold change of CEBPA RNA at C1 D2 normalized to pre-treatment values (C1 D1). Bar is at median with 95% confidence error bars, each point is one patient. Significance was determined by one sample Wilcoxon test with hypothetical median value of 1. (B) Fast gene set enrichment analysis (FGSEA) analysis of differentially expressed genes between pre- and post-treatment time points across all patients, testing Nanostring pathways of the IO Pan-cancer panel. Pathways in red reached significance ( p adjust <0.05), with significance determined and adjusted for multiple testing by the fgsea algorithm. (C) Quantification of CD3 + CD8 + T cell numbers (left) and CD3 + CD8+Ki67+GZMB+ T cell numbers (right) between pre- (screening) and post treatment (C2D16) of 23 paired biopsies as determined by IHC. p values are non-adjusted and are determined by paired two-sided Wilcoxon test. (D) Quantification of CD11b+CD68 + CD64 − CD206+CD163 − cells as a proportion of total macrophages (CD11b+CD68 + cells) (left) and stromal CD11b+CD68 + CD64 − CD206+CD163 − cell numbers (right) between pre- (screening) and post-treatment (C2D16) of 23 paired biopsies as determined by IHC. p values are non-adjusted and are determined by paired two-sided Wilcoxon test. (E) Quantification of parenchymal CD11b+CD15 + CD14 − HLA-DR − LOX1+ (PMN-MDSC) cells as a proportion of total myeloid cells (CD11b+ cells) (left), between pre- (screening) and post treatment (C2D16) of 23 paired biopsies as determined by IHC. Right, parenchymal PMN-MDSC cell numbers in patients with at least 1 PMN-MDSC per cell/mm 2 ( N = 18). p values are non-adjusted and are determined by paired two-sided Wilcoxon test. The boxplots in (C), (D), and (E) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient. (F) Flow cytometric detection of PMN-MDSCs in the blood of TIMEPOINT patients. % change refers to change of PMN-MDSC as a proportion of total live cells (see gating strategy in Figure S2 D) compared to C1 D1. Each point is one patient, line is at mean with SD error bars. Significance was determined by Wilcoxon test compared to expected value of 100.

Article Snippet: For MDSCs, this cocktail includes CD11b FITC (Miltenyi 130-110-552, clone REA713), CD14 APC-Vio770 (Miltenyi 130-110-522, clone REA599), CD15 PerCP-Vio700 (Miltenyi 130-113-487, clone VIMC6), LOX-1 PE (BioLegend 358604, clone 15C4), HLA-DR APC (Miltenyi 130-111-790 clone REA805), lineage markers: CD3 V500 (BD Horizon 561416, clone UCHT1), CD56 eFluor506 (eBiosciences 15520677, clone TULY56), CD19 V500 (BD Horizon 561121 clone HIB19), Dead cell exclusion dye eFluor 506 (Thermo Fisher Scientific 65-0866-14).

Techniques: Activation Assay, Paraffin-embedded Immunohistochemistry

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: MTL-CEBPA/pembrolizumab combination treatment is associated with immunomodulatory changes in the immune-desert TME, converting to immune-inflamed (A) Fold change in CD3 + CD8 + T cells (left) and CD3 + CD8 + GZMB+ T cells (right) at C2D16 compared to screening, between patients with low or high levels of TIL at baseline using the median as threshold at screening. p value was determined by an unpaired two-sided Wilcoxon test. (B) Quantification of Immunosign, IS21 between pre- (screening) and post treatment (C2D16) between cold and hot tumors, left and right, respectively. p values are non-adjusted and were determined by paired two-sided Wilcoxon test. (C) IHC quantification of parenchymal CD3 + CD8 + Ki67+ T cells (left) and CD3 + CD8 + Ki67+GZMB+ T cells (right) between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10). p value is determined by paired two-sided Wilcoxon test. (D) IHC quantification of CD11b+CD14 + CD15 − HLA-DR+ cells in the whole tumor (left) or stroma (right) between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10). p value is determined by paired two-sided Wilcoxon test. The boxplots in (A)–(D) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient. (E) Differential gene expression calculated between pre- and post-treatment time points of 9 paired immune-desert patient tumor biopsies. p values on y axis refer to adjusted p values by DESeq2 algorithm. (F) FGSEA analysis of differentially expressed gene sets between pre- (screening) and post treatment (C2D16) of 9 paired biopsies with an immune-desert TME (IS21 < 10), using supplied Nanostring pathways of the IO Pan-cancer panel. Pathways in red reached significance ( p adjust <0.05), adjusted by fgsea algorithm.

Article Snippet: For MDSCs, this cocktail includes CD11b FITC (Miltenyi 130-110-552, clone REA713), CD14 APC-Vio770 (Miltenyi 130-110-522, clone REA599), CD15 PerCP-Vio700 (Miltenyi 130-113-487, clone VIMC6), LOX-1 PE (BioLegend 358604, clone 15C4), HLA-DR APC (Miltenyi 130-111-790 clone REA805), lineage markers: CD3 V500 (BD Horizon 561416, clone UCHT1), CD56 eFluor506 (eBiosciences 15520677, clone TULY56), CD19 V500 (BD Horizon 561121 clone HIB19), Dead cell exclusion dye eFluor 506 (Thermo Fisher Scientific 65-0866-14).

Techniques: Gene Expression

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet: Characteristics linking to MTL-CEBPA mode of action are enriched in patients with stable disease after combination treatment (A) Fold change of CEBPA RNA at C1 D2 compared to pre-treatment, C1 D1 in patients with HCC from the OUTREACH clinical trial. Bar is at median with 95% confidence interval error bars, each point is one patient. Significance was tested with Kruskal-Wallis test with p value determined 0.1375. (B) Fold change of CEBPA RNA at C1 D2 normalized to pre-treatment, C1 D1 in iCCA patients from TIMEPOINT. Bar is at median with 95% confidence interval error bars, each point is one patient. Significance was not tested due to low patient numbers. For (A) and (B), clinical outcome refers to clinical response by RECIST or if unavailable, at C2 D22 determined by site. (C) Change in stromal CD11b+CD14 + CD15 − HLA-DR+ APC-like cells between pre- (screening) and post treatment (C2D16) calculated independently for patients with disease stabilization and progressive disease. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (D) Change in numbers of parenchymal CD8 T cells (CD3 + CD8 + ) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease independently calculated. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (E) Change in proportion of CD11b+CD68 + CD64 − CD206+CD163 − cells as a proportion of total macrophages (CD11b+CD68 + cells) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease independently calculated. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. (F) Change in proportion of parenchymal CD11b+CD14 − CD15+HLA-DR-LOX1+ cells (PMN-MDSCs) as a percentage of total myeloid cells (CD11b+) between pre- (screening) and post treatment (C2D16) in patients with disease stabilization and progressive disease calculated independently. Significance was determined by paired two-sided Wilcoxon test and p values are unadjusted. The boxplots in (C)–(F) show the data distribution, where the line denotes the median, the box edges show the interquartile range, and each dot is one patient ( N = 23). Clinical outcome of PD or non-PD refers to the clinical response determined by site at C2 D22.

Article Snippet: For MDSCs, this cocktail includes CD11b FITC (Miltenyi 130-110-552, clone REA713), CD14 APC-Vio770 (Miltenyi 130-110-522, clone REA599), CD15 PerCP-Vio700 (Miltenyi 130-113-487, clone VIMC6), LOX-1 PE (BioLegend 358604, clone 15C4), HLA-DR APC (Miltenyi 130-111-790 clone REA805), lineage markers: CD3 V500 (BD Horizon 561416, clone UCHT1), CD56 eFluor506 (eBiosciences 15520677, clone TULY56), CD19 V500 (BD Horizon 561121 clone HIB19), Dead cell exclusion dye eFluor 506 (Thermo Fisher Scientific 65-0866-14).

Techniques:

Journal: Cell Reports Medicine

Article Title: TIMEPOINT, a phase 1 study combining MTL-CEBPA with pembrolizumab, supports the immunomodulatory effect of MTL-CEBPA in solid tumors

doi: 10.1016/j.xcrm.2025.102041

Figure Lengend Snippet:

Article Snippet: For MDSCs, this cocktail includes CD11b FITC (Miltenyi 130-110-552, clone REA713), CD14 APC-Vio770 (Miltenyi 130-110-522, clone REA599), CD15 PerCP-Vio700 (Miltenyi 130-113-487, clone VIMC6), LOX-1 PE (BioLegend 358604, clone 15C4), HLA-DR APC (Miltenyi 130-111-790 clone REA805), lineage markers: CD3 V500 (BD Horizon 561416, clone UCHT1), CD56 eFluor506 (eBiosciences 15520677, clone TULY56), CD19 V500 (BD Horizon 561121 clone HIB19), Dead cell exclusion dye eFluor 506 (Thermo Fisher Scientific 65-0866-14).

Techniques: Purification, Polymer, Derivative Assay, Clinical Proteomics, Extraction, Immunohistochemistry, Gene Expression, Inhibition, Software