rea623 1ul 2e5 cells cd8 apc vio770 miltenyi  (Miltenyi Biotec)

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: Scientific Reports

Article Title: Long-term exposure to “low-dose” bisphenol A decreases mitochondrial DNA copy number, and accelerates telomere shortening in human CD8 + T cells

doi: 10.1038/s41598-020-72546-x

Figure Lengend Snippet: Short-term effect of “low dose” BPA exposure on telomerase activity and DNA repair capacity in activated human CD8 + T lymphocytes. ( A ) CD4 + and CD8 + T cell subsets were stimulated with anti-CD3/CD28 and exposed to BPA or solvent control (SC = DMSO 0.01%) for 24 h. Telomerase activity was analysed using the TRAP-ELISA assay. ( B , C ) Isolated CD8 + T cells were stimulated with anti-CD3/28 and treated with BPA or SC for 24 h. After that, the cells were washed with PBS before challenging with 5 µM H 2 O 2 or 2.5% PBS (SC) for 10 min at 37 °C. 3 µM APC was used to block DNA damage repair in the cells. The % tail DNA was analysed as parameter for DNA damage and is given in ( B ), the calculated DNA repair capacity is given in ( C ). ( D ) Gene expression analysis of human DNA repair genes using a real—time PCR array. The scatter blot compares expression of 84 DNA repair pathway genes between the solvent control (0.01% DMSO) and 0.3 nM BPA treated cells. The solid diagonal line represents no change in expression. Any data point above the upper line represents genes that are upregulated > twofold. Any data point below the lower line represents genes that are downregulated > twofold. Bars are mean values; results were presented as means + SD. Significance of difference was calculated relative to the respective control, * p < 0.05; ** p < 0.01.

Article Snippet: The following primary human antibodies labelled with fluorophore were used for flow cytometry: CD8‐PE/APC (clone BW135/80), CD4‐PE/APC (clone REA623), CD3-FITC/PE/APC (clone REA 613), CD27-PerCP (clone M-T271), CD28-FITC/PE (clone REA 612), CD45RA-PE (clone T6D11), CD197-FITC (clone REA 108), IFN-γ-FITC (clone REA600) (Miltenyi Biotec, Bergisch Gladbach, Germany).

Techniques: Activity Assay, Solvent, Enzyme-linked Immunosorbent Assay, Isolation, Blocking Assay, Expressing, Real-time Polymerase Chain Reaction