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: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Flow cytometry stainings per panel.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Flow Cytometry, Conjugation Assay, Functional Assay, Blocking Assay

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Phenotypic characterization of MSLN-CAR T cells after CAR production . A) Frequency of CD4 + and CD8 + cells within CD3 + EGFRt + M28z and MBBz CAR T cells and within CD3 + untransduced T cells (UT). N = 10 donors. B) Ratio of CD4 + /CD8 + within EGFRt + (CAR + ) and EGFRt- (CAR − ) M28z and MBBz transduced T cells. N = 10 donors. C) Maturation phenotype determined by CD45RA and CCR7 expression in CD3 + EGFRt + M28z and MBBz CAR T cells and within CD3 + UT cells. Gated on lymphocytes→ single cells→ viable cells (7AAD − )→ CD3 + EGFRt + (M28z and MBBz) or CD3 + EGFRt − (UT)→ CD4 + /CD8 + and ultimately CD45RA/CCR7. Median of n = 9 donors displayed. D) IFNy, TNF, IL2 cytokine production and CD107a degranulation by CD4 + and CD8 + M28z and MBBz transduced T cells following 6 hours of co-culture with OVCAR-3 MSLN + , determined by intracellular cytokine staining. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Wilcoxon tests were performed to detect differences between and within M28z and MBBz CAR constructs. For comparison of three T cell subsets (M28z vs MBBz vs UT) Friedman tests were used. * = P < .05 and ** = P < .01. Each dot represents 1 donor.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Expressing, Co-Culture Assay, Staining, Construct, Comparison

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Superior lysis and infiltration of MSLN high SKOV-3 spheroids by M28z CAR T cells as compared to MBBz CAR T cells . A) Lysis of MSLN high OVCAR-3 cells as determined by LDH release following co-culture with CD4 + enriched, CD8 + enriched and unsorted CD4 + /CD8 + MSLN-CAR T cells at a 1:5 or 2:1 effector to target ratio for 24 hours. N = 6 donors, each dot represents one donor. B, C) Lysis of MSLN high SKOV-3 spheroids in response to co-culture with UT cells, M28z or MBBz transduced T cell was monitored for 24 hours by detection of Caspase3/7 activation using the IncuCyte S3 live cell imaging system. B) Caspase3/7 activation as depicted by the green signal in representative spheroid. C) Caspase3/7 activation illustrated by the integrated GCU over time. Dots represent the mean+SEM of 3 technical replicates of 6 donors. GCU = green calibrated unit. D) Non-linear regression revealed EC50 of Caspase3/7 activation. E) Frequency of total T cells (%CD4 + +CD8 + ) within SKOV-3 MSLN high spheroids as detected by confocal microscopy following 6 hours of co-culture. F) CD4 + or CD8 + T cells within MSLN high SKOV-3 spheroids as detected by confocal microscopy after 6 hours of co-culture. Each dot represents the mean of 3 technical replicates from 1 donor, n = 6 donors. G) Representative confocal image of T cell infiltration of one donor. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Friedman tests were performed to detect differences in target cell lysis by CD4 + , CD8 + or CD4 + /CD8 + T cells (OVCAR-3 vs SKOV-3). Wilcoxon was performed to compare OVCAR-3 vs SKOV-3 per T cell condition and M28z vs MBBz. One-way ANOVA was used to compare caspase3/7 signal over a 24 hour time course in response to UT, M28z or MBBz treatment. N = 6. * = P < .05, ** = P < .01, *** = P < .001.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Lysis, Co-Culture Assay, Activation Assay, Live Cell Imaging, Confocal Microscopy

Journal: Oncoimmunology

Article Title: Trogocytosis and fratricide killing impede MSLN-directed CAR T cell functionality

doi: 10.1080/2162402X.2022.2093426

Figure Lengend Snippet: Dynamic expression of PD-1, LAG-3 and TIM-3 exhaustion markers by M28z and MBBz CAR T cells after co-culture with target cells . A) Prior to start of experiment (t = 0), CAR surface expression on M28z and MBBz transduced T cells was determined by hFAB staining. During co-culture with MSLN high OVCAR-3 and SKOV-3 hFAB expression was monitored. B) Kinetics of PD-1, LAG-3 and TIM-3 frequency (top) and MFI (bottom, logarithmic y-axis) within CD4 + or CD8 + CAR + M28z and MBBz transduced T cells prior (0 h) and during co-culture (4 h and 24 h) with MSLN high OVCAR-3 cells. Each dot represents one donor, n = 6 donors. C) Frequency of PD-1/LAG-3/TIM-3 triple positive (TP or Triple+) within CAR + M28z and MBBz transduced T cells overtime. Median of 6 donors is displayed. D) Comparison between CIM (co-)expression following 24 hours of co-culture with MSLN high OVCAR-3 and SKOV-3 cells. Each dot represents one donor, n = 6 donors. Grey symbols represent M28z CAR T cells and green symbols represent MBBz CAR T cells. Wilcoxon tests were performed to compare between two time points, CAR constructs and cell lines (delta hFAB 4 hours vs 24 hours, M28z vs MBBz, and OVCAR-3 vs SKOV-3). Friedman tests were used per CAR construct over time (≥3 time points). * = P < .05, ** = P < .01.

Article Snippet: Primary anti-human CD8a (Biolegend) and anti-human CD4 (R&D Systems) antibodies were added and incubated before DNAHoechst (Invitrogen), secondary rat-anti-mouse AF647 (Biolegend) and donkey-anti-goat AF488 (Thermo Scientific) antibodies were added and incubated for 24 h. For mesothelin staining, spheroids were incubated with PE-conjugated anti-human MSLN antibody (R&D Systems) for 24 h. All stainings were performed in PBS 1% BSA at 4°C.

Techniques: Expressing, Co-Culture Assay, Staining, Comparison, Construct

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: Gating strategy for the identification of human T SCM . T cells were identified by gating on lymphocytes (SSC versus FSC), singlets (FSC-H versus FSC-A) and live T cells (SSC versus LIVE/DEAD). CD4 + and CD8 + T cells were simultaneously identified with anti-CD4 and anti-CD8 antibodies. a Gating strategy of fresh blood cells. After gating on CD4 + and CD8 + cells, T CM and T EM subpopulations were identified based on CCR7 and CD45RO expression. In the gated CCR7 + CD45RO − population, cells expressing CD45RA and CD27 were further analyzed. In this later population (CCR7 + CD45RO − CD45RA + CD27 + ), T N and T SCM were identified based on the CD95 expression. T N is defined as CCR7 + CD45RO − CD45RA + CD27 + CD95 − whereas T SCM subpopulation is defined as CCR7 + CD45RO − CD45RA + CD27 + CD95 + . Red arrows indicate the sequential gating strategy. b Gating strategy of 10 days culture cells. After gating on CD4 + and CD8 + cells, T CM and T EM subpopulations were identified based on CCR7 and CD45RO expression. In the gated CCR7 + CD45RO − population, cells expressing CD45RA and CD27 were further analyzed. In this later population (CCR7 + CD45RO − CD45RA + CD27 + ), T SCM were identified based on the CD95 expression. T SCM subpopulation is defined as CCR7 + CD45RO − CD45RA + CD27 + CD95 + . Similarly, in the gated CCR7 + CD45RO + population, cells expressing CD45RA, CD27 and CD95 + identify a T SCM- like subpopulation, which is defined as CCR7 + CD45RO + CD45RA + CD27 + CD95 + . Red arrows indicate the sequential gating strategy

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: Short CD3/CD28 costimulation increases CD4 + and CD8 + T SCM frequencies compared with long costimulation. Naïve T cells from healthy donors (n = 6) were cultured for 10 days with short (48 h) ( solid black line ) or long ( solid grey line ) costimulation. a , b Frequency of CD4 + ( a ) and CD8 + ( b ) T SCM cell subset (mean ± SEM). c , d Frequencies of total T SCM (T SCM + T SCM -like) CD4 + ( c ) and CD8 + ( d ) (mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques: Cell Culture

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: IL-21 enhances CD4 + and CD8 + T SCM frequencies. Naïve T cells from healthy donors (n = 6) were cultured for 10 days with short ( solid black line ) or long ( solid grey line ) costimulation and in the presence ( black dashed line ) or absence ( grey dashed line ) of IL-21. a , b Frequencies of CD4 + and CD8 + T SCM (mean ± SEM) are shown in ( a ) and ( b ), respectively. c , d Frequencies of CD4 + and CD8 + Total T SCM (T SCM + T SCM -like) (mean ± SEM) are shown in ( c ) and ( d ), respectively. *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques: Cell Culture

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: Analysis of CD4 + and CD8 + T cell subsets composition. a , b Column graphs showing the relative frequencies (mean ± SEM) of CD4 + ( a ) and CD8 + ( b ) T cell subsets respectively, for each condition tested (long costimulation, short costimulation, long + IL-21, and short + IL-21). T N (T Naïve), T CM (T central memory), T EM (T effector memory), T EMRA (T effector memory-RA+ cells)

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques:

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: IL-21 enhances CD4 + and CD8 + T SCM expansion. Naïve T cells from healthy donors (n = 3) were cultured for 10 days and fold expansion were analyzed in short costimulation ( solid black line ), long costimulation ( solid grey line ) short + IL-21 ( black dashed line ) and long + IL-21 ( grey dashed line ) conditions. a , b Absolute fold expansion of CD4 + ( a ) and CD8 + ( b ) (mean ± SEM), expressed in increment of cells from the starting culture time point is shown. c , d Absolute fold expansion of CD4 + ( c ) and CD8 + ( d ) total TSCM (TSCM + TSCM-like) (mean ± SEM). *p < 0.05; **p < 0.01; ***p < 0.001

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques: Cell Culture

Journal: Journal of Translational Medicine

Article Title: A short CD3/CD28 costimulation combined with IL-21 enhance the generation of human memory stem T cells for adoptive immunotherapy

doi: 10.1186/s12967-016-0973-y

Figure Lengend Snippet: Analysis of transduction efficiency with a GFP-expressing lentivirus. Lentivirus transduction efficiency measured in CD4 + and CD8 + T SCM and T CM subsets by flow cytometry (% of cells expressing GFP; mean ± SEM)

Article Snippet: Cells were thawed and labeled with fluorescent antibodies against human CD4-APC-Vio-770 (clone VIT4), CD8-Viogreen (clone BW135/80), CD45RA-PerCP-Vio700 (clone T6D11), CD45RO-PE (clone UCHL1), CD27-Vioblue (clone M-T271), CD95-FITC (clone DX2) and CCR7-PE-vio770 (clone REA108) (all from Miltenyi Biotech, Germany).

Techniques: Transduction, Expressing, Flow Cytometry