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: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Flow cytometry contour plots demonstrating the loss of the endogenous splenic CD4 + Foxp3 -GFP + Treg population in Foxp3 GFP-DTR mice following administration of diphtheria toxin (DTx). (C) Lung and spleen Foxp3 -GFP + Treg cell quantification in Foxp3 GFP-DTR mice that received influenza A virus but no DTx (No DTx + flu, n = 3) or influenza and DTx (DTx + flu, n = 4) at 13 DPI compared with mice that received DTx but no influenza (DTx No flu, n = 3). (D) Mass over time of Foxp3 GFP-DTR mice receiving no DTx or DTx until pre-specified timepoints (DTx withdrawal) following intra-tracheal inoculation of 5 plaque-forming units (PFU) of influenza A/WSN/33 H1N1 virus (n = 5 per group). (E) Representative lung histopathology (H&E staining) at 60 DPI of control (No DTx) and DTx withdrawal mice following influenza A virus infection. Original magnification x10, scale bar = 1 mm. (F) Metagene analysis of DNA methylation across Treg-SE of Tregs recovered from the mice described in 1D compared with splenic nTregs and iTregs from culture. Data presented as mean and SD. * q < 0.05 according to one-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C).* q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (D).

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Virus, Histopathology, Staining, Control, Infection, DNA Methylation Assay

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Flow cytometry contour plots for phenotyping of the CD4 + Foxp3 -GFP + tdTomato + iTreg population following culture in the presence of αCD3/αCD28, IL-2, TGF-β, and tamoxifen. (B) Mass over time of Foxp3 GFP-DTR mice receiving adoptive transfer of nTreg (n = 27), PBS (n = 21), Tconv (n = 25), or iTreg (n = 18) cells 5 days following intra-tracheal inoculation of 6.5 PFU of influenza A/WSN/33 H1N1 virus. Negative controls included mice that received influenza but no DTx (no DTx + flu, n = 9) and DTx but no influenza (DTx no flu, n = 3). (C) Total number of lung CD45 + cells at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10). (D-H) Total number of lung CD326 + CD31 − CD45 − cells (D), CD326 + MHCII + T1A − cells (E), KRT5 + CD326 + cells (F), Ki-67 + CD326 + MHCII + cells (G), and CD326 − CD31 + cells (H) at 24 DPI in mice that received No DTx (n = 3), nTreg (n = 15), PBS (n = 11), or Tconv (n = 10) cells. * q < 0.05 according to mixed-effects model (REML) with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (B). Data presented as mean and SD (C, F-H) with * q < 0.05 according to multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% (C). UHRF1 is dispensable for iTreg FOXP3 induction and stability but is required to maintain transcriptional and epigenetic programs in vitro .

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Flow Cytometry, Adoptive Transfer Assay, Virus, MANN-WHITNEY, In Vitro

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) Schematic of experimental design. (B) Frequency of Foxp3 -GFP + and Foxp3 -GFP − tdTomato + (ex-FOXP3) cells in culture of iTregs with UHRF1 deleted concurrent with (early) or 5 days after (delayed) FOXP3 induction compared with Uhrf1 +/+ controls on days 5 and 12 of culture (n = 3 per group). (C) Division index of CD4 + CTV + Foxp3 -GFP − splenic responder T cells co-cultured for 72 hours with varying ratios of Uhrf1 +/+ CD4 + Foxp3 -GFP + iTregs, Uhrf1 fl/fl CD4 + Foxp3 -GFP + iTregs, or Uhrf1 +/+ nTregs (n = 3 per group). (D) Principal component analysis of 6,978 differentially expressed genes from sorted cells described in A, identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (E) K-means clustering of 127 genes with an FDR q < 0.05 comparing the Uhrf1 fl/fl and Uhrf1 +/+ iTreg populations on day 12 in which UHRF1 was deleted after FOXP3 induction (delayed) with k = 2. (F) MA plot comparing gene expression of delayed Uhrf1 +/+ and Uhrf1 fl/fl iTregs on day 12 of culture. Genes of interest are annotated. (G) Enrichment plot of the GSE14415_INDUCED_TREG_VS_TCONV_UP, GSE14415_INDUCED_TREG_VS_TCONV_DN, GSE14415_INDUCED_TREG_VS_FAILED_INDUCED_TREG_UP, GSE37605_TREG_VS_TCONV_C57BL6_FOXP3_FUSION_GFP_UP, GSE13306_TREG_VS_TCONV_UP gene sets ( p < 0.05, FDR q < 0.25) generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture. (H) PCA of 81,179 differentially methylated cytosines identified from ANOVA-like testing with FDR q < 0.05. Ellipses represent normal contour lines with 1 standard deviation probability. (I) Cumulative distribution function plot of differentially methylated cytosines expressed as β scores, with 0 representing unmethylated and 1 representing fully methylated; a shift in the cumulative distribution function up and to the left represents relative hypomethylation. * q < 0.05 according to two-way ANOVA with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Cell Culture, Standard Deviation, Gene Expression, Generated, Control, Methylation

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A) MA plot comparing gene expression of Uhrf1 +/+ iTregs (control) with CD4 + Foxp3 -GFP + nTregs on day 5 of culture. Genes of interest are annotated. (B) Enrichment plots of the HALLMARK_MYC_TARGETS_V1, HALLMARK_E2F_TARGETS, HALLMARK_TGF_BETA_SIGNALING, HALLMARK_WNT_BETA_CATENIN_SIGNALING, HALLMARK_TNFA_SIGNALING_VIA_NFKB, HALLMARK_MTORC1_SIGNALING, HALLMARK_IL2_STAT5_SIGNALING, and HALLMARK_KRAS_SIGNALING_UP gene sets generated through GSEA preranked testing of the expressed genes of delayed Uhrf1 +/+ iTregs (control) and delayed Uhrf1 fl/fl iTregs on day 12 of culture.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: Gene Expression, Control, Generated

Journal: bioRxiv

Article Title: Maintenance DNA methylation is required for induced regulatory T cell reparative function following viral pneumonia

doi: 10.1101/2025.02.25.640199

Figure Lengend Snippet: (A-F) Total numbers of CD45 + (A), CD3 + (B), CD4 + CD8 − (C), CD4 − CD8 + (D), CD11b − CD64 + CD11c + SiglecF + (E), and CD11b + Ly6G + (F) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 16), or U hrf1 fl/fl iTregs. (n = 16) (G) Total number of CD326 + CD31 − CD45 − (epithelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 7), Uhrf1 +/+ iTregs (n = 15), or U hrf1 fl/fl iTregs (n = 16). (H) Total number of KRT5 + CD326 + epithelial cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 7), Tconv (n = 7), PBS (n = 7), Uhrf1 +/+ iTregs (n = 13), or U hrf1 fl/fl iTregs (n = 14). (I) Total number of CD326 − CD31 + (endothelial) cells in the lungs at 11 DPI of Foxp3 GFP-DTR mice that received nTreg (n = 8), Tconv (n = 8), PBS (n = 8), Uhrf1 +/+ iTregs (n = 14), or U hrf1 fl/fl iTregs (n = 15). Data presented as mean and SD. ns = not significant by multiple Mann-Whitney tests and correcting for multiple comparisons using the two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli with Q = 5% test.

Article Snippet: For iTreg cell sorting, the single-cell suspension was enriched using a CD4 + antibody (Miltenyi cat. no. 130-101-962) and the lungs were stained using the reagents listed in .

Techniques: MANN-WHITNEY

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: A technical control of non-stimulated (NST) PMBCs was included. a, b, c) Percentage CD4 T cells in PBMCs stimulated with S peptides in the H donors (a) and in O patients (b). c) Comparison between H donors and O patients. d, e, f) Percentage CD4 T cells in PBMCs stimulated with M peptides in H donors (d) and in O patients (e). f) Comparison between H donors and O patients. g, h, i) Percentage CD4 T cells in PBMCs stimulated with N peptides in the H donors (g) and in O patients (h). i) Comparison between H donors and O patients. a-i) Krustal-Wallis test followed by Dunn’s test for pair-wise comparisons was employed. j) Dot plot of the percentage of S and M-specific CD4 T cells. The U of Mann-Whitney was used to test differences. k) Dot plot of S- and M-specific CD4 T cells in vaccinated H donors, from samples collected at the indicated timelines. Pair-wise comparisons were performed using the U of Mann-Whitney. l, m) Relative percentages of CD4 T cell differentiation phenotypes in H donors (l) and O patients (m) . N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L- CD45RA-) and effector (CD62L-CD45RA+) phenotypes. Relevant statistical comparisons are detailed in . *, **, ***, **** indicate P <0.05, <0.01, <0.001 and <0.0001, respectively.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Control, Comparison, MANN-WHITNEY, Cell Differentiation

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: Representative flow cytometry density plots of CD154 and CD137 co-expression profiles in CD4 T cells from donors before and after stimulation with S-peptides, as indicated.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with CD62L-CD45RA co-expression profiles in CD4 T cells before and after the stimulation with S-peptides. Quadrants were established with unstained controls. Percentages of the corresponding populations are shown within the quadrants. b , c) CD4 T cell phenotypic changes in H-V (b) and H-CoV-V (c) donors. (-) and (+S), non-stimulated and S-peptide stimulation. N, CM, EM and E, indicate naïve-stem cell (CD62L+ CD45RA+), central memory (CD62L+ CD45RA-), effector memory (CD62L- CD45RA-) and effector (CD62L-CD45RA+) phenotypes. d) Phenotypic changes in CD4 T cells within O-CoV-V before and after stimulation with S-peptides. e , f) Effects of previous CoV infection in vaccinated H donors and O patients over T cell phenotypes after stimulation with S-peptides. b-f) Relevant statistical comparisons are indicated by ANOVA followed by pair-wise comparisons with Tukey’s test. g , h) Relative percentages of CD4 T cell differentiation phenotypes in H-V and O-V (g) and in H-CoV-V and O-CoV-V (h) CD27+ CD28+, CD27-CD28+ and CD27+ CD28+ indicate poorly differentiated, intermediate differentiated and highly differentiated T cell phenotypes. U of Mann-Whitney was used to test for significance.*, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing, Infection, Cell Differentiation, MANN-WHITNEY

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a) Representative flow cytometry density plots with the expression of INFγ and IL-17 before and after stimulation with S peptides in CD4 T cells. b) In CD8 T cells.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Flow Cytometry, Expressing

Journal: medRxiv

Article Title: Profiling of immune responses to COVID-19 and vaccination uncovers potent adjuvant capacities of SARS CoV-2 infection to vaccination leading to memory T cell responses with a Th17 signature in cancer patients

doi: 10.1101/2022.05.27.22275672

Figure Lengend Snippet: a, b, c) Percentage of IFNγ-CD4 T cells specific for the S protein in H and O groups. d, e, f) Percentage of IFNγ-CD4 T cells specific for the M protein in H and O groups. g, h, i) Percentage of IL17-CD4 T cells specific for the S protein in H and O groups. j, k, l) Percentage of IL17-CD4 T cells specific for the M protein in H and O groups. a-l) Statistical significance was evaluated with Kruskal-Wallis followed by Dunn’s pair-wise comparisons. m, n) Percentage of CD4 T cells expressing IL-17 and IFNγ expression in H-V donors that completed the vaccine regime before sample collection in the indicated timelines, for both S and M proteins. Significance was tested with the U of Mann-Whitney test. NST, technical control of non-stimulated PMBCs. *, **, *** indicate significant (P<0.05), very significant (P<0.01) and highly significant (P<0.001) differences.

Article Snippet: The following fluorochrome-conjugated antibodies were used: CD14-Violet Fluor 450 (Ref 75-0149-T100, TONBO), CD11b-PerCP-Cy5-5 (Ref 65-0112-U1, TONBO), CD62L-APC (Ref 130-113-617, Miltenyi), CD66b-APC-Cy7 (Ref 130-120-146, Miltenyi), CD54-FITC (Ref 130-104-214, Miltenyi), CD19-PE (Ref 130-113-731, Miltenyi), CD3-APC (Ref 130-113-135, Miltenyi), CD8-APC-Cy7 (Ref 130-110-681, Miltenyi), CD4-FITC (Ref 130-114-531, Miltenyi), CD27-PE (Ref 50-0279-T100, TONBO), CD28-PE-Cy7 (Ref 130-126-316, Miltenyi).

Techniques: Expressing, MANN-WHITNEY, Control

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