Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: A, sort parameters for isolation of the human CD4+CD25− (up) and CD4+CD25+CD127− (down) T cells also called regulatory T cells. The left panels show the gate eliminating the CD4− T cells and selected specifically the CD4+ T cells (FSC mean Forward Scatter). The right top panel shows the gate for selecting the CD4+CD25− T cells from the CD4+ fraction previously selected. The right bottom panel shows the gate for selecting the CD4+CD25+CD127− T cells from the CD4+ fraction previously selected. B, sort parameters for isolation of the mouse CD4+CD25− and CD4+CD25high T cells (int mean intermediate). The different populations were isolated after a CD4+ T cell enrichment of mouse splenocytes. The panel displays only CD4+ T cells. CD4− T cells were eliminated from this population by the enrichment procedure and then the population was gated for CD4+ T cells. The left gate shows the sorting of the CD4+CD25− T cells. The right gate shows the sorting of the CD4+CD25high T cells.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Isolation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: A, quantification of the TRIB1 and Foxp3 mRNA in human CD4+CD25− and CD4+CD25+CD127− T cells. Real-time quantitative RT-PCR analysis for quantification of TRIB1 mRNA (left) and Foxp3 mRNA (middle) in CD4+CD25− and CD4+CD25+CD127− human T cells from seven healthy individuals. Statistical analyses were performed using a non parametric Mann-Whitney test. Analysis of correlation between TRIB1 and FOXP3 in CD4+CD25+CD127− T cells and statistical analysis of correlation were performed with a nonparametric Spearman test (right). B, quantification of the TRIB1 and Foxp3 mRNA in mouse CD4+CD25− and CD4+CD25high T cells. Real-time quantitative RT-PCR analysis of TRIB1 (left) and FOXP3 (right) mRNA in CD4+CD25− and CD4+CD25high mouse T cells of six normal C57Bl6 mice. Statistical analyses were performed using a nonparametric Mann-Whitney test.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

Article Title: Identification of Tribbles-1 as a Novel Binding Partner of Foxp3 in Regulatory T Cells *

doi: 10.1074/jbc.M112.448654

Figure Lengend Snippet: Quantification of TRIB1 and Foxp3 mRNA in human CD4+CD25+CD127− T cells freshly isolated or after activation. Real-time quantitative RT-PCR analysis for quantification of TRIB1 mRNA (A) and FOXP3 mRNA (B) in CD4+CD25+CD127− human T cells from healthy individuals after 0 to 24 h of culture in complete medium associated with recombinant IL-2 (rIL-2), anti-CD3, and anti-CD28 antibodies. Analysis of correlation between TRIB1 and Foxp3 expression with statistical analyses performed using a parametric Pearson test.

Article Snippet: CD4+ T cells were isolated using the biotinylated mouse CD4 T lymphocyte enrichment mixture (BD Biosciences) according to the manufacturer's instructions.

Techniques: Isolation, Activation Assay, Quantitative RT-PCR, Recombinant, Expressing

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. ( A ) Percentage of CD4 + and CD8 + T cells in PBLs. ( B ) Ratios of CD4 + and CD8 + T cells (left) and the frequency of CD4 + Foxp3 + Treg (right). The horizontal lines represent the median of all analyzed samples in each group.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Isolation, Staining, Flow Cytometry

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: PBMCs isolated from the whole blood of both diabetic patients and healthy controls were stained with indicated antibodies and analyzed by flow cytometry. ( A ) Percentage of CD45RA + or CD45RO + cells detected in the CD4 + or CD8 + T cells. ( B ) Percentage of CD45RA + or CD45RO + cells in CD4 + Foxp3 + Treg. ( C ) Representative results from FACS analyses of CD45RA, CD45RO and Foxp3 expression in the CD4 + T cells in PBLs. The cells were electronically gated for FACS analyses. * p <0.05, ** p <0.01, *** p <0.001.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Isolation, Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: Expanded CD4 + CD25 + CD127 lo/− T cells from healthy controls and T1D patients are highly enriched Foxp3 + Treg * .

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques:

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: CD4 + CD25 + CD127 −/lo Treg, electronically sorted using FACS from diabetic patients and healthy subjects, were activated in vitro using anti-CD3/anti-CD28-coated beads plus recombinant human IL-2. Expanded Treg were stained with CD4, CD25 and Foxp3 antibodies and analyzed by FACS on day 7 (D7) and day 14 (D14) following activation. ( A ) FACS analyses of CD4 and Foxp3 expression of expanded Treg on D7 and D14. The representative results were from cells obtained from one individual of each subject group. ( B ) Collective analyses of the purity and expansion fold of expanded Treg cells in each group on D7 and D14.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: In Vitro, Recombinant, Staining, Activation Assay, Expressing

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: PBMCs and expanded Treg were stained with indicated antibodies and analyzed by FACS. ( A ) Percentage of Helios + cells in total CD4 + T cells (CD4 + Helios + ) or in Foxp3 + CD4 + T cells (CD4 + Foxp3 + Helios + ) in PBLs of T1D patients or healthy controls. ( B ) FACS analyses of Helios and Foxp3 expression in PBLs and expanded Treg on D14 from one representative individual of each subject group. The cells were electronically gated on CD4 + T cell population. ( C ) Collective analyses of the percentage of Helios + cell frequency in PBLs or expanded Foxp3 + Treg on D14 in each subject group. ( D ) Paired comparison analyses of Helios expression on Foxp3 + Treg in PBLs vs. its expression on expanded Foxp3 + Treg in each subject group.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Staining, Expressing, Comparison

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: PBMCs and expanded Treg were stained with indicated antibodies and analyzed by FACS. FACS analyses of ( A ) TNFRII, ( C ) GARP expression on Foxp3 + Treg in PBLs and expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4 + T cell population. Collective analyses of the percentage of ( B ) TNFRII + or ( D ) GARP + cell frequency in PBLs or expanded Foxp3 + Treg.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Staining, Expressing

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: PBMCs were stained with indicated antibodies following activation with PMA/ionomycin for 5 h in vitro and analyzed by FACS. ( A ) Percentage of IFN-γ + , TNF-α + ,or IL17 + cells present in CD4 + T cells of PBLs from T1D patients or healthy controls. ( B ) The ratios of Th1/Treg, Th1/Th17, and Th17/Treg in CD4 + T cells from PBLs of diabetic patients or healthy controls.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Staining, Activation Assay, In Vitro

Journal: PLoS ONE

Article Title: Foxp3 + Treg Expanded from Patients with Established Diabetes Reduce Helios Expression while Retaining Normal Function Compared to Healthy Individuals

doi: 10.1371/journal.pone.0056209

Figure Lengend Snippet: Cells from PBMCs and expanded Treg on Day 14 after anti-CD3/CD28 activation were stained with indicated antibodies following stimulation with PMA/ionomycin for 5 h and analyzed by FACS. ( A – B ) IFN-γ, TNF-α, and IL17 expression in Foxp3 + Treg of ( A ) PBLs and ( B ) expanded Treg from one representative individual of each subject group. Cells were electronically gated on CD4 + T cell population. (C – D ) Collective analyses of the percentage of IFN-γ + , TNF-α + , or IL17 + in ( C ) Foxp3 + Treg of PBLs or ( D ) expanded Foxp3 + Treg.

Article Snippet: For some subjects, CD4 + T cells were enriched with a human CD4 + lymphocyte enrichment kit (BD) prior to staining.

Techniques: Activation Assay, Staining, Expressing

Journal: Cancer Immunology Research

Article Title: Conditional PD-1/PD-L1 Probody Therapeutics Induce Comparable Antitumor Immunity but Reduced Systemic Toxicity Compared with Traditional Anti–PD-1/PD-L1 Agents

doi: 10.1158/2326-6066.CIR-21-0031

Figure Lengend Snippet: C5H9v2 mAb is a high-affinity blocker of PD-L1 and can potentiate T-cell responses. A, The capacity of C5H9v2 hIgG4 mAb to bind and block immobilized PD-L1 was evaluated in an ELISA format. Serial dilutions of C5H9v2 mAb were incubated with recombinant plate-bound PD-L1-Fc fusion protein of human, mouse, rat, or cynomolgus monkey origin. Bound antibody was detected using an anti-human HRP-conjugated secondary antibody and is shown in relation to the concentration. Data shown are representative of 3 independent experiments. Serial dilutions of C5H9v2 mAb were added to wells containing biotinylated hPD-1 ( B ) or hB7-1 (CD80; C ) protein and plate-bound PD-L1-Fc to measure blocking capacity. The binding of hPD-1 or hB7-1 to PD-L1 was detected using a streptavidin–HRP conjugate. Data shown are representative of 3 independent experiments. D, Blockade of PD-L1 by C5H9v2 mAb enhanced IFNγ production in a CMV recall assay. CMV-positive hPBMCs were plated at 350,000 cells/well in media containing serial dilutions of C5H9v2 mAb or isotype control and stimulated with 4 μg/mL CMV lysate for 4 days, at which point IFNγ was measured in the supernatants by ELISA. Data shown are representative response from one donor ( n = 3 donors tested) of 3 independent experiments. All data points represent the mean ± SD. Abs, absorbance; Cyno, cynomolgus monkey; hB7-1, human B7-1; hPBMC, human peripheral blood mononuclear cell.

Article Snippet: Binding of hPD-L1 mAb (C5H9v2), CX-072 Pb-Tx, and preactivated CX-072 Pb-Tx to PD-L1–positive cells of human (SAS human cell line), mouse (MC38 mouse cell line), rat (Sprague Dawley rat peripheral blood CD4 + T lymphocytes, BioIVT, catalog #RATWBLIHPO-M), and cynomolgus monkey (peripheral blood CD4 + T lymphocytes, BioIVT, catalog #CYNWBLIHP-M) origin was evaluated by flow cytometry.

Techniques: Blocking Assay, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant, Concentration Assay, Binding Assay, Control

Journal: PLOS ONE

Article Title: Immunocapture of cell surface proteins embedded in HIV envelopes uncovers considerable virion genetic diversity associated with different source cell types

doi: 10.1371/journal.pone.0296891

Figure Lengend Snippet: The “Total” sequence at the bottom is the bulk genotype of the sample. Series 1 (S1) antibody order was anti-CD2 → CD36 → CD44 → CD15; series 2 (S2) order was anti-CD15 → CD36 → CD44 → CD2. IDs with “CAP” in the title are the sequences obtained when the single capture was applied to the sample to compare to the sequence obtained when the marker was used within a series. CD3 and CD68 are pan lymphocyte and macrophage markers, respectively. Only mutations associated with drug resistance are shown and differences in mutations are highlighted by amino acids in color. The relative proportion of amino acid at codons with mixtures are indicated as majority (capital letter), minority (lower-case letter), or equivalent proportions (both capital letters). Inferred using the Neighbor-Joining method and distances computed using the Tamura 3-parameter method with 500 bootstrap replicates. Rate variation was modeled with gamma distribution (5-parameter) (MEGA v6). Bootstrap values >60 are indicated at nodes.

Article Snippet: Lymphocyte sorts were first enriched for CD4 cells by magnetic removal of CD8, CD16, CD11b, CD36, CD56, CD123 and CD235a cells (Beckton Dickinson iMag Human CD4 T Lymphocyte Enrichment) then applied to a cell sorter (Sony SH800Z) gated for CD3+ and CD45+.

Techniques: Sequencing, Marker