Journal: Frontiers in Immunology

Article Title: Phosphoproteomics Reveals Regulatory T Cell-Mediated DEF6 Dephosphorylation That Affects Cytokine Expression in Human Conventional T Cells

doi: 10.3389/fimmu.2017.01163

Figure Lengend Snippet: Experimental setup and quality controls for phosphoproteomics in primary human T cells. (A) Conventional CD4 + CD25 – T cells (Tcons) were cocultured either with allogeneic Tcons or regulatory T cells (Tregs), and cocultures were stimulated for 5 min with cross-linked anti-CD3/anti-CD28 antibodies. Stimulation was stopped on ice. Tstim (blue) and Tsup (red) were obtained after separation of T cell receptor (TCR)-stimulated Tcon:Tcon or Tcon:Treg cocultures, respectively. Unstimulated Tcons (Trest; gray) from the same donor were processed in parallel. Proteins were digested, peptides dimethyl-labeled and mixed, before phosphopeptides were enriched and measured by mass spectrometry (MS). Relative abundance of phosphopeptides was quantified by calculating the intensity ratios between the different samples as indicated. (B) An aliquot of cells used for phosphoproteomics was stimulated for 3 h before coculture separation, and suppression of cytokine mRNA was measured in re-isolated responder Tcons [Trest, Tstim, and Tsup as in panel (A) ]. As additional control, responder Tcons were stimulated without allogeneic Tcons (control Tstim). IL2 and IFNG mRNA were measured by quantitative RT-PCR, normalized to GAPDH mRNA. Results are presented as fold change compared to Trest (set to 1). The upper panel shows a representative donor (mean ± SD of technical PCR duplicates). Percentage suppression of respective cytokines in Tsup as compared to Tstim was calculated and is summarized for the three phosphoproteomics donors (lower panel). T cells were processed in three independent experiments (one experiment/donor) and phosphopeptide enrichment was performed in two independent experiments. (C) The number of unique phosphopeptides detected in each donor was determined, and the overlap is depicted as Venn diagram.

Article Snippet: We first isolated CD25 high cells with CD25-specific MACS beads (2 μl/10 7 cells, Miltenyi Biotec; cat. no. 130-092-983) as described previously ( ).

Techniques: Phospho-proteomics, Labeling, Mass Spectrometry, Isolation, Control, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A ) Schematic diagram for high-throughput luciferase complementation screening. ( B ) Screening of FDA-approved drugs library for compounds capable of inhibiting luciferase activity in 293T-NF + RC. ( C ) Lac suppresses transcription of Foxp3, Il10, Cd25, Ifnγ and Ctla4 in Tregs. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Cd25 , * P = 0.0157; Ctla4 , ** P = 0.0065; Foxp3 , ** P = 0.0021; Il10 , *** P = 0.0001; Ifnγ , P = 0.1926. ( D – G ) Flow cytometry analysis of the impact of Lac on marker protein expression by Tregs. ( H ) Lac suppresses IL10 expression by Tregs. Tregs (2 × 10 5 ) were isolated from lymph nodes which were treated with DMSO or Lac for 48 or 72 h before ELISA analysis. Data are representative of three independent experiments. **** P < 0.0001 by Student’s t test. Error bars denote mean ± SD. ( I ) Lac suppresses the proliferation of Tregs in vitro. CFSE-labeled Tregs in lymph nodes were (2 × 10 5 ) treated with DMSO or Lac for 48 h. ( J ) Statistics analysis of ( I ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: PI, *** P = 0.0004; DI, *** P = 0.0006. .

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: High Throughput Screening Assay, Luciferase, Activity Assay, Flow Cytometry, Marker, Expressing, Isolation, Enzyme-linked Immunosorbent Assay, In Vitro, Labeling

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A ) Tregs-purifying efficiency from mouse lymph nodes. Conventional CD4 + T (CD4 + CD25 - ) or Treg (CD4 + CD25 + ) cells were purified from lymph nodes by negative selection with MACS before flow cytometry assay. ( B – I ) Flow cytometry analysis of the impact of Lac on marker protein expression by Tregs. The gating of FOXP3 + CD25 + ( B ), FOXP3 + CTLA4 + ( D ), FOXP3 + Perforin + ( F ) and IL10 + ( H ) populations were determined against their FMO; Statistics analysis of Fig. and EV1B (EV1C), Fig. and EV1D (EV1E), Fig. and EV1F (EV1G), Fig. and EV1H (EV1I). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: FOXP3 + CD25 + (Lymph nodes), *** P = 0.0006; FOXP3 + CD25 + (Spleen), ** P = 0.0024; FOXP3 + CTLA4 + (Lymph nodes), ** P = 0.0035; FOXP3 + CTLA4 + (Spleen), ** P = 0.0017; FOXP3 + Perforin + (Lymph nodes), ** P = 0.0014; FOXP3 + Perforin + (Spleen), *** P = 0.0008; FOXP3 + IL10 + (Lymph nodes), *** P = 0.0006; FOXP3 + IL10 + (Spleen), **** P < 0.0001. ( J ) Lac suppresses Tregs differentiation in vitro. Representative flow plots were gated on CD4 + population for analysis. The FOXP3 + CD25 + (upper panel) or FOXP3 + CTLA4 + (lower panel) populations were determined against their FMO, respectively. ( K ) Statistics analysis of Fig. EV1J. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: FOXP3 + CD25 + , ** P = 0.0010; FOXP3 + CTLA4 + , ** P = 0.0016. ( L ) Lac suppresses the proliferation of Tregs in vitro. Proliferation of Tregs were determined by dilution of CFSE through flow cytometry analysis. ( M ) Statistics of the percentages of proliferating Tregs of Fig. EV1L. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: PI, ** P = 0.0014; DI, ** P = 0.0041. ( N ) Impact of Lac on apoptosis of Tregs. Cells were stained with PI and Annexin V-FITC for flow cytometry analysis. ( O ) Statistics of apoptosis population of Fig. EV1N. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: PI + , P = 0.1653; Annexin V + , P = 0.4850; Annexin V + PI + , P = 0.4890.

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Purification, Selection, Flow Cytometry, Marker, Expressing, In Vitro, Staining

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A ) Lac alleviates inhibition of CTLs proliferation by Tregs. ( B ) Statistics analysis of ( A ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: PI (DMSO-CD25 − vs DMSO-CD25 + ), *** P = 0.0007; PI (DMSO-CD25 + vs Lac-CD25 + ), *** P = 0.0003; DI (DMSO-CD25 − vs DMSO-CD25 + ), *** P = 0.0007; DI (DMSO-CD25 + vs Lac-CD25 + ), *** P = 0.0002. ( C – F ) Lac alleviates Tregs-induced suppression of CTLs expression of GRZB and IFNγ. Expression of GRZB ( C ) and IFNγ ( E ) were detected by flow cytometry. Statistics analysis of ( C , D ) and ( E , F ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: GRZB (DMSO-CD25 − vs DMSO-CD25 + ), **** P < 0.0001; GRZB (DMSO-CD25 + vs Lac-CD25 + ), *** P = 0.0003; IFNγ (DMSO-CD25 − vs DMSO-CD25 + ), **** P < 0.0001; IFNγ (DMSO-CD25 + vs Lac-CD25 + ), * P = 0.0174. ( G ) Lac alleviates the inhibition of cytotoxicity of CTLs by Tregs. ( H ) Statistics analysis of ( G ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: DMSO-CD25 − vs DMSO-CD25 + , *** P = 0.0004; DMSO-CD25 + vs Lac-CD25 + , *** P = 0.0005. .

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Inhibition, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A – C ) Lac shrinks LLC allograft tumor. The allografts ( n = 6 mice for each group) were dissected to photograph ( A ), weigh ( B ) by the end of experiments. Tumor growth ( C ) was recorded every day. Values represent the mean ± SD by Student’s t test. P value: Rel.tumor weight, ** P = 0.0056; Rel.tumor volume, **** P < 0.0001. ( D – G ) Lac treatment suppresses the infiltration of FOXP3 + CD25 + ( D , E ), FOXP3 + CTLA4 + ( F , G ) Tregs. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: FOXP3 + CD25 + , **** P < 0.0001; FOXP3 + CTLA4 + , **** P < 0.0001. ( H ) Lac treatment shrinks autochthonous lung cancer in EGFR-DEL mice. EGFR-DEL mice ( n = 3 mice for each group) were irradiated with X-ray (left panel) or control (right panel). ( I ) Statistics of alteration of tumor burden of ( H ). Data are representative of three independent experiments. ** P < 0.01, *** P < 0.001, and **** P < 0.0001 were analyzed by Student’s t test. Error bars denote mean ± SD. The exact P values are provided in Dataset . ( J ) H&E and Ki67 staining of lung sections of mice in ( H ). Representative images of Hematoxylin and eosin (H&E) (left panel) staining or Ki67 (right panel) expression of the lung tissue obtained from ( H ). Scale bar: 200 μm. ( K ) Statistics of relative tumor burden and Ki67-positive cells of ( J ). Data are representative of three independent experiments. ** P < 0.01 and *** P < 0.001 were analyzed by Student’s t test. The exact P values are provided in Dataset . ( L ) Impact of DT or Lac treatment on tumor-infiltrating Tregs population. Representative images of flow cytometry on FOXP3 + or GFP + population in tumors of mice in ( H ). ( M ) Statistics of alteration of GFP + or FOXP3 + population of ( L ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: GFP + (Veh. vs DT), **** P < 0.0001; GFP + (Veh. vs Lac.), ** P = 0.0016; FOXP3 + (Veh. vs DT), P = 0.1511; FOXP3 + (Veh. vs Lac.), ** P = 0.0061. .

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Irradiation, Control, Staining, Expressing, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A ) Lac treatment synergizes with PD-1 antibody to shrink lung cancers. Lung cancer-bearing KRAS G12D mice ( n = 3 mice for each group) were treated with PBS, Lac (6 mg/kg), PD-1 antibody (10 mg/kg) or Lac+PD-1 antibody (Combo) through IP injection. Tumor burden was monitored through CT scanning before and 2-week after treatment. ( B ) Statistics of tumor burden changes of ( A ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh, P = 0.0630; PD-1 Ab, P = 0.0935; Lac, ** P = 0.0012; Combo, *** P = 0.0002. ( C ) Representative images of H&E staining of the lung tissues from mice in ( A ). Scale bar: 200 μm. ( D ) Statistics of relative tumor burden of ( C ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh vs PD-1 Ab, P = 0.9065; Veh vs Lac, *** P = 0.0002; Veh vs Combo, **** P < 0.0001. ( E ) Representative images of Ki67-positive cells of the lung tissues from different treatment groups. Scale bar: 200 μm. ( F ) Statistics analysis Ki67-positive cells of ( E ). Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh vs PD-1 Ab, P = 0.8925; Veh vs Lac, ** P = 0.0052; Veh vs Combo, ** P = 0.0033. ( G , H ) Impact of drug treatment on tumor-infiltrating Tregs. FOXP3 + CD25 + ( G , upper panel), FOXP3 + CTLA4 + ( G , lower panel), FOXP3 + IL10 + ( H , upper panel), FOXP3 + PER + ( H , lower panel) populations in KRAS G12D tumors were analyzed by flow cytometry. ( I , J ) Impact of drug treatment on tumor-infiltrating conventional CD8 + or CD4 + T cells. CD8 + IFNγ + ( I , upper panel), CD8 + PER + ( I , lower panel), CD4 + IFNγ + ( J ) populations in KRAS G12D tumors were analyzed by flow cytometry. .

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Injection, Staining, Flow Cytometry

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: ( A ) Statistics of the proportion of FOXP3 + CD25 + , FOXP3 + CTLA4 + , FOXP3 + IL10 + and FOXP3 + PER + Tregs in tumor of Fig. 7G, H . Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh. vs PD-1 Ab, * P = 0.0127, Veh. vs Lac, ** P = 0.0015, Veh. vs Combo, *** P = 0.0004 for FOXP3 + CD25 + ; Veh. vs PD-1 Ab, * P = 0.0218, Veh. vs Lac, ** P = 0.0016, Veh. vs Combo, *** P = 0.0007 for FOXP3 + CTLA4 + ; Veh. vs PD-1 Ab, P = 0.0624, Veh. vs Lac, ** P = 0.0010, Veh. vs Combo, **** P < 0.0001 for FOXP3 + IL10 + ; Veh. vs PD-1 Ab, P = 0.6317, Veh. vs Lac, ** P = 0.0027, Veh. vs Combo, ** P = 0.0049 for FOXP3 + PER + . ( B ) Immunofluorescence analysis of Tregs infiltration. Scale bar: 100 μm. ( C ) Statistics of the Tregs infiltration (CD4 + ; FOXP3 + ) in tumor of Fig. EV5B. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh. vs PD-1 Ab, P = 0.6870, Veh. vs Lac, * P = 0.0210, Veh. vs Combo, * P = 0.0412 for CD4 + ; Veh. vs PD-1 Ab, * P = 0.0246, Veh. vs Lac, * P = 0.0151, Veh. vs Combo, ** P = 0.0021 for FOXP3 + . ( D ) Lac or PD-1 antibody treatment did not significantly alter the infiltration of CD4 + and CD8 + T cells into tumors. Representative flow plots were gated on CD45 + population for analysis. ( E ) Statistics of the proportion of CD8 + (right panel) and CD4 + (left panel) in tumor of Fig. EV5D. Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh. vs PD-1 Ab, * P = 0.0191, Veh. vs Lac, * P = 0.0195, Veh. vs Combo, * P = 0.0129 for CD4 + ; Veh. vs PD-1 Ab, P = 0.9277, Veh. vs Lac , P = 0.5793, Veh. vs Combo , P = 0.7526 for CD8 + . ( F ) Statistics of the proportion of CD8 + IFNγ + (left panel) and CD8 + PER + (right panel) in tumor of Fig. . Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh. vs PD-1 Ab, * P = 0.0124, Veh. vs Lac, * P = 0.0318, Veh. vs Combo, ** P = 0.0013 for IFNγ + ; Veh. vs PD-1 Ab, *** P = 0.0005, Veh. vs Lac, *** P = 0.0007, Veh. vs Combo, **** P < 0.0001 for PER + . ( G ): Statistics of the proportion of CD4 + IFNγ + in tumor of Fig. . Data are representative of three independent experiments and were analyzed by Student’s t test. Error bars denote mean ± SD. P value: Veh. vs PD-1 Ab, ** P = 0.0013, Veh. vs Lac, ** P = 0.0090, Veh. vs Combo, *** P = 0.0004 for IFNγ + . ( H ) Representative images of H&E staining of the indicated organs of treated mice. Scale bar: 100 μm.

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Immunofluorescence, Staining

Journal: EMBO Molecular Medicine

Article Title: Lanatoside C activates the E3 ligase STUB1 to inhibit FOXP3 transcriptional activity and promote antitumor immunity

doi: 10.1038/s44321-025-00200-y

Figure Lengend Snippet: Reagents and tools table

Article Snippet: CD4 + CD25 + regulatory T cell isolation kit , Miltenyi , 130-091-041.

Techniques: Recombinant, Sequencing, Software, Computed Tomography, Microscopy, Mass Spectrometry, Flow Cytometry, Luciferase, Protein Purification, Cell Isolation, Enzyme-linked Immunosorbent Assay, Staining

Journal: Scientific Reports

Article Title: Staphylococcus aureus -derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3 + CD161 + T-helper cells in a partly monocyte-dependent manner

doi: 10.1038/srep22083

Figure Lengend Snippet: (a–c) The percentages and representative facs-plots of CD4 T-cells expressing IL-10 ( a ), IFN-γ ( b ) and IL-17A ( c ) after 24-hour stimulation with SEA (n = 6–10), S. aureus 161:2-CFS (n = 16–19), S. aureus 139:3-CFS (n = 5), S. carnosus TM300-CFS (n = 5) and S. epidermidis KX293A1-CFS (n = 5). The horizontal line represents the median within each group.

Article Snippet: The human CD4 + CD25 high T-cell isolation kit (Stemcell Technologies) was used to positively select for CD4 + CD25 high cells (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 55.6%) or to deplete CD25 + cells from the CD4 T-cell population (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 1.64%) according to the instructions from the manufacturer.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Staphylococcus aureus -derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3 + CD161 + T-helper cells in a partly monocyte-dependent manner

doi: 10.1038/srep22083

Figure Lengend Snippet: Lymphocytes were gated according to FSC and SSC properties. The further gating strategy is displayed in (a) : Live CD4 + T-cells were gated either as CD25 + FOXP3 + and CD127 low/neg (left panel) or as FOXP3 − (right panel). These two populations are referred to as FOXP3 + and FOXP3 − cells respectively in the text and figures. (b) The percentage of CD25 + FOXP3 + CD127 low cells among CD4 + T-cells in PBMC-cultures after 24-hour stimulation with SEA (n = 21), S. aureus 161:2-CFS (n = 36), S. aureus 139:3-CFS (n = 5), S. carnosus TM300-CFS (n = 5) or S. epidermidis KX293A1-CFS (n = 5) (2b, top, left) and mean fluorescence intensity (MFI) of FOXP3-expression (2b, top, right). The dot plot shows one representative staining of CD25 vs FOXP3-expression (2b, bottom). (c) CTLA-4-expression in FOXP3 + cells after 24-hour stimulation with S. aureus 161:2-CFS (n = 8). (d) Representative stainings of CD25 vs FOXP3-expression in purified CD4 + CD25-depleted T-cell-cultures either unstimulated or after stimulation with SEA or S. aureus 161:2-CFS. Boxes cover data values between the 25 th and 75 th percentiles, with the central line as median. For scatter dot plots, the horizontal lines represent the median within each group.

Article Snippet: The human CD4 + CD25 high T-cell isolation kit (Stemcell Technologies) was used to positively select for CD4 + CD25 high cells (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 55.6%) or to deplete CD25 + cells from the CD4 T-cell population (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 1.64%) according to the instructions from the manufacturer.

Techniques: Fluorescence, Expressing, Staining, Purification

Journal: Scientific Reports

Article Title: Staphylococcus aureus -derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3 + CD161 + T-helper cells in a partly monocyte-dependent manner

doi: 10.1038/srep22083

Figure Lengend Snippet: ( a) The percentages of FOXP3 + cells and (b) the percentages of FOXP3 − cells that express IL-10 (upper panel), IFN-γ (middle panel) or IL-17A (lower panel) after 24-hour stimulation with SEA (n = 6–10) or S. aureus 161:2-CFS (n = 16–18). ( c ) Representative facs-plots of cytokine-expression in CD25 + FOXP3 + cells in isolated CD4 + CD25 high T-cell-cultures after 40-hour stimulation with S. aureus 161:2-CFS. ( d ) Representative facs-plots of cytokine-expression in cells with de novo expression of CD25 and FOXP3 in CD4 + CD25-depleted T-cell-cultures after 40-hour stimulation with S. aureus 161:2-CFS. For scatter dot plots, the horizontal lines represent the median within each group.

Article Snippet: The human CD4 + CD25 high T-cell isolation kit (Stemcell Technologies) was used to positively select for CD4 + CD25 high cells (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 55.6%) or to deplete CD25 + cells from the CD4 T-cell population (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 1.64%) according to the instructions from the manufacturer.

Techniques: Expressing, Isolation

Journal: Scientific Reports

Article Title: Staphylococcus aureus -derived factors induce IL-10, IFN-γ and IL-17A-expressing FOXP3 + CD161 + T-helper cells in a partly monocyte-dependent manner

doi: 10.1038/srep22083

Figure Lengend Snippet: ( a) The percentage of CD161 + cells within the FOXP3 + population and CD161-expression on FOXP3 + cells in PBMC-cultures after 24-hour stimulation with SEA (n = 19) or S. aureus 161:2-CFS (n = 31). Facs-plot shows one representative staining of CD161-expression on FOXP3 + cells in PBMC-cultures. (b) Representative facs-plot of CD161-expression on CD25 + FOXP3 + cells in stimulated CD4 + CD25-depleted T-cell-cultures. (c) The percentage of CD161 + cells within the FOXP3 − population as described in ( a ). (d , e) The percentages of IL-10 + , IFN-γ + and IL-17A + cells within the CD161 − subpopulation (grey bars) or the CD161 + subpopulation (black bars) both for FOXP3 + cells ( d ) and FOXP3 − cells ( e ) after 24-hour stimulation with S. aureus 161:2-CFS (n = 8–19). For scatter dot plots, the horizontal line represents the median within each group. Boxes cover data values between the 25 th and 75 th percentiles, with the central line as median. Bars show medians with interquartile range.

Article Snippet: The human CD4 + CD25 high T-cell isolation kit (Stemcell Technologies) was used to positively select for CD4 + CD25 high cells (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 55.6%) or to deplete CD25 + cells from the CD4 T-cell population (average percentage of CD25 + cells among live CD4 + T-cells for 2 donors: 1.64%) according to the instructions from the manufacturer.

Techniques: Expressing, Staining

Journal: Thyroid

Article Title: Impairment of Regulatory T-Cell Function in Autoimmune Thyroid Disease

doi: 10.1089/thy.2012.0514

Figure Lengend Snippet: Flow cytometric analysis of peripheral blood mononuclear cells (PBMCs). (A) PBMCs were stained with antigen-presenting cell (APC)-conjugated anti-CD4 (y-axis) and phycoerythrin (PE)-conjugated anti-CD25 (x-axis) and analyzed by flow cytometry. The CD4+CD25high cells exhibiting a slightly reduced expression of CD4 were considered regulatory T cells (Tregs). (B, C) PMBCs were stained either with PE-conjugated antihuman FoxP3 or an isotype control as well as fluorescein isothiocyanate (FITC)-conjugated anti-CD4 and APC-conjugated anti-CD25 and analyzed by flow cytometry. (B) Histogram demonstrating FoxP3 expression in gated CD4+CD25high cells (Tregs). (C) Histogram demonstrating absence of FoxP3 expression in gated CD4+CD25− cells (T effector [Teff] cells). ---, FoxP3 stain; ---, isotype control.

Article Snippet: To confirm that CD4 + CD25 high T cells were FoxP3 positive, PE-conjugated antihuman FoxP3, fluorescein isothiocyanate (FITC)–conjugated anti-CD4, and APC-conjugated anti-CD25 antibodies were obtained from eBioscience (San Diego, CA).

Techniques: Staining, Flow Cytometry, Expressing, Control

Journal: Thyroid

Article Title: Impairment of Regulatory T-Cell Function in Autoimmune Thyroid Disease

doi: 10.1089/thy.2012.0514

Figure Lengend Snippet: Tregs are immunosuppressive in healthy controls (HC), but not AITD. (A) Representative T-cell proliferation. CD4+ T cells from one HC subject, one GD patient, and one HT patient were activated at various ratios of Teff to Treg. Effector cell density was maintained at 20,000 cells/well, and Treg populations were titered from 10,000 to 2500. [3H]-thymidine was added for the final 15–18 hours of culture, and proliferation was measured by scintillation counting. (B, C) Percentage proliferation of activated Teff cells when cocultured with various concentrations of Tregs. (B) A 20,000:10,000 ratio of Teff to Treg. (C) A 20,000:5000 ratio of Teff to Treg. Horizontal bars show the mean with SEM. A one-way ANOVA Bonferonni test was used for multiple group comparisons in (B) and (C).

Article Snippet: To confirm that CD4 + CD25 high T cells were FoxP3 positive, PE-conjugated antihuman FoxP3, fluorescein isothiocyanate (FITC)–conjugated anti-CD4, and APC-conjugated anti-CD25 antibodies were obtained from eBioscience (San Diego, CA).

Techniques:

Journal: Thyroid

Article Title: Impairment of Regulatory T-Cell Function in Autoimmune Thyroid Disease

doi: 10.1089/thy.2012.0514

Figure Lengend Snippet: The method of T-cell stimulation affects the ability to detect a difference between Treg suppression in HC (●) versus AITD (▲) patients. CD4+CD25− T cells and Tregs were cultured at a 20,000:10,000 ratio. Cells from AITD patients and HC were stimulated with auto- or allo-PBMCs plus anti-CD3 and anti-CD28 antibodies or allo-PBMCs alone. Proliferation was measured by [3H]-thymidine incorporation at 4 days. Impairment in suppression of AITD patients was only observed when auto-PBMCs plus anti-CD3 and anti-CD28 antibodies were used. A two-tailed Mann–Whitney t test was used to compare the groups.

Article Snippet: To confirm that CD4 + CD25 high T cells were FoxP3 positive, PE-conjugated antihuman FoxP3, fluorescein isothiocyanate (FITC)–conjugated anti-CD4, and APC-conjugated anti-CD25 antibodies were obtained from eBioscience (San Diego, CA).

Techniques: Cell Stimulation, Cell Culture, Two Tailed Test, MANN-WHITNEY