Journal: Cellular and Molecular Life Sciences

Article Title: Phenotype and regulation of immunosuppressive Vδ2-expressing γδ T cells

doi: 10.1007/s00018-013-1467-1

Figure Lengend Snippet: γδ T cells suppress responder T cell ( Resp ) proliferation. 10 4 negatively isolated Resp (CD4 + CD25 - ) were co-cultured at the indicated ratio with positively isolated γδ T cells ( γδ ) or regulatory T cells ( Treg ). The cells were stimulated with T cell A/E beads coated with 0.5 μg/mL anti-CD2, 10 μg/mL anti-CD3, and 10 μg/mL anti-CD28. a Proliferation was measured by tritiated thymidine ( 3 H-TdR ) incorporation after 3 days of stimulation. Results are shown as relative proliferation in comparison to solo-cultured Resp, whose proliferation was set to 100 %. Results are presented as the mean ± standard deviation (SD) of four independent experiments with triplicate determinations. b Microscopic analysis of 7-day cell cultures (magnification ×50). c Absolute cell number of viable Resp or γδ T cells in solo- or co-culture (1:1 ratio) was analyzed in 18 different donors by the standard cell dilution assay (SCDA) after 7 days of stimulation. Medium of solo cultivated γδ T cells was supplemented with 50 U interleukin (IL)-2. The mean value of quadruplicate (solo-culture, co-culture) or triplicate (γδ solo-culture + IL-2) determination for each donor is depicted as one symbol . Black bars Mean values of the different experiments, asterisks statistical significance according to Students t test (* p ≤ 0.05; ** p ≤ 0.01). d Cell death was measured 3 days after stimulation in 13 different donors by propidium iodide ( PI ) incorporation into cells. Relative responder suppression was defined as percentage reduction of absolute Resp number in co-culture compared to solo-culture on day 7

Article Snippet: PBMC with γδ T cells which consisted of only Vδ2 T cells were used; CD4 + CD25 − responder T cells and CD4 + CD25 high Treg were purified from the PBMC using a magnetic cell separation system (Miltenyi Biotec, Bergisch-Gladbach, Germany). γδ T cells were isolated by positive selection (anti-TCRγ/δ MicroBead Kit; Miltenyi Biotec), and responder T cells by negative selection (CD4 + T Cell Isolation Kit II; Miltenyi Biotec) followed by the positive selection of Treg by Dynabeads (Life Technologies, Carlsbad, CA).

Techniques: Isolation, Cell Culture, Comparison, Standard Deviation, Co-Culture Assay, Dilution Assay

Journal: Cellular and Molecular Life Sciences

Article Title: Phenotype and regulation of immunosuppressive Vδ2-expressing γδ T cells

doi: 10.1007/s00018-013-1467-1

Figure Lengend Snippet: Expression of Treg-associated markers by γδ T cells. a 10 5 purified Resp, Treg, or γδ T cells were stained with the indicated antibodies. The expression of Helios and FoxP3 was determined intracellulary and CD25 expression on the cell surface by flow cytometry. For FoxP3 staining, two different anti-FoxP3 monoclonal antibodies (mAb) were applied, as indicated. The small-sized dot-blots located in the upper left edge of the large-sized dot-blots show the isotype controls. The numbers in the dot-blots represent the relative proportion. One representative out of five experiments is shown. b Co-expression of CD27 and Helios was determined in a time-course study on/in A/E beads-stimulated γδ T cells. Mean values of the relative co-expression from three different donors are depicted in a bar chart. The co-expression on day 0 or 8 after stimulation is depicted for one representative donor in a dot-blot and for five different donors in a scatter plot. c , d . Changes in the expression of the analyzed transcription factors in solo- or co-cultured T cells after A/E beads-stimulation are displayed in a time-course study ( c ) or 7 days after stimulation ( d ). Solid lines Mean values of the mean fluorescence intensity of the indicated γδ- or responder T cells from at least three different donors cultured alone, dashed lines co-cultured cells. The mean fluorescence intensity of the appropriate isotype control was subtracted from that of the analyzed transcription factors [= difference ( diff. ) of median fluorescence intensity]

Article Snippet: PBMC with γδ T cells which consisted of only Vδ2 T cells were used; CD4 + CD25 − responder T cells and CD4 + CD25 high Treg were purified from the PBMC using a magnetic cell separation system (Miltenyi Biotec, Bergisch-Gladbach, Germany). γδ T cells were isolated by positive selection (anti-TCRγ/δ MicroBead Kit; Miltenyi Biotec), and responder T cells by negative selection (CD4 + T Cell Isolation Kit II; Miltenyi Biotec) followed by the positive selection of Treg by Dynabeads (Life Technologies, Carlsbad, CA).

Techniques: Expressing, Purification, Staining, Flow Cytometry, Bioprocessing, Dot Blot, Cell Culture, Fluorescence, Control

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: Science (New York, N.Y.)

Article Title: Engineering synthetic suppressor T cells that execute locally targeted immunoprotective programs

doi: 10.1126/science.adl4793

Figure Lengend Snippet: ( A ) Synthetic suppressor T cells that act as a source for inhibitory cytokines and a sink for inflammatory cytokines drove stronger suppression of CAR T cells in vitro. Synthetic suppressor T cells that induced a combination of TGFβ1 and CD25 were more potent at suppressing CD8 + CAR T cell activity compared with each individual payload alone. Cell counts are normalized to the 0 hour time point ( n = 3 replicates, error bars = standard error, filled markers indicate two-tailed t test, P < 0.05, comparison to no-suppressor cell control). ( B ) Synthetic suppressor T cells depleted IL-2 produced by activated CD4 + T cells in vitro. Human CD4 + T cells activated by anti-CD3/CD28 beads for 24 hours were cocultured with synthetic suppressor T cells activated with synNotch activating beads (anti-Myc beads). The IL-2 levels in the supernatant were measured by ELISA ( t = 48 hours, n = 3 replicates, error bars = standard error, two-tailed t test comparing TGFβ1 and CD25 to each payload alone, * P < 0.05). ( C ) Synthetic suppressor T cells required both TGFβ1 and CD25 to be produced by the same cell for effective suppression in vitro. Separation of TGFβ1 and CD25 into two separate cells led to weaker suppression of CD8 + CAR T cell killing (reduced target-cell proliferation) than a one-cell system where both payloads are produced by the same suppressor T cell in vitro ( n = 3 replicates, error bars = standard error, filled markers indicate two-tailed t test, P < 0.05, comparison to no-suppressor cell control). ( D ) CD25 drives increased TGFβ1 production by synthetic suppressor T cells in vitro. Suppressor cells that induced a combination of TGFβ1 and CD25 led to more TGFβ1 accumulation than suppressor cells inducing TGFβ1 alone. Suppressor cells were activated in vitro with synNotch activation beads (anti-Myc beads). TGFβ1 levels were measured by ELISA of supernatant ( t = 72 hours, n = 3 replicates, error bars = standard error, two-tailed t test between TGF β1 circuit with and without CD25, * P < 0.05). ( E ) CD25 can enhance suppressor cell activity by two mechanisms. CD25 depletes IL-2 from the local microenvironment and drives preferential proliferation of suppressor cells. An increase in suppressor cell number can yield higher TGFβ1 accumulation.

Article Snippet: Human polyclonal T reg cells were isolated by sorting CD4 + (BioLegend, SK3 clone), high CD25 + (Thermo Fisher, 4E3 clone), and CD127 − (BD Biosciences, HIL-7R-M21 clone) immediately after isolation of primary human CD4 + T cells from leukapheresis packs.

Techniques: In Vitro, Activity Assay, Two Tailed Test, Comparison, Control, Produced, Enzyme-linked Immunosorbent Assay, Activation Assay

Journal: Science (New York, N.Y.)

Article Title: Engineering synthetic suppressor T cells that execute locally targeted immunoprotective programs

doi: 10.1126/science.adl4793

Figure Lengend Snippet: ( A ) Two-tumor mouse model was used to assess local immune suppression. Two tumors were injected subcutaneously into immunocompromised NSG mice, such that the right flank had a dual-antigen tumor (Her2 + CD19 + K562 tumor) and the left flank had a single-antigen tumor (Her2 + K562 tumor). Anti-Her2 CAR T cells and anti-CD19 synNotch suppressor T cells were injected intravenously. Tumor volumes were measured by calipers. ( B ) Synthetic suppressor T cells can block CAR T cell killing locally without systemic suppression. Suppressor T cells (anti-CD19 synNotch→TGFβ1+CD25) are effective at blocking CAR T cell killing of the dual-antigen tumor (CD19 + ) without compromising killing of the single antigen tumor (CD19 − ). Suppressor cells producing each payload alone were not sufficient to protect the dual-antigen tumor from CAR T cell killing. Tumor measurements shown as time after T cell injection ( n = 5 replicates, solid line = mean, shading = standard error, two-tailed t test, * P < 0.001 on day 28). Dashed gray line indicates tumor growth with no T cell injection. See for tumor growth curves for individual mice. ( C ) Synthetic suppressor T cells reduced CAR T cell proliferation in dual-antigen tumor in vivo. Flow profiling of isolated tumors at day 14 showed reduced accumulation of both CD4 + and CD8 + CAR T cells (GFP + ) and an increased accumulation of suppressor cells (BFP + ) in the dual-antigen tumor. Cell counts normalized to tumor weight after isolation ( n = 3 replicates, error bars = standard error, two-tailed t test, * P < 0.05). ( D ) Multicellular NOT gate tumor-killing circuit combining CAR T cells and synthetic suppressor T cells drove robust local suppression. Multicellular NOT gate circuit leads to more-robust local suppression than iCAR NOT circuit in two-tumor model in vivo . iCAR NOT gate circuit (anti-Her2 CAR + anti-CD19 PD-1 iCAR) fails to block killing of the dual-antigen tumor. In the multicellular NOT gate tumor-killing circuit, anti-Her2 CAR T cells recognize and kill both tumors, whereas anti-CD19 synthetic suppressor T cells block killing in the CD19 + dual-antigen tumor ( n = 5 replicates, solid line = mean, shading = standard error, two-tailed t test, * P < 0.001 day 21). Dashed gray line indicates tumor growth with no T cell injection. Additional replicates shown in , and tumor growth curves for individual mice shown in .

Article Snippet: Human polyclonal T reg cells were isolated by sorting CD4 + (BioLegend, SK3 clone), high CD25 + (Thermo Fisher, 4E3 clone), and CD127 − (BD Biosciences, HIL-7R-M21 clone) immediately after isolation of primary human CD4 + T cells from leukapheresis packs.

Techniques: Injection, Blocking Assay, Two Tailed Test, In Vivo, Isolation

Journal: Science (New York, N.Y.)

Article Title: Engineering synthetic suppressor T cells that execute locally targeted immunoprotective programs

doi: 10.1126/science.adl4793

Figure Lengend Snippet: ( A ) eBC organoids were generated from hPSCs. eBC organoids were differentiated from hPSCs as previously described . eBC organoids were engineered to express model antigen CD19 by lentiviral transduction on day 19 of differentiation. eBC organoids were HLA-A2 + and expressed GFP under the control of the insulin promoter. Confocal microscopy (maximum projection) of an eBC is shown on day 23 of differentiation. Coculture with T cells was performed on day 26 of differentiation. Scale bar, 100 μm. ( B ) Cytotoxic T cells can kill eBC organoids. Human anti-HLA-A2 CAR CD8 + T cells cocultured with HLA-A2 + eBC organoids effectively killed eBC organoids in vitro. Confocal microscopy (maximum projection) showed eBC organoid destruction mediated by CAR T cells in vitro after 48 hours ( n = 3 replicates, error bars = standard error). ( C ) Synthetic suppressor T cells protected beta cells from cytotoxic T cell killing. eBC organoids were cocultured with T cells as in (B). Anti-HLA-A2 CAR T cell killing of eBCs was blocked by synthetic suppressor T cells (anti-CD19 synNotch→TGFβ1+CD25 circuit) but not by no-payload control cells (anti-CD19 synNotch→mCherry). Dashed lines indicate CAR-only control (blue) and no T cell control (gray) ( n = 3 replicates, error bars = standard error, two-tailed t test, * P < 0.001 at 70 hours comparing control cells to suppressor cells). Confocal microscopy (maximum projection) shows protection of an eBC organoid with suppressor T cells. Caspase 3/7 dye was used to label apoptotic cells and imaged (maximum projection) at the 48-hour time point. ( D ) Synthetic suppressor T cells self-organized around cytotoxic T cells during suppression in vitro. Suppressor T cells spatially self-organized around individual activated CAR T cells during suppression ( t = 48 hours), blocking the formation of CAR T cell clustering that is normally observed in target killing in the absence of suppression. Scale bars, 100 μm (zoomed-out image) and 25 μm (zoomed-in image).

Article Snippet: Human polyclonal T reg cells were isolated by sorting CD4 + (BioLegend, SK3 clone), high CD25 + (Thermo Fisher, 4E3 clone), and CD127 − (BD Biosciences, HIL-7R-M21 clone) immediately after isolation of primary human CD4 + T cells from leukapheresis packs.

Techniques: Generated, Transduction, Control, Confocal Microscopy, In Vitro, Two Tailed Test, Blocking Assay

Journal: Science (New York, N.Y.)

Article Title: Engineering synthetic suppressor T cells that execute locally targeted immunoprotective programs

doi: 10.1126/science.adl4793

Figure Lengend Snippet: ( A ) Transplant rejection was modeled by cytotoxic T cell rejection of transplanted eBC organoids under the kidney capsule of immunocompromised NSG mice. Fourteen days after transplantation, T cells were coinjected intravenously. eBC organoids express luciferase, allowing for noninvasive imaging of transplant survival. ( B ) Synthetic suppressor T cells blocked cytotoxic T cell killing of eBC organoid transplants. Bioluminescence imaging was used to track eBC organoid survival. Human anti-HLA-A2 CAR T cells alone cleared transplants within 2 weeks. However, transplants remained intact when synthetic suppressor T cells (anti-CD19 synNotch→TGFβ1+CD25 circuit) were coinjected along with CAR T cells. ( C ) Synthetic suppressor T cells protect eBC organoid transplants with synNotch priming antigen (CD19 + ). Survival of CD19 + eBC organoid transplants as in (B) is assessed by noninvasive imaging ( n = 6 to 8 replicates, two-tailed t test, * P < 0.001 comparing CAR T cell condition with and without suppressor T cells). Increased survival of eBC organoid transplants was observed with synthetic suppressor T cells (anti-CD19 synNotch→TGFβ1+CD25 circuit), but all transplants were cleared by anti-HLA-A2 CAR T cells alone. Dashed line indicates no–T cell control ( n = 3 replicates, mean). ( D ) Synthetic suppressor T cells did not protect eBC organoid transplants that lack the synNotch priming antigen. Survival of CD19 − eBC organoid transplants is assessed as in (B). No survival advantage was observed in the presence or absence of suppressor T cells in all cases ( n = 5 replicates, two-tailed t test, * P < 0.001 comparing CAR T cell condition with and without suppressor T cells). Dashed line indicates no–T cell control ( n = 3 replicates, mean). ( E ) Transplanted eBC organoids (CD19 + ) maintain their structure in the presence of synthetic suppressor T cells but are cleared by CAR T cells alone. eBC organoids were transplanted as in (B). Anti-human CD19 staining was used to identify transplanted eBC organoids in isolated mouse kidneys from transplanted mice 5 days after T cell injection. Staining shows survival of transplants in no–T cell control and CAR T cell in the presence of suppressor cells. Minimal human CD19 staining was observed in the CAR T cell–only condition. Scale bars, 100 μm (zoomed-in images) and 500 μm (zoomed-out image). See for anti-human CD19 and insulin staining of adjacent tissue section. ( F ) Transplanted eBC organoids retain endocrine function after synthetic suppressor T cell protection. Glucose challenge test was performed on NSG mice with eBC organoid transplants 21 days after injection of T cells (35 days after transplantation). Human C-peptide during fasting conditions and 30 min after intraperitoneal glucose injection ( n = 3 or 4 replicates, error bars = standard error) was measured by ELISA of blood serum. Glucose challenge showed that eBC organoids in mice injected with synthetic suppressor T cells remain functional and can secrete human C-peptide after glucose stimulation. P = 0.0018, two-tailed t test between CAR T cells with and without suppressor cells after glucose injection.

Article Snippet: Human polyclonal T reg cells were isolated by sorting CD4 + (BioLegend, SK3 clone), high CD25 + (Thermo Fisher, 4E3 clone), and CD127 − (BD Biosciences, HIL-7R-M21 clone) immediately after isolation of primary human CD4 + T cells from leukapheresis packs.

Techniques: Transplantation Assay, Luciferase, Imaging, Two Tailed Test, Control, Staining, Isolation, Injection, Enzyme-linked Immunosorbent Assay, Functional Assay