Journal: bioRxiv

Article Title: Natural killer cell division regulates FcεRIγ expression downstream of mTOR activity

doi: 10.1101/2021.08.03.454985

Figure Lengend Snippet: (A) NKG2C vs. FcRγ in ex vivo CD56 dim CD 16 + NK cells. Donor# I (upper), Donor #2 (lower). N = FcRγ negative , L = FcRγ low , H = FcRγ high NK cells. (B) Representative histograms of indicated KIRs or CD85J on ex vivo CD56 dim CD16 + NK cell subsets (X-axis log 10 fluorescence). (C) Percentage of CDS + cells in ex vivo CD56 dim CD16 + NK cell subsets. Box and whiskers: min to max. Integrated results from 3 independent measurements. (D-F) Intracellular levels for informative adaptive NK cell proteins FcRγ, PLZF, Syk (D), STATs signaling molecules (pSTATl Y701 , pSTAT4 Y963 ,pSTAT5 Y964 (E), pAKT T308 , mTORCl/mTORC2 activity (pS6 S235/236 and pAKT S473 respectively), mTOR, or Bcl2 (F), in the defined NK cell subsets. Values were normalized to CD56 dim CD16 + NKG2C-FcRγ + CD57 - NK cells (index = 1). Each measurement represents independent staining of an independent blood sample collected at an independent time point. (G) Single cell expression of (left to right) pS6 S2351236 ,pAKT S473, mTOR, or PLZF, log 2 values. Red bar = geometric mean with 95% confidence interval, unpaired t-test, two tails, *p<0.05, *** p<0.001.

Article Snippet: Becton Dickinson: APC-conjugated anti-CD122 (Mik-β3, cat. Cat# 566620, RRID:AB_2869796), AF647-conjugated anti-pSTAT4-Y693 (Cat# 562074, RRID:AB_10896660), AF647-conjugated anti-pSTAT5-Y694 (Cat# 612599, RRID:AB_399882), AF647-conjugated anti-pSTAT1-Y701 (Cat# 612597, RRID:AB_399880), AF647-conjugated anti-Bcl-2 (Cat# 563600, RRID:AB_2738306), AF647-conjugated anti-Ki-67 (Cat# 558615, RRID:AB_647130).

Techniques: Ex Vivo, Fluorescence, Activity Assay, Staining, Expressing

Journal: PLOS One

Article Title: The ubiquitin-like modifier FAT10 does not affect IL-12 expression and signaling

doi: 10.1371/journal.pone.0323005

Figure Lengend Snippet: (A-D) CD4 + T cells were magnetically purified from spleens of C57BL/6 wild type mice or FAT10 -/- mice. (A, B) CD4 + T cells were treated with TNF (400 U/ml)/IFN-γ (200 U/ml) (indicated +) for 1 day or were left untreated (indicated -). pSTAT1, STAT1, and GAPDH were analyzed by western blot. (B) Quantification of pSTAT1 normalized to GAPDH. Data are depicted as mean ± SDs derived from 7 different experiments (n = 7). (C, D) CD4 + T cells were treated with TNF (400U/ml)/IFN-γ (200 U/ml) for 1 day or left untreated, followed by a treatment with IL-12 (10 ng/ml) for 2 h. Treatment regime is indicated. pSTAT4 and GAPDH were analyzed by western blot. (D) Quantification of pSTAT4 normalized to GAPDH. Data are depicted as mean ± SDs derived from 5 different experiments (n = 5).

Article Snippet: Protein was analyzed by the following antibodies: anti-pSTAT1 antibody (Cell Signaling, 7649S), anti-STAT1 antibody (Cell Signaling, 9172S), and anti-pSTAT4 antibody (Cell Signaling, 4134S).

Techniques: Purification, Western Blot, Derivative Assay

Journal: Frontiers in Pharmacology

Article Title: Saikosaponin A Inhibits Breast Cancer by Regulating Th1/Th2 Balance

doi: 10.3389/fphar.2019.00624

Figure Lengend Snippet: The effects of SSa on gene expression and protein amounts of IL-12, IL-12R, and STAT4. (A) Gene expressions of IL-12, IL-12R, and STAT4 in tumors. (B) Protein amounts of IL-12, IL-12R, and pSTAT4 in tumors. Data were expressed as mean ± SD ( n = 12). * P < 0.05, ** P < 0.01 vs. control group. # P < 0.05 vs. TAM group.

Article Snippet: The primary antibodies, goat polyclonal anti-IL-12 antibody (1:1,000, Santa Cruz, CA, USA), rabbit polyclonal anti-IL-12R antibody (1:500, Santa Cruz, CA, USA), rabbit polyclonal anti-pSTAT4 antibody (1:200, Santa Cruz, CA, USA), or mouse monoclonal anti-β-actin (1:2,000, Santa Cruz, CA, USA) was used to probe the blots overnight at 4°C, respectively.

Techniques: Gene Expression, Control

Journal: Frontiers in Pharmacology

Article Title: Saikosaponin A Inhibits Breast Cancer by Regulating Th1/Th2 Balance

doi: 10.3389/fphar.2019.00624

Figure Lengend Snippet: The effects of SSa on in situ expressions of IL-12, IL-12R, and pSTAT4 in tumors. (A) Immunohistochemistry staining of IL-12, IL-12R, and pSTAT4 in rat tumors. (B) Morphometric analysis of IL-12, IL-12R, and pSTAT4 expression levels in rat tumors. Data were expressed as mean ± SD ( n = 12). * P < 0.05 vs. control group. ## P < 0.01 vs. TAM group.

Article Snippet: The primary antibodies, goat polyclonal anti-IL-12 antibody (1:1,000, Santa Cruz, CA, USA), rabbit polyclonal anti-IL-12R antibody (1:500, Santa Cruz, CA, USA), rabbit polyclonal anti-pSTAT4 antibody (1:200, Santa Cruz, CA, USA), or mouse monoclonal anti-β-actin (1:2,000, Santa Cruz, CA, USA) was used to probe the blots overnight at 4°C, respectively.

Techniques: In Situ, Immunohistochemistry, Staining, Expressing, Control

Journal:

Article Title: Preemptive HMG-CoA reductase inhibition provides graft-versus-host disease protection by Th-2 polarization while sparing graft-versus-leukemia activity

doi: 10.1182/blood-2007-08-106005

Figure Lengend Snippet: Differential impact of AT on RAS, RAP-1, and Rho-B prenylation, T-bet, GATA3, and c-MAF expression. (A) CD4+ T cells (H-2Kq) were exposed to irradiated allogeneic APCs (H-2Kd) of each cell type, 2 × 106 T cells/flat-bottomed, 6-well plate for 48 hours in the presence or absence of AT (10 μM), harvested and analyzed by intracellular cytokine staining with anti–IL-10, –IL-4, –TNF-α, or –IFN-γ. Mean fluorescence for the representative cytokines of CD4+H-2Kq+ gated cells is presented. (B) Cells cultured under the conditions described in panel A were then washed and responder T cells (H-2Kq) were re-isolated by MACS with anti–H-2Kq-FITC and anti-FITC beads. Protein samples from in vitro–treated T cells were subject to Western blot analysis. AT is effective at inhibiting the prenylation of Ras, Rap-1, and Rho-B in the allogeneic activation culture. Increased amounts of nonprenylated proteins are found when AT is present (solid arrow). (C) T cells were harvested from AT-treated donors on day 0 (left panel) or on day 3 (right panel) after BMT from the recipients' secondary lymphoid organs and were analyzed by Western blot for the indicated total or phosphorylated proteins. (D) Mean fluorescence for the representative pSTATs is analyzed by phospho-flow analysis of CD4+H-2Kq+ gated cells, presented for the respective day prior to or after BMT. MFI for pSTAT4 was 11.4 (± 0.7) versus 47.4 (± 3.4) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .05). MFI for pSTAT4 was 97.2 (± 2.1) versus 541.4 (± 11.3) for the AT versus the PBS group, respectively, on day 3 after BMT (P < .01). (E) Donor type T cells derived from AT-treated donors are presented for the respective day prior to or after BMT. Mean fluorescence for c-MAF, T-bet, and GATA-3 within CD4+H-2Kq+ gated cells is presented. MFI for T-bet was 12.4 (± 0.8) versus 98.4 (± 6.3) for the AT versus the PBS group, respectively, prior to transfer into the irradiated host (P < .01). MFI for T-bet was 137.2 (± 4.1) versus 21.4 (± 5.2) for the PBS versus the AT group, respectively, on day 3 after BMT (P < .01).

Article Snippet: Anti-phospho STAT1 (Y701, clone 4a), STAT4 (pY693, clone 38/pSTAT4), and STAT6 (pY641, clone 71-773.58.11) antibodies were obtained from BD Pharmingen.

Techniques: Expressing, Irradiation, Staining, Fluorescence, Cell Culture, Isolation, In Vitro, Western Blot, Activation Assay, Derivative Assay

Journal: Nature immunology

Article Title: TGF-β co-opts STAT3-STAT4 signaling to promote human T follicular helper cell differentiation

doi: 10.1038/ni.2947

Figure Lengend Snippet: (a) Ranking of the differentiation conditions inducing the expression of CXCR5 and ICOS on activated (FSC hi SSC hi ) human adult blood naïve T H cells following 4 d stimulation with CD3-CD28 mAbs in the presence of the indicated cytokines (CK) during the last 3 days. Each dot represents a result from 13 sets of 4 d-culture experiments. Black bars show the mean value and the blue boxes show the range of ± 1 s.d. (b) Expression of CXCR5 and ICOS on activated T H cells stimulated with the indicated cytokines as in (a) . Results are from one representative of 13 sets of 4 d-culture experiments. (c) Ranking of the differentiation conditions inducing IL-21 expression by activated human naïve T H cells cultured as in (a). (d) IgG ELISA at day 14 in the supernatants of co-cultures of activated naïve T H cells (after 3 d culture with the indicated cytokines) and autologous memory B cells in the presence of the superantigen Staphylococcal enterotoxin B. Mean, n=4. A representative experiments out of 3 experiments.

Article Snippet: For the pSmad2, pSTAT4, CD3 staining, the primary staining was done with rabbit polyclonal anti-pSmad2, mouse IgG1 anti-pSTAT4 (E-2, Santa Cruz) and mouse IgG2a anti-CD3 (HIT3a, BD) followed by a secondary staining with goat anti-rabbit A488, anti-mouse IgG1 A568 and anti-mouse IgG2a A647.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Nature immunology

Article Title: TGF-β co-opts STAT3-STAT4 signaling to promote human T follicular helper cell differentiation

doi: 10.1038/ni.2947

Figure Lengend Snippet: (a) Ranking of the differentiation conditions inducing BCL6 and suppressing PRDM1 by human adult blood naïve T H cells cultured as in . The data were normalized to the values in the culture with no cytokines in each experiment. Each dot represents a result from 11 sets of 4 d culture experiments. Black bars show the mean value and blue boxes indicate ± 1 s.d. (b) Immunoblot of Bcl-6 and Blimp-1 with naïve T H cells cultured with the indicated cytokines for 3 days. The band density after normalization with GAPDH is shown in number. Note that human Blimp-1 has three isoforms. A representative out of 3 independent experiments. (c) Correlation between BCL6 and MAF , BATF , and PRDM1 expressed by human naïve T H cells cultured as in (a) . Pearson R values are indicated. P values were all <0.0001. (d) Kinetics of BCL6 , PRDM1 , and BATF expression by naïve T H cells cultured with cytokines for 1–4 days as in (a) . Arrows indicate days when cytokines (CK) were added. (e) The chromatin fraction of human naïve T H cells cultured with IL-23+IL-6+IL-1β+TGF–β for 3 days were immunoprecipitated with anti-BATF or Rabbit IgG, followed by quantitative PCR analysis of the two BATF binding sites within BCL6 promoter. Results were normalized to input. IGX1A region represents a negative control. A representative out of 5 independent experiments. (e) Correlation between BCL6 and JUN and JUNB expressed by cord blood naïve T H cells cultured as in (a) . Pearson R values are indicated. P values were both <0.0001.

Article Snippet: For the pSmad2, pSTAT4, CD3 staining, the primary staining was done with rabbit polyclonal anti-pSmad2, mouse IgG1 anti-pSTAT4 (E-2, Santa Cruz) and mouse IgG2a anti-CD3 (HIT3a, BD) followed by a secondary staining with goat anti-rabbit A488, anti-mouse IgG1 A568 and anti-mouse IgG2a A647.

Techniques: Cell Culture, Western Blot, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction, Binding Assay, Negative Control

Journal: The Journal of Experimental Medicine

Article Title: Cdc42-mediated MTOC polarization in dendritic cells controls targeted delivery of cytokines at the immune synapse

doi: 10.1084/jem.20100007

Figure Lengend Snippet: Antigen-specific synapse formation induces STAT4 signaling and IFN-γ neosynthesis in T cells. (A) DCs were pretreated with TLR agonist, loaded with 10 nM of OVA peptide, and mixed with OT-I cells. After 30 min of incubation, cells were lysed and analyzed by Western blotting using an antibody against pSTAT4. Control lanes (1–4) are T cells alone (1) or incubated with soluble IL-12 (2) and DCs alone not stimulated (3) or stimulated (4) with TLR agonist. Lanes 5–8 are DCs coincubated with T cells in different conditions as indicated in the table. (B) Detection of pSTAT4 by intracellular FACS analysis. DCs were either left untreated or stimulated with TLR agonist (TLR), loaded (pep) or not with OVA peptide, and mixed with OT-I for 30 min. Cells were fixed and labeled intracellularly with anti-pSTAT4 Alexa Fluor 488 antibody. The MFI was determined by gating on isolated T cells (gate on T cells) or on DC–T cell doublets (gate on DC–T cell) as described in experimental procedure. Data represent the mean values ± SEM (subtracted for the isotype control values) obtained in three independent experiments. (C) Control DCs or DCs treated with colchicine were used in the assay described in B. Data show the MFI ± SEM of pSTAT4 signal determined on T cells engaged in doublets in four independent experiments (P < 0.01; Student’s t test). (D) DC–T cell antigen-specific interactions trigger IFN-γ neosynthesis in T cells. DCs activated by TLR agonist and loaded with peptide were allowed to interact with OT-I for 2 h, fixed, and labeled. Confocal images show polarized IL-12 (red) and DC-MTOC (blue) facing IFN-γ staining around the T cell MTOC (green). (E) Quantification of IFN-γ neosynthesis in T cells engaged in antigen-specific synapses was measured by intracellular FACS analysis under different conditions (as specified). Values plotted indicate the percentage of IFN-γ + /CD8 + T cells gated in the region of DC–T cell doublets (isolated T cells show no IFN-γ signal), subtracted for the isotype control (one of three experiments with identical results is presented). (F) Early expression of IFN-γ in T cells correlates to DC-MTOC polarization at the IS. Cells were labeled as in (D) and quantified. Bars show the distribution of IFN-γ–positive T cells in synapse with DCs that present (polarized) or not (not polarized) the MTOC facing the T cell membrane. The data comes from quantification of >100 cells in three independent experiments (**, P = 0.052).

Article Snippet: Membranes were blocked in TBS-5% BSA and developed using anti pSTAT4-ser721 (sc-22160; Santa Cruz Biotechnology, Inc.), followed by anti–rabbit horse radish peroxidase (Sigma-Aldrich).

Techniques: Incubation, Western Blot, Control, Labeling, Isolation, Staining, Expressing, Membrane

Journal: The Journal of Experimental Medicine

Article Title: Cdc42-mediated MTOC polarization in dendritic cells controls targeted delivery of cytokines at the immune synapse

doi: 10.1084/jem.20100007

Figure Lengend Snippet: Cdc42 depletion in DCs selectively impairs IL-12–dependent events in T cells . (A) Control and Cdc42-silenced cells activated by TLRs agonist and loaded (peptide) or not loaded (no peptide) with 10 nM of class I peptide were mixed with OT-I cells for 30 min and lysed to analyze the levels of activated STAT4 by W.B. Numbers indicate the intensity values (ratio pSTAT4 peptide/pSTAT4 no peptide normalized for actin levels) obtained from density scans. (B) Neosynthesis of IFN-γ in T cells engaged in synapse with control or Cdc42-silenced cells. DCs treated as in A were incubated for 2 h, fixed, and analyzed for intracellular IFN-γ in T cells engaged in synapse (gated on DC–T cell doublets) by FACS. Values represent average of three independent experiments (*, P = 0.0133). (C) Conjugate formation is not affected by Cdc4 silencing. 10 5 control or Cdc42-silenced DCs were loaded with graded doses of peptide as indicated, labeled with SNARF and mixed with 10 5 CFSE labeled OT-I for 20 min at 37°C. The number of DC–T cell doublets was measured by FACS gating on the double-positive events and normalized to the number of T cells. Values shown are mean ± SD of three experiments. (D) T cells proliferation and survival are affected by Cdc42 silencing in DCs. 10 4 control (control-siRNA) or Cdc42-depleted (Cdc42-siRNA) DCs were TLR stimulated, loaded with graded doses of peptide, and mixed with OVA-specific OT-I cells that had been prelabeled with CFSE. (left) Day 3 CFSE dilution profiles at the indicated doses of peptide. (right) The plot shows the total number of cells recovered at day 3. One of three independent experiments with similar results is shown. (E) Dot plots show the percentage of IFN-γ–producing cells at day 3 after priming, when T cells were primed using control or Cdc42-depleted DCs. Data show one representative of two experiments. Cell culture supernatants were harvested at day 3, and the levels of IFN-γ were measured by ELISA (values are mean ± SD of three independent experiments (**, P = 0.001). IFN-γ levels secreted by T cells are inhibited by DCs expressing dominant-negative Cdc42. Cdc42WT or the dominant-negative Cdc42N17 were stimulated as in D and mixed with OT-I. Bars show the levels of IFN-γ in day 3 cell culture supernatants. Data are the means ± SD of three independent experiments (**, P = 0.0017). (G) Expression of a rescue construct partially restores IFN-γ levels. DCs were cotransfected with control or Cdc42-specific siRNA, plus a plasmid encoding a Cdc42 resistant to silencing (rescue). Cells were treated as in F. Bars shows values of IFN-γ in day 3 cell culture supernatants from 2 independent experiments (means ± SD; *, P = 0.0385, **, P = 0.0068).

Article Snippet: Membranes were blocked in TBS-5% BSA and developed using anti pSTAT4-ser721 (sc-22160; Santa Cruz Biotechnology, Inc.), followed by anti–rabbit horse radish peroxidase (Sigma-Aldrich).

Techniques: Control, Incubation, Labeling, Cell Culture, Enzyme-linked Immunosorbent Assay, Expressing, Dominant Negative Mutation, Construct, Plasmid Preparation

Journal: Cell reports

Article Title: Mass Cytometry Reveals Global Immune Remodeling with Multi-lineage Hypersensitivity to Type I Interferon in Down Syndrome

doi: 10.1016/j.celrep.2019.10.038

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Mouse monoclonal anti-human phospho-STAT4 (Tyr693) (clone 38/p-Stat4) , Fluidigm , Cat#3148006A; RRID:AB_2811098.

Techniques: Recombinant, Mass Cytometry, Software