Journal: iScience

Article Title: Atypical B cells consist of subsets with distinct functional profiles

doi: 10.1016/j.isci.2023.108496

Figure Lengend Snippet:

Article Snippet: One day prior to sorting B cells, wells of a 96-well plate were each seeded with 30,000 adherent, CD40L-expressing 3T3 cells (kind gift from Dr. Mark Connors, NIH) in 100 μl IMDM/Glutamax/ 10% FBS containing 2× MycoZap Plus-PR (Lonza #VZA-2021), 100 ng/ml human IL-2 (GoldBio #1110-02-50), and 100 ng/ml human IL-21 (GoldBio #1110-21-10) to promote expansion and differentiation of B cells into antibody-secreting cells.

Techniques: Staining, Recombinant, Cell Isolation, Multiplex Assay, Sequencing, Plasmid Preparation, Software

Journal: International Journal of Molecular Sciences

Article Title: D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms20030742

Figure Lengend Snippet: 2-HG modulates LPS-stimulated cytokine secretion by DCs. Monocyte-derived DCs were plated in 6-well plates. DCs were activated with 100 ng/mL LPS and in parallel exposed to D-2-HG or L-2-HG for 24 h. Supernatants were collected and measured for IL-12 ( a , b ), IL-10 ( c ), IL-6 ( d ), and TNF ( e ). The measurements were performed by commercially available ELISA’s. Data represent the median of four independent experiments. Statistical analysis was tested using Dunnet’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001). ( f ) Antigen presentation was analyzed in a mixed lymphocyte reaction. DCs were cultured in flasks in the absence or presence of LPS and 2-HG for 24 h, harvested, and cocultured with allogeneic T cells for another five days. Proliferation was determined by [ 3 H]-thymidine incorporation.

Article Snippet: After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStop TM , BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/Cytoperm TM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls.

Techniques: Derivative Assay, Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms20030742

Figure Lengend Snippet: D-2-HG reduces intracellular IL-12 levels and mRNA expression in DCs. ( a ) Monocyte-derived immature dendritic cells (iDCs) were activated with 100 ng/mL LPS with or without D-2-HG in the presence of monensin. Intracellular levels of IL-12 p35 and IL-12 p40 were analyzed by flow cytometry. One representative experiment out of three is shown. ( b , c ) Monocyte-derived iDCs were activated with 100 ng/mL LPS with or without D-2-HG or L-2-HG for 4 h ( b , c ) or 24 h ( d , e ). RNA was isolated from cell lysates. After reverse transcription, the samples were analyzed by RT-qPCR. Gene expression of IL-12p70 subunits IL-12A and IL-12B relative to 18S RNA are shown. Data represent the median of three independent experiments. Statistical analysis was tested using an Ordinary One-Way Test with Dunnet’s multiple comparison test (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStop TM , BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/Cytoperm TM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls.

Techniques: Expressing, Derivative Assay, Flow Cytometry, Isolation, Quantitative RT-PCR

Journal: International Journal of Molecular Sciences

Article Title: D-2-Hydroxyglutarate and L-2-Hydroxyglutarate Inhibit IL-12 Secretion by Human Monocyte-Derived Dendritic Cells

doi: 10.3390/ijms20030742

Figure Lengend Snippet: 2-HG reprograms DC metabolism and thereby reduces IL-12 secretion. ( a ) Lactate secretion was determined in supernatants of DCs incubated in the presence or absence of 2-HG. ( b , c ) Monocyte-derived DCs were placed in oxygraph chambers in culture medium. After stabilization of respiration, 2-HG or medium was added to the chambers. After 10 min, cells were activated with 100 ng/mL LPS and oxygen consumption was monitored for one hour. ( b ) One representative experiment out of eight is shown. ( c ) Data represent the median of 11 independent experiments. Statistical testing was performed using a RM One-Way ANOVA test with Holm Sidak’s multiple comparison test (* p ≤ 0.05). ( d ) To evaluate the effect of oligomycin on IL-12 production by DCs, 0.2 × 10 6 monocyte-derived DCs were plated in 24-well plates and treated with 100 ng/mL LPS and the indicated concentrations of oligomycin. IL-12 was measured in supernatants by commercially available ELISA. Data represent the median of at least four independent experiments. Statistical significance was tested using the Kruskal Wallis test. (* p ≤ 0.05). ( e ) Monocyte-derived DCs were plated in 24-well plates and treated with 100 ng/mL LPS and with 2-HG in combination with oligomycin. IL-12 was measured in supernatants by commercially available ELISA. Data represent the median of at least four independent experiments. Statistical significance was tested using the Kruskal Wallis test. (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001).

Article Snippet: After seven days of culture, cells were treated with 100 ng/mL LPS with or without 10 mM D-2-HG in the presence of a protein transport inhibitor containing Monensin (BD GolgiStop TM , BD Bioscience, Franklin Lakes, NY, USA) for 16 h. DCs were washed, permeabilized, and fixed using the BD Cytofix/Cytoperm TM Kit (BD Biosciences), followed by staining with anti-IL-12 p40 (R&D), anti-IL-12 p35 (R&D), and the respective isotype controls.

Techniques: Incubation, Derivative Assay, Enzyme-linked Immunosorbent Assay

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: IL-33 and ST2L are overexpressed in human GC tissues and predict poor prognosis. a QRT-PCR analysis of IL-33 mRNA expression in GC and corresponding normal tissues ( n = 18). b Histogram displaying the relative mRNA expression of IL-33 in 18 GC tissues. Data are shown as −ΔΔCt and 2 −ΔCt . c IHC staining of α-SMA, IL-33 and ST2L in GC tissues (200×; scale bar = 100 μm). d Histogram displaying the number of α-SMA, IL-33 and ST2L positive cells/field in GC tissues. e Histogram displaying the correlation between IL-33 expression and ST2L expression determined by IHC ( r = 0.6503). f , g Survival of patients with low or high levels of IL-33 and ST2L protein expression. h – j Immunofluorescence staining of IL-33, α-SMA, FAP, ST2L and Cytokeratin in GC tissues (200×; scale bar = 400 μm). k – m Histogram displaying the percentage of IL-33 + /α-SMA + , IL-33 + /FAP + and ST2L + /Cytokeratin ++ cells in GC tissues. Data are represented as the mean ± SD; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Quantitative RT-PCR, Expressing, Immunohistochemistry, Immunohistochemistry-Resin, Immunofluorescence, Staining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Relationship between IL-33 and ST2L expression levels and clinicopathologic parameters in 134 GC tissues

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Expressing, Immunostaining

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: CAFs-derived IL-33 promotes the migration, invasion and EMT of GC cells. a – d The migration and invasion ability of SGC7901 and MKN45 cells were analyzed after culture in medium alone (blank) or treatment with: exogenous IL-33 (300 ng/ml); co-culture with CAFs supplemented with IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). Histograms show the average cell number/field (100×; scale bar = 100 μm). e – h The migration and invasion ability of SGC7901 and MKN45 cells was detected after culture in medium alone (blank), co-culture with CAFs transfected with IL-33/siRNA or nc/siRNA. Histograms display the average cell number/field (100×; scale bar = 100 μm). i – l The migration and invasion ability of SGC7901 and MKN45 cells was analyzed after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) plus IgG isotype antibody or ST2L neutralizing antibody (3 μg/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). Histograms show the average cell number/field. (100×; scale bar = 100 μm). m , n QRT-PCR of the genes for EMT in SGC7901 and MKN45 cells after culture in medium alone (blank); or activation with exogenous IL-33 (300 ng/ml); co-culture with CAFs in the presence of IgG isotype antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). o – r Western blot of EMT markers in SGC7901 and MKN45 cells cultured in medium alone (blank); or stimulated with exogenous IL-33 (300 ng/ml) or co-culture with CAFs or co-cultured with CAFs in the presence of DMSO, U0126 (20 μM), IL-33 neutralizing antibody (3 μg/ml) or ST2L neutralizing antibody (3 μg/ml). p and r Densitometric analysis shows the expression level of EMT markers in GC cells stimulated by the above factors. Data are represented as the mean ± SD of three independent experiments; * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Migration, Co-Culture Assay, Transfection, Quantitative RT-PCR, Activation Assay, Western Blot, Cell Culture, Expressing

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: The metastatic potential of GC cells induced by CAFs-derived IL-33 is mediated by the activation of ERK1/2-SP1-ZEB2 pathway via ST2L. a – d Western blot analysis showing the phosphorylation of ERK1/2 in SGC7901 and MKN45 cells treated with medium alone or stimulated as follows: exogenous IL-33 (300 ng/ml), U0126 (20 μM), CAFs supernatants (CAFsu) supplemented with IgG isotype control antibody (3 μg/ml), DMSO; co-cultured with CAFs transfected with IL-33/siRNA or NC/siRNA; CAFsu supplemented with IL-33 neutralizing antibody (3 μg/ml). e Western blot analysis of pERK1/2 in SGC7901 and MKN45 cells incubated with medium alone; exogenous IL-33 (300 ng/ml); U0126 (20 μM); co-culture with CAFs; IL-33 neutralizing antibody (3 μg/ml); and/or ST2L neutralizing antibody (3 μg/ml). f , g The migration and invasion ability of SGC7901 and MKN45 cells after culture in medium alone (blank) or stimulation with exogenous IL-33 (300 ng/ml) and/or U0126 (20 μM); co-culture with CAFs and/or U0126 (20 μM). Histograms show the average cell number per field. (100×; scale bar = 100 μm). h , i QRT-PCR analysis of SP1 mRNA expression in SGC7901 and MKN45 cells incubated in medium alone or stimulation with exogenous IL-33 (300 ng/ml) or CAFs in the presence of IgG isotype control antibody (3 μg/ml) or IL-33 neutralizing antibody (3 μg/ml). j , k Western blot analysis of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells incubated in medium alone or stimulated by co-culture with CAFs in the presence of exogenous IL-33 (300 ng/ml) or U0126 (20 μM). l Western blot analysis showing the protein expression of pERK1/2, ERK1/2, SP1 and ZEB2 in SGC7901 and MKN45 cells cultured in medium alone (Mock); or transfected with control plasmid (nc) or SP1-expressing plasmid. m Dual luciferase reporters containing four different lengths of the ZEB2 promoter were co-transfected with SP1-expressing plasmid into 293T cells. Firefly luciferase activity was assessed relative to Renilla luciferase activity. Data are representative of three independent experiments. Densitometry shows relative protein expression. * P <0.05, ** P <0.01, *** P <0.001

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Derivative Assay, Activation Assay, Western Blot, Phospho-proteomics, Control, Cell Culture, Transfection, Incubation, Co-Culture Assay, Migration, Quantitative RT-PCR, Expressing, Plasmid Preparation, Luciferase, Activity Assay

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Knockdown of IL-33 expression in CAFs or ST2L expression in GC cells inhibits peritoneal dissemination of GC cells in nude mice. a , c In vivo tumor peritoneal dissemination was examined by co-injection of SGC7901 or MKN45 cells and CAFs transfected with control siRNA (NC) or IL-33 siRNA into nude mice. Metastatic nodules are indicated by the red arrows. b , d Histograms displaying the number of peritoneal nodules in nude mice. e – h In vivo tumor peritoneal dissemination was examined by co-injection of CAFs and GC cells with ST2L knockdown (KD) into nude mice. Metastatic nodules are indicated by the red arrows. f , h Histograms displaying the number of peritoneal nodules in nude mice. Data are representative of three independent experiments. * P <0.05, * P <0.05, ** P <0.01

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Knockdown, Expressing, In Vivo, Injection, Transfection, Control

Journal: Oncogene

Article Title: The reciprocal interaction between tumor cells and activated fibroblasts mediated by TNF-α/IL-33/ST2L signaling promotes gastric cancer metastasis

doi: 10.1038/s41388-019-1078-x

Figure Lengend Snippet: Model for the epithelial–stromal interactions in the tumor microenvironment of GC. a IL-33 released from CAFs promotes the migration, invasion and EMT of GC cells via the ST2L-ERK1/2-SP1-ZEB2 pathway. b GC cell-derived TNF-α upregulates IL-33 expression in CAFs via the TNFR2-NF-κB-IRF-1 pathway

Article Snippet: Anti-human IL-33 (no. AF3625), ST2L (no. AF523), TNF-α (no. AF-410-NA), TNFR1 (no. MAB225-100) and TNFR2 (no. MAB726-100) neutralizing antibodies were purchased from R&D Systems.

Techniques: Migration, Derivative Assay, Expressing

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: MDSCs identification. A, the indicated myeloid cell subsets were tested for suppressive activity against CFSE-labeled autologous T cells stimulated with beads coated with anti-CD3/anti-CD28 antibodies. Data normalized on the control (no MDSC) are cumulative of five independent experiments using PBMCs from 5 patients. P value for the ANOVA test (Pa) and the Tukey post hoc test are reported. B, example of multicolor FACS analysis for MDSC phenotype CD33+IL4Rα+ cells are highlighted in blue. C, intratumoral CD33+ IL4Rα+ cells were retrospectively evaluated in the tumor specimen of recurrent or nonrecurrent OSCC patients by immunofluorescence microscopy. P value for t test is reported.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Activity Assay, Labeling, Control, Immunofluorescence, Microscopy

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: An intermediate tadalafil dose modulates most effectively tumor immunity. The ratio between the MDSCs (A) or the log2-ratio of the CD8 proliferation (B) after (t2) and before (t1) pharmacologic treatment was plotted against the weight-normalized tadalafil dose. Best-fitting quadratic curve and confidence interval (gray area) are reported. cGMP (C) and cAMP (D) were measured by ELISA in the following FACS-sorted cell population from patients (n = 3) treated with intermediate or high dosage of tadalafil: CD33+IL4Rα+ (MDSCs), HLADRhigh (APC), or CD3+ (T cells). Pt, paired t test; BDL, below detection limit.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Tadalafil Reduces Myeloid-Derived Suppressor Cells and Regulatory T Cells and Promotes Tumor Immunity in Patients with Head and Neck Squamous Cell Carcinoma

doi: 10.1158/1078-0432.CCR-14-1711

Figure Lengend Snippet: Tadalafil modulates tumor microenvironment. CD33/IL4Rα (A), CD4/FoxP3 (B), or CD8/CD69 (C) intratumoral concentration was evaluated by immune-fluorescence microscopy. Pa, P ANOVA test.

Article Snippet: FACS sorting For the suppressive assay, cryoconserved PBMCs were thawed and stained with Percp-Cy5.5–conjugated anti-human HLADR, FITC-conjugated anti-human CD33 (BD) and PE-conjugated anti-human IL4Rα (R&D Systems).

Techniques: Concentration Assay, Fluorescence, Microscopy

Journal: BMC Immunology

Article Title: Increased circulating Tfh to Tfr ratio in chronic renal allograft dysfunction: a pilot study

doi: 10.1186/s12865-019-0308-x

Figure Lengend Snippet: The frequency of cTfh and cTfr cells and the level of associated factors between CAD group and stable group . Squares refer to chronic allograft dysfunction (CAD) group, cycles refer to stable renal function group; a cTfh: CXCR5 + Foxp3 − on CD4 + cells; b cTfr: CXCR5 + Foxp3 + on CD4 + cells; c cTfh to cTfr ratio; d Tregs: CD25 + Foxp3 + on CD4 + cells; e CXCR5 + PD-1 + on CD4 + cells; f PD-1 + on CXCR5 + CD4 + cells; g CXCR5 + ICOS + on CD4 + cells; h ICOS + on CXCR5 + CD4 + ; i CXCR5 + STAT3 + on CD4 + cells; j CXCR5 + STAT5 + on CD4 + cells; k STAT3 + on CXCR5 + CD4 + cells; l STAT5 + on CXCR5 + CD4 + cells; m CXCR5 + STAT4 + on CD4 + cells; n STAT4 + on CXCR5 + CD4 + cells; o CXCR5 + IL-21 + on CD4 + cells; p IL-21 + on CXCR5 + CD4 + cells; q The serum level of CXCL13; r The serum level of TGF-β

Article Snippet: Then samples were stained with IL-21-PE (BD Bioscience, New Jersey, US).

Techniques: