Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.M112.448654

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

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

Techniques: Isolation

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.M112.448654

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

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

Techniques: Quantitative RT-PCR, MANN-WHITNEY

Journal: The Journal of Biological Chemistry

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

doi: 10.1074/jbc.M112.448654

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

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

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

Journal: International Journal of Molecular Sciences

Article Title: Lipopolysaccharide-Induced Acute Kidney Injury Is Dependent on an IL-18 Receptor Signaling Pathway

doi: 10.3390/ijms18122777

Figure Lengend Snippet: Lipopolysaccharide (LPS) affects the expression levels of interleukin (IL)-18, IL-18Rα and IL-18Rβ in the mouse kidney. C57BL/6 mice were injected intraperitoneally with 30 mg/kg LPS and sacrificed at 18 and 120 h after LPS injection. At 18 and 120 h, CD4 + T cells and F4/80 + cells were isolated using a BD FACSAria TM special-order research product with purities of 90–95% from splenocytes in C57BL/6 WT mice. Gene expressions of IL-18, IL-18Rα and IL-18Rβ were measured by real-time PCR. In each experiment, the expression levels were normalized to the expression of 18SrRNA and were expressed relative to the values of saline-treated control mice. The data are the mean fold-increase ± SEM: ** p < 0.01, **** p < 0.0001, the mice without and with LPS injection at 0 and 120 h ( n = 4 and n = 6) vs. 18 h ( n = 4) after LPS injection.

Article Snippet: CD4 + T cells and F4/80 + cells were isolated using a BD FACSAria special-order research product (Becton Dickinson) for purities of 90–95% from splenocytes in C57BL/6 WT mice for the real-time PCR and adoptive transfer.

Techniques: Expressing, Injection, Isolation, Real-time Polymerase Chain Reaction, Saline, Control

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Top panel: cDCs were rested 16 h and stimulated as indicated for 15 min. pSTAT3 was evaluated by flow cytometry. cDCs were gated as CD11c hi cells. Bottom panel: cDCs were rested 1 h, treated with 20 ng ml −1 IL-21, IL-6, IL-10 or Flt3L for 4 h, and intracellular pro-IL-1β analysed by flow cytometry. Shown are data representative of three experiments. ( b ) Summary of three experiments from lower panel of a . ** P values of the untreated sample compared with IL-21, IL-6 and IL-10 treated samples are 0.0002, 0.0017 and 0.0049, respectively; NS, P =0.4; error bars are means±s.e.m. ( c – e ) cDCs were stimulated with 100 ng ml −1 IL-21 or IL-10 for 1 h, then stimulated with 100 ng ml −1 LPS for 4 h, and the expression of Il1b ( c ), Il6 ( d ), and Tnf ( e ) mRNA were determined. Shown are combined results of 3 independent experiments; error bars are means±s.e.m. ( f ) Top panel: BMMs were rested without M-CSF for 16 h, treated with IL-21 or LPS for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: BMMs (gated as CD11c + F4/80 + cells) were rested and treated with IL-21 or LPS for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of two experiments (total of 6 individual samples). ( g ) Top panel: CD4 + T cells were pre-activated with 5 μg ml −1 plate-bound anti-CD3+2 μg ml −1 soluble anti-CD28 for 3 days, washed, rested 16 h, treated with IL-21 for 4 h, and intracellular pro-IL-1β assessed by flow cytometry. Bottom panel: Rested CD4 + T cells were treated with IL-21 for 15 min. pSTAT3 was evaluated by flow cytometry. Data are representative of three experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a – g ), cDCs were rested 1 h, treated with IL-21 for 1 h; pre-activated CD4 + T cells were washed, rested 16 h, treated with IL-21 for 1 h, and ChIP-Seq performed for STAT3, H3K4me1, and H3K27ac. ( a ) Venn diagram showing overlapping and distinctive STAT3 binding sites in cDCs and CD4 + T cells. ( b – g ) For IL-21-induced-STAT3 binding sites that differentially exist in cDCs or CD4 + T cells, there were cell type-specific binding profiles of STAT3 ( b versus c ) and H3K4me1 ( d versus e ) and H3K27ac ( f versus g ) enhancer marks. Shown are normalized read densities near peak summits for cDC- or CD4 + T-cell specific STAT3 binding sites. ‘Dips' at the plot centres ( d – g ) represent open chromatin corresponding to nucleosome depletion. Data are representative of two experiments.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: ChIP-sequencing, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) Freshly isolated cDCs were rested 1 h, then stimulated with IL-21 for 4 h; CD4 + T cells were pre-activated for 3 days, then washed and rested 16 h, and stimulated with IL-21 for 4 h. Shown are genes differentially regulated by IL-21 in cDCs versus pre-activated CD4 + T cells. For cDCs, gene expression profiling was performed by microarray analysis, where cDCs were pooled from three independent experiments, as described in ref. . For pre-activated CD4 + T cells, gene expression profiling data were generated by RNA-Seq analysis. Shown are data from one of two similar experiments. ( b ) Il1b and Il21 expression in cDCs and CD4 + T cells not treated or stimulated with IL-21 as in a . Data are representative of 3 experiments. Error bars are technical duplicates of the representative experiment. ( c , d ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3, and H3K27me3 marks at the Il1b locus in cDCs ( c ) and CD4 + T cells ( d ). Arrows in c indicate STAT3 binding sites at GAS1, GAS2 and GAS3 regions (GAS1: TTAgggGAA (−155 bp), TACcctGAA (−175 bp), TCCctgGAA (−195 bp); GAS2: TTTgggGAA (−2,452 bp), TTCctcCAA (−2,525 bp), TTCttcAAA (−2,549 bp); GAS3: TTGtgtGAA (−9,761 bp)). Arrows in d indicate the STAT3 binding sites identified in cDCs, but no STAT3 binding was seen at these sites in CD4 + T cells. ( e , f ) STAT3 binding, H3K4me1, H3K27ac, H3K4me3 and H3K27me3 marks at the Il21 gene locus in cDCs ( e ) and CD4 + T cells ( f ). Arrow in f indicates the STAT3 binding site at the GAS motif in the Il21 promoter region. Arrow in e indicates this same site, but no STAT3 binding was seen at this site in cDCs. Data are representative of two experiments. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Isolation, Expressing, Microarray, Generated, RNA Sequencing Assay, Binding Assay

Journal: Nature Communications

Article Title: IL-21-mediated non-canonical pathway for IL-1β production in conventional dendritic cells

doi: 10.1038/ncomms8988

Figure Lengend Snippet: ( a ) cDCs were rested 1 h, treated with 100 ng ml −1 IL-21 or LPS at the indicated time points, and intracellular pro-IL-1β expression was determined. β-actin was used as control. Shown is one of two similar experiments. ( b ) cDCs were treated as in a , with 5 mM ATP added 1 h prior indicated time points in LPS-stimulated samples, and the secretion of IL-1β was determined by enzyme-linked immunosorbent assay (ELISA). Shown are combined results of two independent experiments; error bars are means±s.e.m. ( c ) CD4 + T cells from WT or Il1r −/− mice were cultured in Th17 cell differentiation conditions for 2 days, then supernatant from a 24 h, IL-21-treated cDC culture was added to the Th17 cells and incubated for 2 days, with or without addition of 10 μg ml −1 of anti-IL-1β. Expression of IL-2Rα (MFI) was determined by flow cytometry. The amount of biologically active IL-1β was determined using a standard curve constructed by assaying recombinant IL-1β. Shown are the combined results of two independent experiments with total of six samples. ( d , e ) WT, Casp1 −/− , Nlrp3 −/− and Pycard −/− cDCs were rested 1 h. In d , cDCs were then treated with 100 ng ml −1 LPS for 20–24 h with 5 mM ATP added in the final 1 h, and IL-1β assessed. Data are from two experiments; error bars are means±s.e.m. In e , cDCs were then treated with IL-21 for 20–24 h and IL-1β protein determined. Data are from five experiments. P values of IL-21-treated WT samples as compared with Casp1 −/− , Nlrp3 −/− and Pycard −/− samples are 0.99, 0.22 and 0.96, respectively; error bars are means±s.e.m. ( f ) cDCs from Ripk3 −/− , Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− mice were treated as in e , and IL-1β assessed. Data shown are from three experiments. P values of IL-21-treated Ripk3 −/− sample compared with Ripk3 +/− Casp8 +/− and Ripk3 −/− Casp8 −/− samples are 0.57 and 0.93, respectively. In b and d – f , IL-1β production in the culture supernatant was determined by ELISA. Pro-IL-1β induced by IL-21 in the culture supernatant was minimal, based on a pro-IL-1β-specific ELISA. Statistical analysis was performed by Student's t -test.

Article Snippet: Total mouse splenic CD4 + T cells were pre-activated with plate-bound anti-CD3+anti-CD28 for 3 days, washed and rested overnight, and not stimulated or stimulated with IL-21 for 4 h. Total RNA was isolated, and 5 μg per sample were used for mRNA purification using Dynal oligo(dT) beads (Invitrogen).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Cell Differentiation, Incubation, Flow Cytometry, Construct, Recombinant

Journal: bioRxiv

Article Title: Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

doi: 10.1101/2020.03.11.987412

Figure Lengend Snippet: (A) Study design using the IL-10 knock-out mouse model. CPZ group dams were treated with CPZ [(0.5 mg/ml) administration in drinking water] during pregnancy from day 14 of gestation until the end of the weaning period (3 weeks of age of offspring). (B) The number of 16S rRNA gene per gram of feces in male offspring from non-treated (NT) and CPZ-exposed dams at 3 and 7 weeks of age ( n = 5 per group). (C) Shannon diversity index in male offspring at 3 and 7 weeks of age. (D) PCoA plots of both unweighted and weighted UniFrac distances of 16S rRNA gene amplicon sequences in NT and CPZ male pups at 3 and 7 weeks of age, respectively. (E) Flow cytometric analyses of live CD4 + T cells expressing Foxp3 (Treg), T-bet (Th1) or RORγt (Th17) in spleens (SPLNs) and mesenteric lymph nodes (MLNs) of NT versus CPZ males at 7 weeks of age ( n = 4-5 per group). Data represent the percentage of live TCRβ + CD4 + cells. (F) IL-12p40 production of dendritic cells obtained from 7-week-old male NT (blue) and CPZ (red) pups with stimulation with fecal slurry ( n = 5 per group). * p < 0.05 and ** p < 0.01. Data represent mean ± SEM for (B), (C), (E), and (F). Female data are shown in the supplementary figure. See also Figure S1.

Article Snippet: DCs and naïve CD4 + T cells were harvested from SPLNs of mice at 7 weeks of age using a CD11c positive selection kit II (STEMCELL Technologies) and a mouse naïve CD4 + T cell Isolation kit (STEMCELL Technologies).

Techniques: Knock-Out, Amplification, Expressing

Journal: bioRxiv

Article Title: Early-life microbial intervention reduces colitis risk promoted by antibiotic-induced gut dysbiosis

doi: 10.1101/2020.03.11.987412

Figure Lengend Snippet: (A) Flow cytometric analyses of live CD4 + T cells expressing IFNγ and IL-17A stimulated via PMA and ionomycin. Cells in spleens (SPLNs) and mesenteric lymph nodes (MLNs) were obtained from NT and CPZ males at 7 weeks of age. Data represent the percentage of live TCRβ + CD4 + cells ( n = 5 per group). (B) IFNγ and IL-17A production of CD4 + T cells obtained from SPLN and MLN of 7-week-old NT and CPZ male pups with co-stimulation via anti-CD3 and anti-CD28 antibodies ( n = 5 per group). (C) The number of 16S rRNA gene per gram of feces of male recipients before CD4 + T cell transfer (NT recipients: n = 12, CPZ recipients: n = 12, Control group: n = 9). (D) Shannon diversity index of male recipients before CD4 + T cell transfer. (E) PCoA plots of both unweighted and weighted UniFrac distances of 16S rRNA gene amplicon sequences in male recipients before CD4 + T cell transfer. (F) Percent weight change (expressed as % of starting weight) of male recipients after CD4 + T cell transfer. (G) Survival rate of male recipients during the 8-week observation period. (H) Fecal lipocalin-2 (LCN-2) levels before CD4 + T cell transfer (week 0) and at 2 weeks after post-transfer (week 2) in male recipients. (I) Histological assessment of colons from the surviving male recipients at week 8 (NT group: n = 10, and CPZ group: n = 9). Representative H&E histological sections of colon are presented for NT recipients, CPZ recipients, and the control group. * p < 0.05, ** p < 0.01, and *** p < 0.001. Data represent mean ± SEM for (A)-(D), (F), (H), and (I). Female data are shown in the supplementary figure. See also Figure S2.

Article Snippet: DCs and naïve CD4 + T cells were harvested from SPLNs of mice at 7 weeks of age using a CD11c positive selection kit II (STEMCELL Technologies) and a mouse naïve CD4 + T cell Isolation kit (STEMCELL Technologies).

Techniques: Expressing, Amplification

Journal: The Journal of Experimental Medicine

Article Title: Interferon-producing Cells Fail to Induce Proliferation of Naive T Cells but Can Promote Expansion and T Helper 1 Differentiation of Antigen-experienced Unpolarized T Cells

doi: 10.1084/jem.20021091

Figure Lengend Snippet: HEL expression and antigen presentation to HEL-specific naive CD4 T cells and 3A9 T cell hybridoma by primary mHEL-transgenic DCs and IPCs. (A) IPCs and Ly-6G/C − DCs from the mHEL mouse were stained with an antibody specific for HEL to measure antigen expression at the cell surface (gray histograms). Open histograms indicate staining with control antibody. (B) Proliferative response of HEL-specific naive 3A9 CD4 T cells (10 4 ) cultured with graded numbers of purified IPCs (open symbols), CD11b + (black filled symbols), and CD8α + (gray filled symbols) DCs derived from mHEL mice. IPCs and DCs were untreated (circles) or treated (squares) in vitro for 12 h with PR8 (1 MOI) before addition of T cells. Supernatants were then collected and type I IFN production was measured (insert to B). Proliferation was measured after 72 h. (C) Expression of costimulatory molecules, MHC class II and HEL48–62/IA k specific complexes in IPCs and DCs derived from mHEL-mice. Mice were either uninfected or challenged with VSV for 12 h. MFI for IPCs and Ly-6G/C − DCs are indicated. (D) Proliferation of 3A9 naive CD4 T cells cocultured with graded numbers of IPCs or DCs purified ex vivo from mHEL mice uninfected or infected with VSV (intravenous, 2 × 10 8 PFU) 12 h before. (E and F) HEL-specific 3A9 cell hybridoma T cells (10 5 ) were cocultured with graded numbers of IPCs and DCs sorted from mHEL mice and stimulated in vitro with influenza virus PR8 (E) or activated in vivo by VSV infection (F). Supernatants were collected after 48 h and the IL-2 produced upon activation was measured as proliferation of CTLL cells after 20 h.

Article Snippet: CD4 + T cells were magnetically isolated with CD4-dynabeads (Dynal) from pooled spleens and lymph nodes of 3A9 TCR transgenic mice (90% purity, <2% MHC class II + cells).

Techniques: Expressing, Transgenic Assay, Staining, Cell Culture, Purification, Derivative Assay, In Vitro, Ex Vivo, Infection, In Vivo, Produced, Activation Assay

Journal: The Journal of Experimental Medicine

Article Title: Interferon-producing Cells Fail to Induce Proliferation of Naive T Cells but Can Promote Expansion and T Helper 1 Differentiation of Antigen-experienced Unpolarized T Cells

doi: 10.1084/jem.20021091

Figure Lengend Snippet: DCs induce expansion of antigen experienced uncommitted T cells which have lymph node homing potential and can respond to IFN-γ–inducing cyto-kines. (A) CD4 + 3A9 T cells expanded by freshly isolated CD11b + or CD8α + mHEL mouse-derived DCs were analyzed for IFN-γ and IL-2 production, 5–7 d after the first stimulation. (B) In vitro migration to CCL19 of 3A9 T cells expanded by CD11b + DCs (black filled symbols) or CD8α + DCs (gray filled symbols) is dose dependent. (C and D) 3A9 T cells expanded by CD11b + DCs express IL-12, IL-23, and IL-18 receptors thus having the capacity to respond to all these IFN-γ–inducing cytokines. IL-12Rβ1, IL-12Rβ2, IL-23R, and β-actin were detected by RT-PCR. (C) Numbers indicate fold dilutions of cDNAs. IL-12Rβ (β1 and β2 chains), and IL18Rα chain expression were measured at the cell surface of 3A9 T cells expanded by CD11b + DCs by flow cytometry at the same time of restimulation. Identical results were obtained with 3A9 T cells expanded by CD8α + DCs.

Article Snippet: CD4 + T cells were magnetically isolated with CD4-dynabeads (Dynal) from pooled spleens and lymph nodes of 3A9 TCR transgenic mice (90% purity, <2% MHC class II + cells).

Techniques: Isolation, Derivative Assay, In Vitro, Migration, Reverse Transcription Polymerase Chain Reaction, Expressing, Flow Cytometry