Journal: Clinical and Translational Medicine

Article Title: The IL‐21‐TET2‐AIM2‐c‐MAF pathway drives the T follicular helper cell response in lupus‐like disease

doi: 10.1002/ctm2.781

Figure Lengend Snippet: IL‐21 positively regulates absent in melanoma 2 (AIM2) expression by promoting ten‐eleven translocation 2 (TET2) enrichment in the Aim2 promoter region of T follicular helper (T FH ) cells. (A) DNA methylation map of naïve CD4 + T cells and T FH cells ( n = 3). Bisulphite sequencing PCR was performed to assess DNA methylation levels in the Aim2 promoter of (B) naïve CD4 + T cells and T FH cells ( n = 8) and (D) peripheral CD4 + T cells from systemic lupus erythematosus (SLE) patients and normal controls ( n = 10). Chromatin immunoprecipitation was performed to assess TET2 enrichment in the Aim2 promoter of (C) naïve CD4 + T cells and T FH cells ( n = 4 or 6) and (E) peripheral CD4 + T cells from SLE patients and normal controls ( n = 3 or 4). Representative flow cytometric profiles and data plots of (F) T FH cells and (G) germinal centre (GC) B cells in draining lymph nodes (dLNs) from Tet2 fl/fl and CD4 cre Tet2 fl/f l mice immunised with keyhole limpet haemocyanin (KLH) ( n = 9 or 10). (H) AIM2 accumulation in CD4 + T cells in wild‐type (WT) and CD4 cre Tet2 fl/fl mice ( n = 3). AIM2 accumulation in response to treatments with T FH ‐related cytokines assessed by (I) Western blot and (J) real‐time PCR ( n = 3). (K) Chromatin immunoprecipitation was performed in human CD4 + T cells with or without IL‐21 treatment after TET2 pull‐down ( n = 3). Bars show the mean ± SEM. * p < .05, ** p < .01, *** p < .001, **** p < .0001

Article Snippet: A total of 2.5 × 105 naïve CD4+ T cells were seeded in anti‐CD3 antibody‐precoated plates (Calbiochem, 5 μg/ml) and further treated with anti‐CD28 antibody (Calbiochem, 2 μg/ml) and different cytokines (PeproTech) as follows.

Techniques: Expressing, Translocation Assay, DNA Methylation Assay, Bisulfite Sequencing, Chromatin Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction

Journal: Cancer immunology research

Article Title: Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy

doi: 10.1158/2326-6066.CIR-14-0038

Figure Lengend Snippet: (a) Flow cytometric analysis of congenically marked OT-I CD8 T cells mixed ∼1:100 with bulk CD8 T cells and expanded with SIINFEKL-pulsed bone-marrow derived dendritic cells in IL-2 or cocktail of small molecules and cytokines (see methods). (b) Representative plots of expression of the phenotypic markers CD62L and CD44 on Thy1.2+ OT-I CD8 T cells. Plots in (a,b) representative of more than five independent experiments. (c) Plots of pre- and post-sort Thy1.2+ OT-I CD8 T cells grown as in (a) and purified by FACS for RNAseq analysis. (d) Unsupervised hierarchical clustering of RNAseq of populations sorted as in (c) showing all genes with a >2 fold difference between groups 1 and 3. Inset: principal component analysis of genes with >2-fold difference between any two groups for populations depicted in (c), and heatmap of selected biologically relevant genes in populations depicted in (c) and at selected time points of the OT-I response to L. monocytogenes Ova (from 21).

Article Snippet: All cytokines except for human IL-2 were from Peprotech.

Techniques: Derivative Assay, Expressing, Purification

Journal: Cancer immunology research

Article Title: Antigen-specific culture of memory-like CD8 T cells for adoptive immunotherapy

doi: 10.1158/2326-6066.CIR-14-0038

Figure Lengend Snippet: (a) Flow cytometric analysis of CD8 T cells cultured from CMV-seronegative HLA-A*0201+ PBMCs after pulldown with CMV-specific HLA-A*0201-pp65 dextramer. Cells were cultured in IL-2 or cocktail of memory-associated cytokines and small molecules. Plots depict dextramer-stained cells at pulldown and after 1, 2 or 3 weeks in culture with autologous antigen-pulsed monocyte-derived dendritic cells. (b) Representative plots of expression of the phenotypic markers of memory CCR7 and CD95 (Fas) on HLA-A*0201-pp65 dextramer positive gated cells. Plots in (a,b) representative of experiments performed with cells from two donors and two separate antigens. (c) Flow cytometric analysis of MACS-isolated CD8 T cells from human PBMCs expanded with CD3/CD28 antibody-coated beads in IL-2- or cocktail-containing media. Cells were labeled with Celltrace violet and divided cells were gated as shown for analysis of phenotypic markers CCR7 and CD95. Plots are representative of five independent experiments with PBMCs from three donors.

Article Snippet: All cytokines except for human IL-2 were from Peprotech.

Techniques: Cell Culture, Staining, Derivative Assay, Expressing, Isolation, Labeling

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) Chemical structure of the SMAC mimetic, compound GT13072. ( B ) Mice with CIA received daily intraperitoneal injections of GT13072 or control compound at 10 mg/kg per day. ( C ) All four paws were assessed daily for signs of arthritis. P values were calculated using two-way analysis of variance (ANOVA) with Tukey’s post hoc test and are not shown in the graph for clarity. ( D ) Histological analysis of arthritic paws from mice with CIA after 10 days of treatment with GT13072 or control compound ( n = 10). ( E ) On day 10 of arthritis, serum samples from CIA mice were analyzed for cytokines by Meso Scale Discovery (MSD). Each data point represents one animal ( n = 19 to 22). Error bars represent ±SEM. The horizontal lines represent the mean. P values were calculated using unpaired Student’s t tests. ( F to I ) GT13072-treated and control CIA mice were culled on day 10, and their draining lymph node and arthritic paw cells were counted and characterized by flow cytometry. P values were calculated using unpaired Student’s t tests. Each data point represents one animal (lymph node, n = 19 to 22; paw, n = 10). (F and G) The percentage of IL-17A–secreting CD4 + T cells in lymph nodes and paws was determined by flow cytometry. One representative dot blot or contour plot is shown. Absolute numbers of CD4 + IL-17A + cells in lymph nodes and arthritic paws are shown. (H) Absolute numbers of TCRγδ cells and IL-17 + TCRγδ T cells in arthritic paws with representative contour plot. (I) Paw cells were examined for RORγT expression.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Flow Cytometry, Dot Blot, Expressing

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) cIAP1 and cIAP2 protein expression in human CD4 + lymphocytes cultured for 10 min with GT13072 (1000 nM) or control compound. Immunoblots are representative of three independent experiments. ( B and C ) Human CD4 + CCR6 + CD161 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. The effect of GT13072 or control compound (1000 nM) on cytokine secretion and RORγT was determined by flow cytometry ( n = 4). Representative dot blots from four donors are shown. Bar graphs represent quantification of flow cytometry data. ( D ) Naïve human CD4 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads plus GT13072 or control compound (1000 nM). After 17 hours, mRNA levels were determined by quantitative reverse transcription polymerase chain reaction. Data are expressed as the mRNA level normalized to RPLPO expression and are shown as means ± SEM from five independent human donors. RPLPO, ribosomal protein, large, P0. (B to D) Error bars represent ±SEM. P values were calculated using unpaired Student’s t tests.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Expressing, Cell Culture, Western Blot, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A ) Phosphorylation of p38 MAPK was determined in human CD4 + T cells cultured with 1000 nM GT13072 or control compound before stimulation with TNF-α at 20 ng/ml. Immunoblots are representative of two independent experiments. p, protein. ( B and C ) Naïve human CD4 + T cells cultured with 1000 nM GT13072 before stimulation with plate-bound anti-CD3 (5 μg/ml), soluble anti-CD28 (2 μg/ml), IL-6 (50 ng/ml), IL-1β (10 ng/ml), and transforming growth factor–β (TGF-β; 1 ng/ml) for the indicated durations. (B) Phospho-p38 MAPK detected by immunoblotting. Results are representative of two independent experiments. (C) NFATc1 protein detected by immunoblotting. Dotted line shows where lanes were removed from the blot. Results are representative of four independent experiments. ( D and E ) Human CD4 + CCR6 + CD161 + T cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. (D) Total number of cells on day 6 of culture. (E) Determination of annexin V and propidium iodide (PI) expression on day 6. Representative dot blots are shown from three donors.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Cell Culture, Western Blot, Expressing

Journal: Science Advances

Article Title: cIAP1/2 inhibition synergizes with TNF inhibition in autoimmunity by down-regulating IL-17A and inducing T regs

doi: 10.1126/sciadv.aaw5422

Figure Lengend Snippet: ( A to C ) Mice with CIA received injections of GT13072 alone, etanercept alone, and GT13072 plus etanercept until day 10. All treatment was stopped on day 10 of arthritis. Animals were culled on day 18 of arthritis ( n = 7). (B) Clinical scores from day 10 to day 18. Error bars represent ±SEM. Representative photomicrographs of arthritic paws from mice on day 18. (C) Human RA patient synovial cells were cultured in the presence of T H 17-differentiating cytokines and anti-CD3/CD28 Dynabeads for 6 days. The effect of GT13072 or control compound (1000 nM) on IL-17A and RORγT was determined by flow cytometry.

Article Snippet: All cytokines were supplied by PeproTech, except IL-23 (R&D Systems).

Techniques: Cell Culture, Flow Cytometry

Journal: The Journal of investigative dermatology

Article Title: Epidermal Dysfunction Leads to an Age-Associated Increase in Levels of Serum Inflammatory Cytokines

doi: 10.1016/j.jid.2017.01.007

Figure Lengend Snippet: 12-month old C57BL/6J mice were treated topically with either petrolatum or glycerol twice-daily for 10 days, followed by collection of blood and skin for analyses of serum cytokines and cutaneous cytokine mRNA. Fig 4a and c: Expression levels of cutaneous pro-inflammatory cytokine mRNA levels in aged mice following treatment with petrolatum or glycerol; Fig 4b and d: Changes in serum cytokine levels in aged mice treated with petrolatum and glycerol, respectively. The data are expressed as % of untreated young mice, setting the level of young mice as 100% (dotted line). One way ANOVA was used to determine the statistical significances. The significances are indicated in the figures. N=8 for all groups. Significances indicated in column bars represent the differences between treated vs. untreated aged mice.

Article Snippet: All primers for cytokines were from Elim Biopharmaceuticals (Hayward, CA, USA).

Techniques: Expressing