Journal: Nature immunology

Article Title: A subset of HLA-DP molecules serve as ligands for the natural cytotoxicity receptor NKp44

doi: 10.1038/s41590-019-0448-4

Figure Lengend Snippet: a, Reporter cell activity of NKp44ζ+ and NKp46ζ+ Jurkat cells was determined as percentage of CD69+ cells after co-incubation with plate-coated anti-NKp44 and anti-NKp46 molecules (1 µg ml−1) as well as four distinct HLA-DP-expressing JE6.1 cell lines (JE6.1-DP). The percentage of CD69+ cells following incubation on non-coated wells (blank) was subtracted from all samples. The percentage of CD69+ cells after co-incubation with non-HLA-DP-transduced JE6.1 cells was subtracted from JE6.1-DP-positive samples. Corrected values are illustrated as median with interquartile range as determined in nine independent experiments. Each dot represents one individual experiment (n = 9). Two-tailed Friedman test with post-hoc Dunn’s multiple comparison was used to determine statistical differences between JE6.1-DP-expressing cell lines. Control conditions (anti-NKp46, anti-NKp44) were not included in the statistical analysis. *P = 0.02 and 0.01. b, Activation of NKp44ζ+ Jurkat reporter cells following co-incubation with CLIP peptide-pulsed JE6.1-DP cell lines is depicted as percentage of CD69+ cells. The percentage of CD69+ cells following incubation on non-coated wells (blank) was subtracted from all samples. The percentage of CD69+ cells after co-incubation with CLIP-pulsed non-HLA-DP-transduced JE6.1 cells was subtracted from JE6.1-DP-positive samples. Corrected values are illustrated as median with interquartile range as determined in seven independent experiments (n = 7). Two-tailed Friedman test with post-hoc Dunn’s multiple comparison was used to determine statistical differences between percentages of CD69+ cells following co-incubation with the four different CLIP peptide-pulsed JE6.1-DP cell lines. *P = 0.04.

Article Snippet: HLA-DP and CLIP surface expression of JE6.1-DP cells was assessed by staining CLIP-pulsed and DMSO-pulsed JE6.1-DP cells with anti-CD3-BUV737 (BD Biosciences), anti-HLA-DP-APC (Leinco Technologies), anti-CLIP-FITC (BD Biosciences) and LiveDead NearIR (Life Technologies) for 30 min at 4 °C.

Techniques: Activity Assay, Incubation, Expressing, Two Tailed Test, Activation Assay

Journal: Immune Network

Article Title: Metabolic Reprogramming by the Excessive AMPK Activation Exacerbates Antigen-Specific Memory CD8 + T Cell Differentiation after Acute Lymphocytic Choriomeningitis Virus Infection

doi: 10.4110/in.2019.19.e11

Figure Lengend Snippet: LCMV-infected mice were treated with various concentrations of IM156 from days −1 to 29 post-infection. (A) Representative flow cytometric analysis of production of IFN-γ, TNF-α, and IL-2 in CD8 + T cells obtained from the spleen at day 30 post-infection after ex vivo re-stimulation with GP33 peptide. (B) Frequency of TNF-α-producing cells (top left) and IL-2-producing cells (top right) among IFN-γ + CD8 + T cells. MFI of TNF-α (bottom left) and IL-2 (bottom right) among IFN-γ + CD8 + T cells as in A. (C) Representative flow cytometric analysis of production of IFN-γ, TNF-α, and IL-2 in CD4 + T cells obtained from the spleen at day 30 post-infection after ex vivo re-stimulation with GP66 peptide. (D) Frequency of TNF-α-producing cells (top left) and IL-2-producing cells (top right) among IFN-γ + CD4 + T cells. MFI of TNF-α (bottom left) and IL-2 (bottom right) among IFN-γ + CD4 + T cells as in C. Data are representative of 3 independent experiments n=4–5 mice per group. Results are the mean ± SEM and statistical significance was determined by 2-tailed unpaired Student's t -test. * p<0.05; ** p<0.01; *** p<0.001.

Article Snippet: For intracellular cytokine staining, splenocytes re-stimulated ex vivo with 0.2 µg/mL of LCMV GP 33-41 peptide for CD8 + activation or GP 66-80 peptide for CD4 + activation in the presence of brefeldin A (GolgiPlug; BD Biosciences) and monensin (GolgiStop; BD Biosciences) for 5 h. Stimulated cells were fixed, permeabilized, and stained with fluorochrome-conjugated antibodies against IL-2 (JE6-5H4), IFN-γ (XMG1.2), and TNF-α (MP6-XT22) (BD Biosciences).

Techniques: Infection, Ex Vivo

Journal: EJHaem

Article Title: RUNX inhibitor suppresses graft‐versus‐host disease through targeting RUNX‐NFATC2 axis

doi: 10.1002/jha2.230

Figure Lengend Snippet: RUNX family‐dependent expression of NFATC2 in human T cells. (A) IGV snapshots showing the alignment data (BigWig or Wig format) around the NFATC2 gene loci for RUNX1, H3K4me2, H3K4me3, and H3K27ac ChIP‐seq experiments in primary human CD4 Tcell and Jurkat T cell. Red arrow indicates a region of active chromatin mark in supporting gene activation. (B) Hierarchical clustering with heat map of human CD4 T cell samples ( n = 376) based on RUN1, RUNX2, RUNX3 and NFATC2 gene expression data. (C) Scatter plots showing the correlation of gene expression between RUNX family genes and NFATC2 in human CD4 T cell samples. p ‐values were calculated by Spearman's correlation. (D and E) Luciferase reporter assay with NFATC2 promoter. (D) HEK293T cells were stably transduced with the lentivirus expressing RUNX1, RUNX2 or RUNX3 , and (E) Jurkat cells were stably transduced with the lentivirus expressing sh PanRUNX , together with the reporter vector expressing luciferase gene under NFATC2 promoter. Cells were incubated with 3 μM doxycycline (Dox+) or the equivalent amount of DMSO (Dox‐) for 48 hours, then the luciferase activity was monitored by a luminometer. Result was normalized to that of the control sample ( n = 3). Error bars indicate the mean ± standard error (SE) * p < 0.05, ** p < 0.01 by two‐tailed Student's t ‐test

Article Snippet: The human Jurkat cell line (clone JE6.1) was obtained from The European Collection of. Authenticated Cell Cultures (ECACC, UK, Cat No: 88042803).

Techniques: Expressing, ChIP-sequencing, Activation Assay, Gene Expression, Luciferase, Reporter Assay, Stable Transfection, Transduction, Plasmid Preparation, Incubation, Activity Assay, Control, Two Tailed Test

Journal: EJHaem

Article Title: RUNX inhibitor suppresses graft‐versus‐host disease through targeting RUNX‐NFATC2 axis

doi: 10.1002/jha2.230

Figure Lengend Snippet: RUNX family knockdown reduces the expression of NFATC2 and cytokine genes. (A) Down‐regulation of NFATC2 expression upon RUNX inhibition. Jurkat cells were transduced with control (sh Luc ) or with RUNX shRNAs (sh RUNX1 _1, sh RUNX1 _2, sh RUNX2 , sh RUNX3 and sh PanRUNX ) and cultured in the presence of 3 μM doxycycline. Twenty‐four hours after treatment, cell lysates were processed for immunoblotting. (B and C) RUNX and NFATC2 ‐depletion‐mediated repression of cytokine gene expression. (B) RUNX ‐depleted Jurkat cells were treated as in (A). (C) Jurkat cells were transduced with NFATC2 shRNAs (sh NFATC2 _1, sh NFATC2 _2 and sh NFATC2 _3) and cultured in the presence of 3 μM doxycycline. Twenty‐four hours after treatment, cells were activated by 50 ng/ml PMA and 1 μM ionomycin. Twelve hours after treatment, total RNA was prepared and analyzed by real‐time RT‐PCR. Values were normalized to that of control vector‐transduced cells ( n = 3). (D) Restoring NFATC2 expression in RUNX‐depleted Jurkat cells reverts RUNX‐depletion‐mediated inhibition of cytokine expression. Non‐RUNX‐depleted (sh Luc ) and RUNX‐depleted (sh RUNX1 _2 and sh PanRUNX ) Jurkat cells transduced with ( NFATC2 ) or without (Empty) lentivirus expressing NFATC2 . Cells were treated with 3 μM doxycycline for 24 hours, then activated by 50 ng/ml PMA and 1 μM ionomycin. Twelve hours after treatment, total RNA was prepared and analyzed by real‐time RT‐PCR. Values were normalized to that of control vector‐transduced cells ( n = 3). Error bars indicate the mean ± standard error (SE) * p < 0.05, ** p < 0.01, compared with the control by two‐tailed Student's t ‐test

Article Snippet: The human Jurkat cell line (clone JE6.1) was obtained from The European Collection of. Authenticated Cell Cultures (ECACC, UK, Cat No: 88042803).

Techniques: Knockdown, Expressing, Inhibition, Transduction, Control, Cell Culture, Western Blot, Gene Expression, Quantitative RT-PCR, Plasmid Preparation, Two Tailed Test

Journal: EJHaem

Article Title: RUNX inhibitor suppresses graft‐versus‐host disease through targeting RUNX‐NFATC2 axis

doi: 10.1002/jha2.230

Figure Lengend Snippet: RUNX inhibitor, Chb‐M’, suppresses T‐cell proliferation and reduces the expression of NFATC2 and cytokine genes. (A) Effect of Chb‐M’ on human T cell proliferation with OKT3‐mediated TCR activation. Human PBMCs (1.0×10 6 /ml) were cultured with OKT3‐coated plate and 100 U/ml rIL2, and treated with 1μM Chb,1 μM Chb‐S, 1μM Chb‐M’ or the equivalent amount of DMSO. Four days after treatment, the viable CD3‐positive T cell number was counted by trypan blue staining, and the percentage of CD4‐positive T cells were analyzed by flow cytometry ( n = 3). (B) Effect of Chb‐M’ on human T cell itself in cell viability and CD4 to CD8 ratio. Human PBMCs (2.0×10 6 /ml) were treated with 1μM Chb‐M’ or the equivalent amount of DMSO. The viable total cell number was counted by trypan blue staining at 24, 48 and 72 hours after treatment, and the percentage of CD3, CD4 and CD8‐positive T cells were analyzed by flow cytometry at 48 hours after treatment ( n = 3). (C and D) Effect of Chb‐M’ on cytokine genes and NFATC2 mRNA expression. Human PBMCs were treated with 1μM Chb,1 μM Chb‐S, 1μM Chb‐M’ or the equivalent amount of DMSO. (C) Six hours after treatment, cells were activated by 50 ng/ml PMA and 1 μM ionomycin, and total RNA was prepared four hours after activation. (D) Simultaneously with treatment, cells were stimulated with OKT3‐coated plates, and total RNA was prepared twenty‐four hours after activation. Total RNA was analyzed by real‐time RT‐PCR for IL2, TNF , IFNG and NFATC2 in (C) and CSF2 and IL17A gene expression in (D). Values were normalized to that of control vector‐transduced cells ( n = 3). (E) Effect of Chb‐M’ on NFATC2 expression. Jurkat cells were treated with 1 or 2 μM Chb‐M′ for 24 and 48 h, and then cell lysates were prepared and immunoblotted. Error bars indicate the mean ± standard error (SE) * p < 0.05, ** p < 0.01 compared with the control (DMSO) by two‐tailed Student's t ‐test. (F) Dose‐response curves of Chb, Chb‐S and Chb‐M’ in mixed lymphocyte reaction of human peripheral blood mononuclear cells. Cells were treated with the indicated concentrations of PI polyamides or Chb. Six days after treatment, cell proliferation was assayed by [ 3 H] thymidine uptake assay. ( n = 3). The table shows IC50 values of Chb, Chb‐S and Chb‐M’

Article Snippet: The human Jurkat cell line (clone JE6.1) was obtained from The European Collection of. Authenticated Cell Cultures (ECACC, UK, Cat No: 88042803).

Techniques: Expressing, Activation Assay, Cell Culture, Staining, Flow Cytometry, Quantitative RT-PCR, Gene Expression, Control, Plasmid Preparation, Two Tailed Test