Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: DTHD1 deficiency increased the cytotoxicity of CD161 − CD8 + T EMRA cells . (a) Heatmap showing the correlation of co-expression modules with different cell subsets. (b) Functional annotations of DTHD1-related genes. (c) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0124. (d) Flow cytometric analysis of mean fluorescence intensity (MFI) of Granzyme B of CD161 − CD8 + T cells electroporated with si-NC or si- DTHD1 siRNAs after coculture with P815 cell. P value is calculated by Unpaired t-test, P = 0.0012. (e) Structural prediction of DTHD1-MYD88 interaction by ZDOCK. Grey, DTHD1; blue, MYD88; red, mutated regions in DTHD1 . (f) Western blotting analysis of HA in HEK293T cells co-transfected with myc-MYD88 and HA-empty vector or HA-DTHD1 (WT) or HA-DTHD1 (Mutant) expressing plasmids after immunoprecipitated with anti-Myc beads. (g) Boxplot indicating the average expression of MYD88 in CD161 − CD8 + T EMRA cells in HCs and SLE samples from our dataset. P value is from Wilcoxon rank-sum test. (h) Luciferase activity analysis of lysates of HEK293T cells co-transfected luciferase reporter plasmid for NF-κB, pRL-TK-renilla-luciferase plasmid, MYD88 plasmid and WT-DTHD1 plasmid or mutant DTHD1 plasmid ( n = 6). P values are determined by Unpaired t-test. (i) Quantification (left) and flow cytometry (right) of the apoptosis percentage of P815 cells after cocultured with primary CD8 + T cells pre-treated with DMSO or TAK-242 for 5 h at the ratio of 1:5. P value is determined by Unpaired t-test, P = 0.0024. One representative experiment of three is shown (f). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Expressing, Functional Assay, Flow Cytometry, Fluorescence, Structural Proteomics, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Immunoprecipitation, Luciferase, Activity Assay

Journal: eBioMedicine

Article Title: Cytotoxic CD161 − CD8 + T EMRA cells contribute to the pathogenesis of systemic lupus erythematosus

doi: 10.1016/j.ebiom.2023.104507

Figure Lengend Snippet: CD161 − CD8 + T EMRA cells contribute to SLE by LIGHT signaling . (a) The number (top) and strength (bottom) of interaction among all cells in HCs and SLE samples. (b) Heatmap of differential interactions between HCs and SLE samples in cell–cell communication network. The top bar indicates the sum of incoming signaling and right bar indicates the sum of outgoing signaling. Red indicates increased signaling and blue indicates decreased signaling in SLE. (c) Number of significant ligand-receptor pairs between CD161 − CD8 + T EMRA cells (outgoing) and other cell subclusters (incoming) in HCs (left) and SLEs (right). The relative number of ligand-receptor pairs is represented by the edge width. (d) Comparison of the significant outgoing signaling from CD161 − CD8 + T EMRA cells between HC and SLE. Empty space means the communication probability is zero. P -values are computed from one-sided permutation test. ∗∗∗ P < 0.001. (e) qPCR analysis of TNFSF14 expression in primary CD8 + T cells electroporated with empty vector, DTHD1-WT or DTHD-mut plasmid cocultured with THP1 cells ( n = 3). P values are determined by unpaired t-test. (f) Receiver operating curve for out-of-sample prediction of case–control state by a logistic regression model trained on DEGs in CD161 − CD8 + T EMRA cells. DEGs include IFI27 , IFI44L , RSAD2 , IFI44 , FAM118A , LGALS9 , MX1 , EPSTI1 , USP18 , OAS3 , LAIR2 , IFIT1 , XAF1 . Inset depicts the changes of DEGs in the public transcriptome profile and CD161 − CD8 + T EMRA cells. (g) Graphic abstract showing the expansion of CD161 − CD8 + T EMRA in patients with SLE and DTHD1 downregulation promotes MYD88-mediated expansion and cytotoxicity of this pathogenic CD161 − CD8 + T EMRA subset in SLE.

Article Snippet: Human MYD88 cDNA was purchased from Genecopoeia Inc. and cloned into the pcDNA3.1-Myc plasmid.

Techniques: Comparison, Expressing, Plasmid Preparation, Control

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet: A) Venn diagram showing overlap of differentially expressed genes (DEGs) between IL-1β, LPS, and PAM after 2 hours of stimulus. B) Heatmap of DEGs for MyD88-dependent ligand treated cells. RNA-sequencing was performed in triplicate. Each row shows the normalized expression (z-score) of a single gene. Dendrogram shows linkage based on Ward’s method. C-D) Volcano plot showing log2(fold change) and -log10(P value) for DEGs between LPS and IL-1β stimulus Among the DEGs with adjusted p-value < 0.01 and fold change > 4, genes annotated as NF-κB negative regulators (GO:0032088) (C) or cytokines (GO:0005125) (D) are colored in red. All differentially expressed regulators and top ten differentially expressed cytokines are labeled. E) qRT-PCR data following for a subset of highly differentially expressed cytokines and NF-κB negative regulators stimulation with 0.2 ng/mL (light blue), 1 ng/mL (dark blue) IL-1β, or 100 ng/mL LPS (red). Gene expression is normalized to basal gene expression for unstimulated cells. Data shown as mean fold change over unstimulated cells +/− S.E.M. from 3 replicates. Benjamini-Hochberg adjusted P value < 0.05 (*) or <0.01 (**). See also Supplemental Figure 7, Supplemental Table 1.

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: RNA Sequencing, Expressing, Labeling, Quantitative RT-PCR, Gene Expression

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: Control, Recombinant, Reverse Transcription, Clinical Proteomics, Purification, cDNA Synthesis, TaqMan Probe Assay, DNA Library Preparation, Plasmid Preparation, Software

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet: A-C) NF-κB response dynamics over 2 hours of stimulus for each ligand normalized to the mean amplitude of the naïve (S1) high dose response. 50 single-cell traces randomly selected for each condition. All sequences of S1 and S2 ligands shown. All 9 sequences shown at high (A), mid (B), and low (C) dose. D) Violin plot comparing the normalized S2 responses of the MyD88-dependent ligands (LPS, PAM, IL-1β) following either TNF-α (blue) or another MyD88-dependent ligand (red) or the TNF-α response following a MyD88 dependent ligand (green) (> 650 cells per condition). Open circle and line show the mean. E) Violin plot comparing the normalized S2 MyD88-dependent responses following IL-1β (blue), PAM (green), and LPS (red) stimulus at high, mid, and low doses (> 340 cells per condition). Bonferroni corrected Wilcoxon rank sum test p-value > 10−2 (n.s.), < 10−2 (*), < 10−3 (**), < 1*10−4 (***). Fold change difference between sample means > 1 (#), > 1.25 (##), or >4 (###). F) Plot of mean trace for conditions where LPS is provided at 0 min (gray arrowhead) and switched to 3 ng/mL (high dose) IL-1β after the indicated time (red arrowhead). Gray region and red regions of trace indicate NF-κB response during LPS stimulus interval and after replacement with IL-1β, respectively. Entirely red traces in the left column show an only IL-1β response and entirely gray traces in the right column show an only LPS response. Each mean trace represents > 100 single cells from 2 biological replicates. G) Plot of mean traces for conditions where 0.2 and 3 ng/mL (mid and high dose) IL-1β are provided at 0 min and switched to 100 and 400 ng/mL (mid and high dose) LPS, respectively, after the indicated time. Gray region and red regions of trace indicate NF-κB response during IL-1β stimulus interval and after replacement with LPS, respectively. As in F), entirely red traces in the left column show an only LPS response and entirely gray traces in the right column show an only IL-1β response. Each mean trace represents > 100 single cells over 2 biological replicates. H) Violin plot comparing the normalized response for 3 ng/mL IL-1β following 100 ng/mL LPS or the response for 100 ng/mL LPS following 0.2 ng/mL IL-1β. Each plot is derived from > 100 cells per condition over 2 biological replicates. Open circle and line show the mean. See also Supplemental Figure 5, 6A–B.

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: Derivative Assay

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet: A) Violin plot comparing IL-1β maximum response following LPS treatment normalized to naïve for untreated (blue) and PS1145 pre-treated (red) cells. Pre-treated cells were exposed to 40 μM PS1145 stimulated with LPS at the indicated concentration, washed and stimulated with 3 ng/mL IL-1β. Each condition shown from > 120 single cells over 2 biological replicates. B) Diagram illustrating the NF-κB network model used for the simulation. Two negative components, IRAK1 autoinhibition and nuclear NF-κB dependent attenuation, are highlighted in red and orange. The TNF-α signaling pathway (green) utilizes different kinases to activate IKK than the MyD88 dependent ligands. C─E) Simulated network responses to different sequences of stimuli. The blue lines show the dynamics of nuclear NF-κB, the red lines for active IRAK1, and the orange lines for the downstream feedback component. Gray dashed vertical line indicates time of simulated replacement of ligands. See also Supplemental Figure 6C–D.

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: Concentration Assay

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: Control, Recombinant, Reverse Transcription, Clinical Proteomics, Purification, cDNA Synthesis, TaqMan Probe Assay, DNA Library Preparation, Plasmid Preparation, Software

Journal: Cell reports

Article Title: NF-κB memory coordinates transcriptional responses to dynamic inflammatory stimuli

doi: 10.1016/j.celrep.2022.111159

Figure Lengend Snippet:

Article Snippet: MyD88 (D80F5) Rabbit mAb , Cell Signaling Technologies , Cat. #4283.

Techniques: Binding Assay, Activation Assay