Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A , B ) Cluster analysis of cell populations in HCC, ICC and metastatic melanoma. ( C , D ) Relative expression levels of the DDX58 , PDCD1 , TIGIT , CTLA-4 , and TOX -associated genes in patients with HCC, ICC ( C ), and metastatic melanoma ( D ). ( E , F ) Relative expression levels of DDX58 , IFIH1 , DHX58 , IPS-1 , and STING in HCC, ICC ( E ) and metastatic melanoma ( F ) tissues. ( G – I ) Multiple immunohistochemistry and statistical analyses of human HCC specimens; white scale bar = 50 μm. ( J ) The expression of granzyme-B secreted by infiltrating CD8 + T cells in the peritumor and tumour tissues of HCC by flow cytometry after stimulating with PMA/ionomycin and GolgiStop. Data information: The data represented different numbers ( n = 5 or 4) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s t -test was used in ( H ) ( P = 0.0419) and ( I ) ( P = 0.0485). Two-tailed paired Student’s test was used in ( J ) ( P = 0.0129). * P < 0.05, ** P < 0.01, and *** P < 0.001 compared with peritumour tissues. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Expressing, Immunohistochemistry, Flow Cytometry, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A – C ) Multiple immunohistochemistry and statistical analysis of human colon cancer specimens. White scale bar = 50 μm. ( D – F ) The expression of Rig-I in CD8 + T cells from the spleens and tumours of different tumour-bearing mice was detected using Western blotting. ( G ) Correlation analysis of DDX58 expression with GZMB and IFNG expression. ( H ) The expression of granzyme-B secreted by infiltrating CD8 + T cells in the peritumor and tumour tissues of CRC by flow cytometry after stimulating with PMA/ionomycin and GolgiStop. Data information: The data represented different numbers ( n = 5) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s test was used in ( B ) ( P = 0.03), ( C ) ( P = 0.0332). The Pearson correlation test was used in ( G ) (Exact p values were reported on graphs). The values of P and R are shown in the figure. Two-tailed paired Student’s test was used in ( H ) ( P = 0.0003). * P < 0.05, *** P < 0.001, compared with the peritumoral group.

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Immunohistochemistry, Expressing, Western Blot, Flow Cytometry, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A ) RNA-seq and KEGG enrichment analysis of signalling pathways (left panel) and key genes associated with leucocyte-mediated cytotoxicity, regulation of cell killing and interferon-gamma production (right panel) in CD8 + T cells from Rig-I +/+ / Rig-I −/ − mouse spleens. ( B , C ) Flow cytometry analysis of the development (including naïve, central memory and effector cells) of CD8 + T cells purified from Rig-I +/+ / Rig-I − / − mouse spleens after α-CD3 and α-CD28 activation ex vivo. ( D – H ) Flow cytometry analysis of CD107a, perforin, granzyme-B, IFN-γ and TNF-α levels in CD8 + T cells purified from Rig-I +/+ / Rig-I −/− mouse spleens after α-CD3 and α-CD28 activation and stimulation with PMA/ionomycin and GolgiStop ex vivo. Data information: A hypergeometric test was used in ( A ). The data represented different numbers ( n = 7) of biological replicates and were shown as the means ± SEMs in ( C – H ). Two-tailed unpaired Student’s test was used in ( C – H ) ( P < 0.0001). **** P < 0.0001 compared with the Rig-I +/+ group. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: RNA Sequencing Assay, Flow Cytometry, Purification, Activation Assay, Ex Vivo, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A – C ) Hepa1-6, MC38 and B16F10 cells were inoculated subcutaneously into Rig-I +/+ and Rig-I −/− mice, and the growth curves of Hepa1-6, MC38 and B16F10 tumours are shown. ( D ) After mice were treated with control immunoglobulin or a CD8 neutralising antibody, MC38 cells were inoculated subcutaneously into Rig-I +/+ and Rig-I − / −mice, and tumour growth curves are shown. ( E ) MC38-OVA cells were inoculated subcutaneously into Rig-I +/+ and Rig-I −/− mice, and the growth curves of MC38-OVA tumours were shown. ( F ) Flow cytometry analysis of the IFN-γ level and proportion of CD8 + T/CD4 + T cells infiltrating tumours formed from the colon cancer cell line MC38 in Rig-I +/+ and Rig-I −/− mice after stimulation with PMA/ionomycin and GolgiStop. ( G – I ) Statistics of the proportions of CD8 + T ( G ) and CD4 + T cells ( H ) and the ratio of CD8 + T/CD4 + T cells ( I ). ( J , K ) Statistics of IFN-γ levels in CD8 + T ( J ) and CD4 + T cells ( K ) after stimulation with PMA/ionomycin and GolgiStop for 4 h. Data information: The data represented different numbers ( n = 6–9) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s t -test was used in ( A ) ( P < 0.0001), ( G ) ( P < 0.0001), ( H ) ( P = 0.2173) and ( J ) ( P = 0.0092). Two-way ANOVA was used in ( D ) (Exact p values were reported on graphs). A two-tailed Mann‒Whitney U -test was used in ( B ) ( P = 0.0022), C ( P = 0.036), ( E ) ( P = 0.0087) and ( K ) ( P = 0.0456). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, and NS not significant compared with the Rig-I +/+ group. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Control, Flow Cytometry, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A ) Survival curves of MC38 tumour-bearing Rig-I +/+ and Rig-I −/− mice. ( B ) Survival curves of MC38 tumour-bearing B-NDG mice after the adoptive transfer of Rig-I +/+ or Rig-I − / − CD8 + T cells. ( C – E ) Flow cytometry was used to detect the proportion ( C , D ) and mean fluorescence intensity ( E ) of CD8 + T cells that produced IFN-γ in the spleens of tumour-bearing Rig-I +/+ and Rig-I − /− mice after stimulation with PMA/ionomycin and GolgiStop. Data information: The data represent different numbers ( n = 7 for ( A ) and n = 9 for ( B ) of biological replicates. The data represent different numbers ( n = 6 or 9 for D and E ) of biological replicates and were shown as the means ± SEMs. The log-rank test was used in ( A ) ( P = 0.0006) and ( B ) ( P = 0.0016). A two-tailed Mann‒Whitney U -test was used in ( D ) ( P = 0.0360). Two-tailed unpaired Student’s test was used in ( E ) ( P = 0.0053). * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the Rig-I +/+ group.

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Adoptive Transfer Assay, Flow Cytometry, Fluorescence, Produced, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A ) Coculture of Rig-I knockout CD8 + T cells from OT-1 mice with MC38-OVA cells at different ratios to determine the specific killing efficiency. ( B ) Flowchart of the transfer of Rig-I +/+ and Rig-I −/− mouse spleen-derived CD8 + T cells for the treatment of MC38 tumours. ( C – E ) Analysis of tumour growth curves, tumour sizes and tumour weights. ( F – I ) Flow cytometry analysis of the absolute number of CD8 + T cells infiltrating tumours, the secretion of granzyme-B and the mean fluorescence intensity after stimulation with PMA/ionomycin and GolgiStop. Data information: The data represented different numbers ( n = 4 or 6) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s test was used in ( A ) (Exact p values were reported on graphs), ( C ) ( P = 0.0001), ( E ) ( P = 0.0006), ( F ) ( P = 0.0043), ( H ) ( P = 0.0257) and ( I ) ( P = 0.0248). * P < 0.05, ** P < 0.01 and *** P < 0.001 compared with the vector or Rig-I +/+ group. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Knock-Out, Derivative Assay, Flow Cytometry, Fluorescence, Two Tailed Test, Plasmid Preparation

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A – E ) Naïve CD8 + T cells were isolated from the spleens of wild-type mice, and after 72 h of costimulation with α-CD3/α-CD28, Rig-I expression was detected using Western blotting ( A ), and the expression of PD-1 ( B ), CD25 ( C ), CD107a ( D ) and IFN-γ ( E ) in CD8 + T cells was detected using flow cytometry after stimulation with PMA/ionomycin and GolgiStop. ( F ) After 72 h of costimulation with α-CD3/α-CD28, the expression of RIG-I, p-AKT (Thr308), AKT, and GAPDH in WT or KO Rig-I CD8 + T cells from mouse spleens was detected using Western blotting. ( G ) Validation of the efficiency of RIG-I knockout in human CD8 + T cells. ( H ) After knocking out RIG-I, the expression of RIG-I, p-AKT (Thr308), AKT, and β-Actin in human CD8 + T cells was detected using Western blotting. ( I – L ) Naïve CD8 + T cells from Rig-I +/+ , Rig-I −/ − mouse spleens were treated with PI3K, AKT and glycolysis inhibitors after α-CD3/α-CD28 stimulation, and the proportions and mean fluorescence intensities of CD69 ( I – J ) and CD25 ( K – L ) in CD8 + T cells were detected using flow cytometry. Data information: The data represented different numbers ( n = 3) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s test was used in ( C ) ( P = 0.0215), ( D ) ( P = 0.0073) and ( E ) ( P = 0.0433). One-way multiple comparisons ANOVA with Tukey’s correction test was used in ( I – L ) (Exact p values were reported on graphs). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 and NS not significant compared with the Rig-I +/+ or Ctrl group. Ctrl control, Wort wortmannin, Ly294 Ly294002, GSK GSK690693, 2-DG 2-deoxy- d -glucose. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Isolation, Expressing, Western Blot, Flow Cytometry, Knock-Out, Fluorescence, Two Tailed Test, Control

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A – F ). Treatment of Rig-I +/+ and Rig-I −/− CD8 + T cells with α-CD3/α-CD28 followed by stimulation with inhibitors of PI3K, AKT and glycolysis. The proportions or mean fluorescence intensities of CD107a ( A , B ) and IFN-γ ( C – F ) were detected using flow cytometry after stimulation with PMA/ionomycin and GolgiStop. Data information: The data represented different numbers ( n = 3) of biological replicates and were shown as the means ± SEMs. One-way ANOVA with Tukey’s correction was used for multiple comparisons in ( A – F ) (Exact p values were reported on graphs). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001, and NS not significant compared with the Ctrl group. figure. Ctrl control, Wort wortmannin, Ly294 Ly294002, GSK GSK690693, 2-DG 2-deoxy- d -glucose.

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Fluorescence, Flow Cytometry, Control

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: ( A , B ) Schematic of adoptive cell therapy targeting RIG-I in an EBV-specific CTL tumour model and growth curves of LCL tumours. ( C , D ) The size and weight of the LCL tumours. ( E – G ) Flow cytometry analysis of the proportions of IFN-γ, granzyme-B and perforin in the control and RIG-I knockout groups after stimulation with PMA/ionomycin and GolgiStop. Data information: The data represented different numbers ( n = 6) of biological replicates and were shown as the means ± SEMs. Two-tailed unpaired Student’s t -test was used in ( B ) ( P < 0.0001), ( D ) ( P = 0.0028), ( E ) ( P = 0.0025), ( F ) ( P = 0.0009). A two-tailed Mann‒Whitney U -test was used in ( G ) ( P = 0.0043). ** P < 0.01, *** P < 0.001 and **** P < 0.0001 compared with the control group. .

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Flow Cytometry, Control, Knock-Out, Two Tailed Test

Journal: EMBO Molecular Medicine

Article Title: RIG-I is an intracellular checkpoint that limits CD8 + T-cell antitumour immunity

doi: 10.1038/s44321-024-00136-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Ionomycin , TargetMol , T11665.

Techniques: Recombinant, Staining, Control, Software

Journal: bioRxiv

Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

doi: 10.1101/2020.10.27.357426

Figure Lengend Snippet: a. tSNE/FlowSOM analyses of CD4 + T cells from 3-month-old (3mo) mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. Heatmaps of the manually gated CD4 + Foxp3 - ( b ) and Foxp3 + CD4 + ( c ) T cell populations indicated at seven, 14 and 21 days after immunisation in the aortic lymph node (right) and spleen (left). Here the frequency of each cell subset in each ChAdOx1 nCoV-19 immunised mouse has been expressed as the log2 fold change over the average frequency in PBS immunised mice (n=5). Bar charts showing the number of CD69 + CD62L + CD44 - CD4 + Foxp3 - ( d ), Ki67 + CD4 + Foxp3 - ( e ) CXCR3 + non-Tfh cells ( f ) and CXCR3 + Th1-like Treg cells ( g ) CD4 + cells in the aortic lymph node of ChAdOx1 nCoV-19 or PBS immunised mice, at the indicated timepoints post immunisation. h . Analysis of cytokine production six hours after PdBu/ionomycin stimulation of aortic lymph node cells from 3-month-old mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. i . Stacked bar plots show the number of CD4 + Foxp3 - cells singly or co-producing IFNγ, IL-2 or TNFα six hours after restimulation with SARS-CoV-2 peptide pools, each bar segment represents the mean and the error bars the standard deviation. In d-g bar height in corresponds to the mean and each circle represents one biological replicate. P-values are calculated using a student’s t-test with Holm-Sidak multiple testing correction.

Article Snippet: For cytokine staining, splenic cells were stimulated with a pool of SARS-CoV-2 spike protein immunodominant domain peptides, (Miltenyi Biotec #130-126-700) at a 0.6 μM concentration (approx.1μg/ml), whilst lymph node cells were stimulated with 0.5μg/ml of Phorbol 12,13 dibutyrate (PdBu, Tocris Bioscience, #), 0.75μg/ml of Ionomycin calcium salt (Tocris Bioscience, #), both in warm complete RPMI (10% FCS, 1% Pen/Strep, 1% glutamine, 1% sodium pyruvate, 1% MEM NAA, 1% HEPES and 55μM-2-mercaptoethanol) for 4hrs at 37°C, 5%CO 2 .

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

doi: 10.1101/2020.10.27.357426

Figure Lengend Snippet: Flow cytometric contour plots showing the gating strategy to identify T cell subsets ( a ), cytokine production in CD4 + Foxp3 - aortic lymph node T cells six hours after PdBu/Ionomycin restimulation ( b ), and in SARS-CoV-2 peptide restimulated CD4 + Foxp3 - splenocytes ( c ).

Article Snippet: For cytokine staining, splenic cells were stimulated with a pool of SARS-CoV-2 spike protein immunodominant domain peptides, (Miltenyi Biotec #130-126-700) at a 0.6 μM concentration (approx.1μg/ml), whilst lymph node cells were stimulated with 0.5μg/ml of Phorbol 12,13 dibutyrate (PdBu, Tocris Bioscience, #), 0.75μg/ml of Ionomycin calcium salt (Tocris Bioscience, #), both in warm complete RPMI (10% FCS, 1% Pen/Strep, 1% glutamine, 1% sodium pyruvate, 1% MEM NAA, 1% HEPES and 55μM-2-mercaptoethanol) for 4hrs at 37°C, 5%CO 2 .

Techniques:

Journal: bioRxiv

Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

doi: 10.1101/2020.10.27.357426

Figure Lengend Snippet: a. tSNE/FlowSOM analyses of CD8 + T cells from 3-month-old (3mo) mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. b . Heatmap of the manually gated CD8 T cell populations indicated at seven, 14 and 21 days after immunisation in the aortic lymph node and spleen. The frequency of each cell subset in each ChAdOx1 nCoV-19 immunised mouse has been expressed as the log2 fold change over the average frequency in PBS immunised mice (n=5). Crossed boxes indicate that there were none of that cell type for that mouse. Bar charts showing the number of Ki67 + ( c ), effector memory CD44 + CD62L - ( d ), CXCR3 + ( e ) and PD-1 + CD44 + ( f ) CD8 cells in the aortic lymph node of ChAdOx1 nCoV-19 or PBS immunised mice, at the indicated timepoints post immunisation. g . Analysis of cytokine production six hours after PdBu/ionomycin stimulation of aortic lymph node cells from 3-month-old mice seven days after immunization with ChAdOx1 nCoV-19 or PBS. h . Stacked bar plots show the number of CD8 + cells singly or co-producing Granzyme B, IFNγ, IL-2 or TNFα six hours after restimulation with SARS-CoV-2 peptide pools, each bar segment represents the mean and the error bars the standard deviation. In c-f bar height in corresponds to the mean and each circle represents one biological replicate. P-values are calculated using a student’s t-test with Holm-Sidak multiple testing correction.

Article Snippet: For cytokine staining, splenic cells were stimulated with a pool of SARS-CoV-2 spike protein immunodominant domain peptides, (Miltenyi Biotec #130-126-700) at a 0.6 μM concentration (approx.1μg/ml), whilst lymph node cells were stimulated with 0.5μg/ml of Phorbol 12,13 dibutyrate (PdBu, Tocris Bioscience, #), 0.75μg/ml of Ionomycin calcium salt (Tocris Bioscience, #), both in warm complete RPMI (10% FCS, 1% Pen/Strep, 1% glutamine, 1% sodium pyruvate, 1% MEM NAA, 1% HEPES and 55μM-2-mercaptoethanol) for 4hrs at 37°C, 5%CO 2 .

Techniques: Standard Deviation

Journal: bioRxiv

Article Title: A booster dose enhances immunogenicity of the COVID-19 vaccine candidate ChAdOx1 nCoV-19 in aged mice

doi: 10.1101/2020.10.27.357426

Figure Lengend Snippet: Flow cytometric contour plots showing the gating strategy to identify T cell subsets ( a ), cytokine and granzyme B production in CD8 + aortic lymph node T cells six hours after PdBu/Ionomycin restimulation ( b ), and in SARS-CoV-2 peptide restimulated CD8 + splenocytes ( c ).

Article Snippet: For cytokine staining, splenic cells were stimulated with a pool of SARS-CoV-2 spike protein immunodominant domain peptides, (Miltenyi Biotec #130-126-700) at a 0.6 μM concentration (approx.1μg/ml), whilst lymph node cells were stimulated with 0.5μg/ml of Phorbol 12,13 dibutyrate (PdBu, Tocris Bioscience, #), 0.75μg/ml of Ionomycin calcium salt (Tocris Bioscience, #), both in warm complete RPMI (10% FCS, 1% Pen/Strep, 1% glutamine, 1% sodium pyruvate, 1% MEM NAA, 1% HEPES and 55μM-2-mercaptoethanol) for 4hrs at 37°C, 5%CO 2 .

Techniques: