Journal:

Article Title: TGF-?-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

doi: 10.1172/JCI34916

Figure Lengend Snippet: (A) Representative pictures of cluster formation of allogeneic DCs with Tregs derived from either Cd18wt mice or Cd18hypo mice are shown. Original magnification, ×40. (B) Cluster formation between allogeneic DCs and Tregs of different genotypes from Cd18wt mice and Cd18hypo mice was assessed by counting aggregated clusters/HPF in 100 randomly selected HPFs. Cluster formation with allogeneic DCs was substantially reduced for Tregs derived from Cd18hypo mice compared with Tregs from Cd18wt control mice. **P = 0.0029, using Student’s t test. (C) Increased neutralizing mAb against CD18 resulted in decreased proliferative response of specific allogeneic Tregs in MLRs. Numbers on the top left of C and F indicate the percentage of CFSE-labeled proliferating cells. Numbers on the top right of C and F indicate the percentage of undivided CFSE-labeled cells. (D) Increased TGF-β1 expression by Cd18wt Tregs was observed in MLRs. Neutralizing mAb against CD18 in MLRs resulted in a dramatic decrease in TGF-β1 expression compared with isotype-matched control antibody. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of D indicate the percentage of CFSE-labeled proliferating cells. CD4+CD25+CD127– Tregs were purified from 4 pooled spleens of Cd18wt PL/J mice and cocultured with irradiated allogeneic DCs in the presence of 500 units/ml recombinant murine IL-2 and various concentrations of anti-CD18 mAb (E), or anti-mouse CD11a mAb (F), or isotype-matched IgG for 7 days. Tregs were then separated from allogeneic DCs by CD11c MACS beads, extensively washed 3 times with PBS, and mixed at a ratio of 1:4 with Cd18wt Tresp cells. After 3 days of culture, cells were harvested and analyzed by flow cytometry. One representative experiment out of 3 or 4 independent experiments is shown.

Article Snippet: Two hundred and fifty micrograms of TGF-β neutralizing mAb (clone 1D11; BioExpress) or isotype control IgG was injected intraperitoneally into recipient mice 1 day after a transfer of 1 × 10 6 Cd18 wt Tregs for 3 consecutive days.

Techniques: Derivative Assay, Control, Labeling, Expressing, Staining, Purification, Irradiation, Recombinant, Flow Cytometry

Journal:

Article Title: TGF-?-dependent suppressive function of Tregs requires wild-type levels of CD18 in a mouse model of psoriasis

doi: 10.1172/JCI34916

Figure Lengend Snippet: (A) Cryosections from affected Cd18hypo mice were double stained with CD25-FITC and TGF-β1–Cy3 mAbs. Original magnification, ×20. (B) FACS analysis of pooled DLNs from affected Cd18hypo mice using TGF-β1 and CD25 mAbs. (C and D) Seven days after adoptive transfer of MACS-sorted Cd18wt Tregs into affected Cd18hypo mice, skin sections were stained with antibody against TGF-β1 and CD18. The overlay (yellow) of CD18-positive Cd18wt Tregs (green) and TGF-β1–expressing cells (red) indicate that most of the Cd18wt Tregs express TGF-β1 in the skin (C) and skin DLNs (D) after transfer into Cd18hypo mice. The dotted line indicates the border between epidermis and dermis. Three independent experiments were performed in total. (E) A total of 1 × 106 Tregs from either Cd18wt or Cd18hypo mice were labeled with CFSE and adoptively transferred into affected Cd18hypo mice. At day 4 after adoptive Tregs transfer, FACS analysis was performed to measure TGF-β1 expression of CFSE-labeled Cd18wt Tregs (left panel) or Cd18hypo Tregs (right panel) from skin DLNs of affected recipients. Gray region, TGF-β1 expression; white region, normal goat IgG control for TGF-β1 staining. Numbers on the top of B and E indicate the percentage of CFSE-labeled proliferating cells. (F) Following adaptive transfer of 1 × 106 Cd18wt Tregs, 250-μg TGF-β1–neutralizing mAb (left panel) or isotype control IgG (right panel) were injected intraperitoneally into affected Cd18hypo recipients. Repetitive injection of TGF-β neutralizing antibody or isotype control IgG after adoptive transfer of Cd18wt Tregs was performed till the end of treatment (21 days). Original magnification, ×40 (C and D). **P = 0.002, using Student’s t test.

Article Snippet: Two hundred and fifty micrograms of TGF-β neutralizing mAb (clone 1D11; BioExpress) or isotype control IgG was injected intraperitoneally into recipient mice 1 day after a transfer of 1 × 10 6 Cd18 wt Tregs for 3 consecutive days.

Techniques: Staining, Adoptive Transfer Assay, Expressing, Labeling, Control, Injection

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma

doi: 10.3389/fcell.2022.821875

Figure Lengend Snippet: Heatmap showing top 30 differentially expressed genes in high/moderate vs low MUC1 PDA samples from TCGA. (A) Top panel shows the color key for MUC1 expression in the 29 PDA samples. Right hand side shows the color key histogram for expression levels of each gene named on the right. Left hand side color key shows the genes associated with each of the three pathways in pink (TGF-β), green (MAPK) and peach (BMP). Genes with a false discovery rate adjusted p < 0.05 are shown. (B) Kaplan-Meier curve for overall survival (OS) in the 29 PDA patients from TCGA in low (blue) vs high/moderate (red) groups are shown.

Article Snippet: Groups 2 and 4 were treated with the monoclonal TGF-β neutralizing antibody (LifeTech) (20ug/100ul per mouse) three times a week for 2 weeks.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma

doi: 10.3389/fcell.2022.821875

Figure Lengend Snippet: Overexpression of MUC1 leads to increased phosphorylation of JNK and c-Myc and knockdown of MUC1 reduces phosphorylation of JNK and c-Myc. (A) Western blot expression of phosphorylation of JNK and c-Myc compared to total JNK and total c-Myc in MiaPaca2 vs MiaPaca2. MUC1 cells in response to 10 ng/ml of TGF-β at 10 min. (B) Western blot expression of phosphorylation of JNK and c-Myc compared to total JNK and total c-Myc in HPAFII cells treated with control siRNA vs MUC1 siRNA in response to 10 ng/ml of TGF-β at 10 min. (C) Densitometric analysis of fold change of expressions of pJNK/Total JNK and p-c-Myc/Total c-Myc normalized to endogenous β-actin is presented in MiaPaca2 cells. (D) Densitometric analysis of fold change of expressions of pJNK/Total JNK and p-c-Myc/Total c-Myc normalized to endogenous β-actin is presented in HPAFII cells. (E) Knockdown efficiency of MUC1 in HPAFII after 72 h of siRNA treatment. Data are presented as means ± SEM of n = 3; Unpaired Student’s t-test and one-way ANOVA were used to analyze the differences between treatment groups. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Groups 2 and 4 were treated with the monoclonal TGF-β neutralizing antibody (LifeTech) (20ug/100ul per mouse) three times a week for 2 weeks.

Techniques: Over Expression, Western Blot, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma

doi: 10.3389/fcell.2022.821875

Figure Lengend Snippet: TGF-β exposure increases viability in cells with high MUC1 and reduces viability in low MUC1 PDA cells. MTT cell viability assay on (A) MiaPaca2.Neo cells with 10 ng/ml of TGF-β for 48 h. (B) HPAFII and (C) MiaPaca2. MUC1 cells with 10 ng/ml of TGF-β for 72 h. (D) HPAFII treated with control or MUC1 siRNA for 72 h followed by treatment with 10ng/ml of TGF-β for 24 h. All data are shown as means ± SEM of n = 3. Unpaired t-test was performed to compare between treated and untreated cells for each one of experiments A-C and two-way ANOVA was used to compare between untreated and treated in HPAFII.controlsiRNA and HPAFII.MUC1siRNA. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: Groups 2 and 4 were treated with the monoclonal TGF-β neutralizing antibody (LifeTech) (20ug/100ul per mouse) three times a week for 2 weeks.

Techniques: Viability Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma

doi: 10.3389/fcell.2022.821875

Figure Lengend Snippet: TGF-β neutralizing antibody treatment significantly reduced high-MUC1 (HPAFII) but not low MUC1 (MiaPaca2) tumor growth in vivo . (A) A schematic of the xenograft study showing the treatment with control IgG and anti-TGF-β antibody (20 ug/100 ul per mouse). (B) On the left: Tumor growth of HPAFII (n = 5 for TGF-β neutralizing Ab and n = 4 for IgG isotype) is shown. On the right: Tumor growth of MiaPaca2 (n = 6 for both groups) is shown. Tumor growth was determined biweekly by caliper measurements and tumor size in mm 3 is plotted. (C) Wet weight of HPAFII tumors (left) and MiaPaca2 tumors (right) respectively are shown. Two-way ANOVA was used to compare between the different treatment groups. * p <0.05, NS: non-significant. (D) Immunohistochemistry showing expression of MUC1 in MiaPaca2 (left) and HPAFII (right) tumors.

Article Snippet: Groups 2 and 4 were treated with the monoclonal TGF-β neutralizing antibody (LifeTech) (20ug/100ul per mouse) three times a week for 2 weeks.

Techniques: In Vivo, Immunohistochemistry, Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Overexpression of MUC1 Induces Non-Canonical TGF-β Signaling in Pancreatic Ductal Adenocarcinoma

doi: 10.3389/fcell.2022.821875

Figure Lengend Snippet: Schematic diagram of the proposed mechanism of TGF-β signaling and functions in high versus low MUC1 PDA . Left panel shows activation of SMAD-dependent canonical pathway in low-MUC1 PDA cells. TGF-β ligands bind to the membranous TGF-β receptor (TGF-βRII) homodimers with high affinity. TGF-βRII binding allows dimerization with TGF-β type I receptor (TGF-βRI) homodimers, activation of the TGF-βRI kinase domain and signal transduction via phosphorylation of the C-terminus of receptor-regulated SMADs (R-SMAD), SMAD2 and SMAD3. The SMAD2/3 dimer then forms a heterotrimeric complex with SMAD4 which translocates in the nucleus ( ; ). This leads to growth inhibition, cell cycle arrest and apoptosis of PDA cells, thus TGF-β acts as a tumor suppressor. Right panel shows activation of SMAD-independent non-canonical pathway in high-MUC1 PDA cells. In this pathway, binding of TGF-β mainly to TGF-β-RII most likely increases phosphorylation of c-SRC which in turn phosphorylates MAPK, followed by JNK and c-Myc . This phosphorylation cascade activates the MAPK/JNK pathway and stabilizes c-Myc which translocates into the nucleus to increase transcription of oncogenic proteins and leads to increased growth, invasion and EMT of PDA cells . MUC1-CT also aids in the process by its oncogenic signaling. Thus, in high-MUC1 PDA cells TGF-β acts as a pro-tumorigenic cytokine. The schematic was created with BioRender.com .

Article Snippet: Groups 2 and 4 were treated with the monoclonal TGF-β neutralizing antibody (LifeTech) (20ug/100ul per mouse) three times a week for 2 weeks.

Techniques: Activation Assay, Binding Assay, Transduction, Inhibition

Journal:

Article Title: The immunomodulator FTY720 and its phosphorylated derivative activate the Smad signalling cascade and upregulate connective tissue growth factor and collagen type IV expression in renal mesangial cells

doi: 10.1038/sj.bjp.0706452

Figure Lengend Snippet: Effect of a TGF-βR type I kinase inhibitor on FTY720-, phospho-FTY- and TGF-β-stimulated CTGF expression in mesangial cells. Cells were pretreated for 30 min with either vehicle (−) or the indicated concentrations (in μM) of the TGF-βRI inhibitor prior to stimulation with either FTY720 (FTY; 3 μM), phospho-FTY720 (p-FTY; 3 μM) or TGF-β2 (20 ng ml−1) for 4 h. Thereafter, cell lysates were subjected to Western blot analysis using antibodies against CTGF or GAPDH at dilutions of 1 : 1000. Bands corresponding to CTGF were densitometrically evaluated. Results are means±s.d. (n=3), *P<0.05, **P<0.01, considered statistically significant when compared to the stimulated values in the absence of the inhibitor.

Article Snippet: FTY720, phospho-FTY720 and TGF- β 2 were kindly donated by Novartis Pharma Ltd, Basel, Switzerland; phospho-specific antibodies against p42/p44-MAPK, JNK, p38-MAPK, phospho-Smad-1(Ser 463/465 ) and phospho-Smad-2(Ser 465/467 ) and total Smad-2 were from Cell signalling, Frankfurt am Main, Germany; the TGF- β RI kinase inhibitor and the total polyclonal anti-human Smad-1 antibody (directed against amino-acid residues 147–258 of human Smad-1) were from Merck Biosciences, Schwalbach, Germany; the total Smad-3 antibody (FL-425) and the collagen type IV (H-234) antibody were from Santa Cruz, Heidelberg, Germany; the neutralizing pan-specific TGF- β antibody and the TGF- β ELISA were from Biosource Deutschland GmbH, Solingen, Germany; [ 35 S]methionine/cysteine pro-mixture (specific activity >1000 Ci mmol −1 ), [ α - 32 P]dCTP (specific activity 3000 Ci mmol −1 ), protein A sepharose 4B, anti-rabbit and anti-mouse horseradish peroxidase-linked IgGs and Hyperfilm were purchased from Amersham Pharmacia Biotech Europe GmbH, Freiburg, Germany; all cell culture nutrients were from Gibco, Karlsruhe, Germany.

Techniques: Expressing, Western Blot

Journal: Scientific Reports

Article Title: Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis

doi: 10.1038/s41598-021-84023-0

Figure Lengend Snippet: Cell sorting strategy, phenotypic and functional characterization of CpG-induced c-kit + Sca-1 + B220 int PDCA-1 + BM cells. ( a ) Total BM cells incubated with PBS or CpG-B (1 µg/ml) for 18 h and magnetically enriched for c-kit + cells were further labelled for Sca-1, B220, PDCA-1 and electronically sorted into c-kit + Sca-1 + B220 int PDCA-1 + cells. Flow cytometry dot plots representative of 30 experiments. ( b ) Further characterization of cell-sorted CpG- as well as PBS-induced BM population was performed using flow cytometry analysis of the expression of various myeloid and dendritic cell markers as well as cell migration receptors. Specific antibody staining is depicted with open histograms (blue line for PBS- and red line for CpG-induced cells). Positive cells are defined using FMO controls (grey histograms). Two experiments. ( c ) FACS analysis of intranuclear expression of transcription factors. Analysis of IRF8 and Id2 expression with specific antibodies compared to FMO controls and of E2.2 and PU.1 by flowRNA, compared with B220 + PDCA-1 − control B cells (grey histograms), in CpG- versus PBS-stimulated c-kit + Sca-1 + B220 int PDCA-1 + population. Two experiments. ( d ) Frequency of c-kit + Sca-1 + B220 int PDCA-1 + cells emerging among total BM cells after 18 h of incubation with different TLR agonists, measured in BM cell cultures of four individual mice. Statistical significance as indicated, analyzed by Mann–Whitney test. ( e ) Cytokine production of cell-sorted PBS- versus CpG-induced c-kit + Sca-1 + B220 int PDCA-1 + cells, measured in the supernatant after 4 h of activation with PMA/ionomycin in the presence of brefeldin A using multiplex ELISA. Biological duplicates. ( f ) Intracytoplasmic TGF-β expression was analyzed by FACS in PBS- and CpG-pre-pDCs. One experiment out of two ( g ) IFN-α production was measured by ELISA in supernatants of CpG- versus PBS-induced cell-sorted c-kit + Sca-1 + B220 int PDCA-1 + BM cells after 18 h incubation with 1 μg/ml TLR-7 (R848), TLR-9 (CpG-B and CpG-P) and TLR-4 (LPS) agonists (biological duplicates). ( h ) Mature spleen pDCs and CpG-pre-pDCs were compared for their IFN-α response after 18-h incubation with CpG-B (1 μg/ml), measured by ELISA in supernatants (biological duplicates).

Article Snippet: 0.1 mg Chariot (Active Motif, La Hupe, Belgium) was combined with 50 μg of either LEAF purified anti-TGF-β neutralizing antibody (clone 2D11.16.8) (BioXCell, West Lebanon, NH) or IgG1 isotype control (Biolegend) according to the manufacturer’s instructions.

Techniques: FACS, Functional Assay, Incubation, Flow Cytometry, Expressing, Migration, Staining, Control, MANN-WHITNEY, Activation Assay, Multiplex Assay, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis

doi: 10.1038/s41598-021-84023-0

Figure Lengend Snippet: Role of TGF-β in CpG-pre-pDC mediated protection against EAE. ( a ) CpG-pre-pDCs were transfected with a neutralizing anti-TGF-β antibody or control isotype antibody using the Chariot protein transfection vector. 80,000 of resulting cells were injected i.v. to MOG 35-55 immunized mice at d-12 after immunization. Clinical score (mean ± s.e.m.) was assessed until d-27, n = 10 mice in control and isotype antibody progenitor-treated groups and n = 9 mice in the anti-TGF-β-progenitor-treated group. Statistical analysis was performed using two-way ANOVA with Bonferroni post-test: controls vs CpG-prepDC + isotype, d-19, * p = 0.0352; controls vs CpG-prep-DC + anti-TGF-β, d-19, *, p = 0.0144, d20–d22, *** p < 0.0025, d27 *, p = 0.0352; CpG-pre-pDCs + isotype vs CpG-pre-pDCs + anti-TGF-β, d17–18, *, p = 0.0119, d19–d27, ****, p < 0.0001. ( b ) CD4 + T-cells isolated at d-27 from the spinal cord of controls or recipients of isotype or neutralizing Ab-transfected CpG-pre-pDCs were analyzed by FACS using FMO controls for their cytokine production after 4 h activation with PMA/ionomycin in presence of brefeldin. Mean ± s.e.m. of percentages of cells expressing a given cytokine, n = 5 mice per group. Statistical analysis was performed using two-way ANOVA with Bonferroni post-tests, for GM-CSF: Controls vs CpG-pre-pDCs + isotype Ab, NS, Controls vs CpG-pre-pDCs + anti-TGF-β, **, p = 0.0032, CpG-pre-pDCs + isotype vs + anti-TGF-β, **, p = 0.008, *; for TNF-α, CpG-pre-pDCs + isotype vs + anti-TGF-β, **, p = 0.0015. ( c , d ) Frequency ( c ) and counts ( d ) of B220 + PDCA-1 − B-cells demonstrating IL-10 production capacity (mean ± s.e.m., n = 5 mice per group, * p = 0.01, **, p = 0.0015, by one-way ANOVA with Bonferroni post-tests). ( e , f ) CD4 + Foxp3 + Tregs frequency ( e ) and cell counts ( f ), (mean ± s.e.m., n = 5 mice per group), N.S. by analysis using Kruskal–Wallis with Dunn’s post-tests.

Article Snippet: 0.1 mg Chariot (Active Motif, La Hupe, Belgium) was combined with 50 μg of either LEAF purified anti-TGF-β neutralizing antibody (clone 2D11.16.8) (BioXCell, West Lebanon, NH) or IgG1 isotype control (Biolegend) according to the manufacturer’s instructions.

Techniques: Transfection, Control, Plasmid Preparation, Injection, Isolation, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Toll-like receptor-9 stimulated plasmacytoid dendritic cell precursors suppress autoimmune neuroinflammation in a murine model of multiple sclerosis

doi: 10.1038/s41598-021-84023-0

Figure Lengend Snippet: Graphical representation of cellular and molecular mechanisms of the protective effect of CpG-pre-pDCs against EAE. Upon adoptive transfer of pDC progenitors at d-12, the onset of clinical signs, TGF-β released only by CpG-pre-pDCs in the first 3 days in the spinal cord (light blue zone) of mice protects against EAE by prompting host pDCs to release in turn TGF-β and promoting accumulation of CD11c + CD11b + cDCs and IL-10 + B cells. CpG-pre-pDCs, once migrated to the spinal cord, additionally release IL-27 that from day-22 onwards, ensures late protection against EAE. BBB blood brain barrier. The figure was created using Biorender.com.

Article Snippet: 0.1 mg Chariot (Active Motif, La Hupe, Belgium) was combined with 50 μg of either LEAF purified anti-TGF-β neutralizing antibody (clone 2D11.16.8) (BioXCell, West Lebanon, NH) or IgG1 isotype control (Biolegend) according to the manufacturer’s instructions.

Techniques: Adoptive Transfer Assay

Journal: iScience

Article Title: Mitoribosome Defect in Hepatocellular Carcinoma Promotes an Aggressive Phenotype with Suppressed Immune Reaction

doi: 10.1016/j.isci.2020.101247

Figure Lengend Snippet: Identification of Molecular Features Linked to Mitochondrial Defects Based on the MDS dn , TCGA-LIHC samples were stratified into the subgroup with higher mitoribosome defect (H-MD) and one with lower mitoribosome defect (L-MD), and molecular features associated with H-MD or L-MD were compared. (A–C) GSEA results based on the OXPHOS (A), mitochondria respiratory chain complex assembly (B), and TGF-β signaling (C) were shown. Normalized enrichment scores (NES) and FDR for each gene set are noted. (D) Overall survival time of H-MD and L-MD was compared based on the Kaplan-Meier survival analysis. (E) Volcano plot indicates fold change (FC) and FDR based on the permutation t test between H-MD and L-MD groups. Differentially expressed genes (DEGs) were marked with red- or blue-colored points (FC > 1 or < −1 & FDR <0.005, respectively). (F) The expression of upregulated (n = 83) or downregulated (n = 161) DEGs in either H-MD or L-MD is shown. Samples are represented in columns, grouped by H-MD or L-MD. (G) Enrichment plots based on the 83 upregulated genes and 161 downregulated genes are shown in the left and right panels, respectively. NES and FDR for each gene set are noted. (H) Gene ontology (GO) analysis was performed based on the up and down DEGs. The -log 10 (p value) is shown in red and blue bars for up- and downregulated genes, respectively. See also ; and .

Article Snippet: Representative images are shown in the right panel. (G) Cell growth rates were monitored by counting the trypan blue-negative viable cells using the Countess automated cell counter. (H–K) TGF-β signaling in H-MD-type HCC cell lines (SNU475 and JHH4) was abolished by exposure to various concentrations of neutralizing antibodies for TGF-β (GTX14052, GeneTex Inc., Irvine, CA) for 24 h. Normal mouse IgG (sc-2025, Santa Cruz Biotechnology, Inc., Dallas, TX) was used as control.

Techniques: Expressing

Journal: iScience

Article Title: Mitoribosome Defect in Hepatocellular Carcinoma Promotes an Aggressive Phenotype with Suppressed Immune Reaction

doi: 10.1016/j.isci.2020.101247

Figure Lengend Snippet: TBRS Induced by MRP Defects Mediates an Aggressive Phenotype in HCC (A and B) The association of mitoribosomal defects in HCC with TBRS was recapitulated in HCC cell lines. For TBRS in HCC cell line, signatures of epithelial cells (Epi_TBRS) were used. The MDS dn shows a significant correlation with Epi_TBRS (A). NES based on Epi_TBRS was compared between H-MD- and L-MD-type HCC cell lines (B). (C) MDS dn shows differential associations with HCC sub-classification signatures (Hoshida_S1, S2, and S3). (D and E) The association of mitoribosomal defect with cancer cell invasiveness was examined. preRanked GSEA was performed based on the invasion signature defined by Anastassiou et al. MDS dn shows a significant association with cancer cell invasiveness (D). NES based on cancer cell invasiveness signature (Anastassiou et al.) was compared between H-MD and L-MD-type HCC cell lines (E). (F) Cell invasion activity was assayed using Matrigel-coated Transwell as described in the section. Representative images are shown in the right panel. (G) Cell growth rates were monitored by counting the trypan blue-negative viable cells using the Countess automated cell counter. (H–K) TGF-β signaling in H-MD-type HCC cell lines (SNU475 and JHH4) was abolished by exposure to various concentrations of neutralizing antibodies for TGF-β (GTX14052, GeneTex Inc., Irvine, CA) for 24 h. Normal mouse IgG (sc-2025, Santa Cruz Biotechnology, Inc., Dallas, TX) was used as control. Phosphorylated SMAD2 (P-smad2) and total SMAD2 (T-smad2) levels were examined by western blot to validate the extent of TGF-β signaling inhibition (H and I). Then, the cell invasion assay was performed (J and K). Representative images for invaded cells are shown in the right panels. Boxplots are shown as first quartile, median, and third quartile (bottom box, middle line, and top box, respectively) with Welch two-sample t test p values. Whiskers represent the minimum and maximum values. Bar plots are represented as mean ± SEM. ∗ p<0.05, ∗∗ p<0.01, ∗∗∗ p<0.005 (Student’s t-test, DOX treated group vs. non-treated group). See also Figure S6 ; .

Article Snippet: Representative images are shown in the right panel. (G) Cell growth rates were monitored by counting the trypan blue-negative viable cells using the Countess automated cell counter. (H–K) TGF-β signaling in H-MD-type HCC cell lines (SNU475 and JHH4) was abolished by exposure to various concentrations of neutralizing antibodies for TGF-β (GTX14052, GeneTex Inc., Irvine, CA) for 24 h. Normal mouse IgG (sc-2025, Santa Cruz Biotechnology, Inc., Dallas, TX) was used as control.

Techniques: Activity Assay, Control, Western Blot, Inhibition, Invasion Assay

Journal: Cell Reports Medicine

Article Title: Tumor-infiltrated double-negative regulatory T cells predict outcome of T cell-based immunotherapy in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102096

Figure Lengend Snippet: Study design and single-cell transcriptomic landscape of TIL infusion products (A) Graphical overview of the experimental design and bioinformatics workflow. In our clinical trial cohort, bulk RNA sequencing was performed on tumor tissue from NPC patients before CCRT+TIL ( n = 22) or CCRT alone ( n = 40), and TIL infusion products were isolated from pretreatment tumor tissue from these patients, expanded ex vivo , and subsequently prepared for single-cell (sc)RNA sequencing ( n = 26) and flow cytometry ( n = 47). (B) Uniform manifold approximation projection (UMAP) plot of 65,978 quality-controlled T cells colored by 14 T cell subsets with the distinct G1 phase, S phase, and G2M phase. The cell-cycle phase of 14 T cell subsets was labeled in the UMAP plot and determined by the cell-cycle phase scores calculated by the Seurat package. (C) Heatmap showing the expression of the top 50 differentially expressed genes among cell subsets obtained by cell type annotation utilizing the expression of canonical marker genes in 14 cell subgroups. Information on the 14 cell subsets is displayed on the right. (D) UMAP plots showing the distribution of indicative T cell subsets between the non-progression group and progression group. (E) Dot plot showing the composition of CD3 + CD4 − CD8 − , CD3 + CD4 + , and CD3 + CD8 + T cells in NPC TIL infusion products according to flow cytometry ( n = 47). Scatter dot plots show individual values and mean ± SEM. (F) Bar plot comparing the frequency of 14 cell subgroups in the TIL infusion products from NPC patients in the non-progression ( n = 15) and progression groups ( n = 11). The data are presented as mean ± SEM. The Wilcoxon rank-sum test was used to determine the significance. ∗ p < 0.05; ns, not significant; adjusted for multiple comparisons using the Benjamini-Hochberg procedure. (G) Boxplot comparing the frequency of (DN) TILs in the non-progression ( n = 28) and progression groups ( n = 19) according to flow cytometry. Median and interquartile were shown in the boxplots. Wilcoxon rank-sum test was used to determine the significance. (H) Kaplan-Meier survival curves for progression-free survival (PFS) and overall survival (OS) in NPC patients stratified according to the abundance (high abundance vs. low abundance) of the C8_DN_ T_cells cluster inferred from (sc)RNA sequencing data ( n = 26). p values were determined based on the two-sided log rank test. Optimal cutoff values were defined using the “survminer” R package. See also and .

Article Snippet: To evaluate the suppression mechanism of DN TILs, neutralizing antibodies targeting TGF-β (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHB215) and IL-10 (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHA915) were added, along with a Fas-FasL signaling antagonist (10 μM; MedChemExpress, HY- P10102 ) and GITR inhibitor (20 nM; KKL Med Inc., KM2426) at an effector-to-target (E:T) ratio of 1:1.

Techniques: RNA Sequencing, Isolation, Ex Vivo, Flow Cytometry, Labeling, Expressing, Marker

Journal: Cell Reports Medicine

Article Title: Tumor-infiltrated double-negative regulatory T cells predict outcome of T cell-based immunotherapy in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102096

Figure Lengend Snippet: Transcriptional characterization and developmental trajectory of CD3 + CD8 − CD4 − (DN) TILs (A) Heatmap showing the expression levels of genes encoding NK cell markers, Treg-related molecules, immune checkpoint proteins, cytokines, and (DN) T cell-related markers among CD8 + , CD4 + , and (DN) TIL clusters. (B) Gene set enrichment analysis (GSEA) showing significant upregulation of TGF-β (left) and IL-10 (right) signaling pathways in (DN) TILs compared with other TIL subsets. (C) Violin plot showing the expression of a regulatory T cell signature across (DN) TIL, CD4 + TIL, and CD8 + TIL clusters. A dashed line indicates the median of the signature score of the (DN) TIL cluster. Median and interquartile were shown in the boxplots. Non-parametric Kruskal-Wallis test was used to determine the significance. (D) Representative overlap histogram for the expression of IL-10, TGF-β, IKZF2, FOXP3, and CTLA4 among (DN) TILs, CD4 + TILs, and CD8 + TILs determined by flow cytometry. (E) Developmental trajectory of 16,978 (DN) T cells from tumor tissues and peripheral blood of naive treated NPC patients ( n = 10) inferred by Monocle 2 and CytoTRACE algorithm. Solid and dotted lines denote distinct cell fates based on expression profiles, with colors indicating the origin of (DN) T cells, pseudo-time, and CytoTRACE score (GEO: GSE162025 ). (F) Dot plot showing the correlation between (DN) TIL signature scores of TMEs in this study and each state of (DN) T cells (GEO: GSE162025 ; PB, Cell Fate_1, and Cell Fate_2). The sizes and colors of the circles represent the strength of the relationship, assessed using Spearman’s correlation test. PB, peripheral blood. (G) Representative overlap histogram for the expression of IL-10, TGF-β, IKZF2, FOXP3, and CTLA4 in the (DN) T cells from TIL infusion products, peripheral blood of NPC patients, or healthy donors determined by flow cytometry. See also and .

Article Snippet: To evaluate the suppression mechanism of DN TILs, neutralizing antibodies targeting TGF-β (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHB215) and IL-10 (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHA915) were added, along with a Fas-FasL signaling antagonist (10 μM; MedChemExpress, HY- P10102 ) and GITR inhibitor (20 nM; KKL Med Inc., KM2426) at an effector-to-target (E:T) ratio of 1:1.

Techniques: Expressing, Protein-Protein interactions, Flow Cytometry

Journal: Cell Reports Medicine

Article Title: Tumor-infiltrated double-negative regulatory T cells predict outcome of T cell-based immunotherapy in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102096

Figure Lengend Snippet: Transcriptomic characterization and association of CD56 − (DN) TILs with clinical outcome (A) UMAP plot depicting 2,609 quality-controlled (DN) TILs colored by six cell subsets. (B) UMAP plot of (DN) TILs, with cells colored based on the relative normalized expression of NCAM1 (encoding CD56), FCGR3A (encoding CD16), and IKZF2 . (C) Volcano plot showcasing differentially expressed genes (DEGs) between CD56 − (DN) TILs and CD56 + (DN) TILs. Red dots and blue dots indicate up-regulated genes ( n = 770) and down-regulated genes ( n = 316) in CD56 − (DN) TILs, respectively. Selected genes were labeled. (D) Heatmap showing the expression of genes encoding TCR proteins, NK cell markers, immune checkpoint proteins, cytokines, and (DN) T cell-related markers among CD56 − and CD56 + (DN) TIL clusters. (E) GSEA plots showing that Treg signaling pathways were significantly up-regulated in CD56 − (DN) TILs compared with CD56 + (DN) TILs. (F) Violin plot showing the expression of an activated DN Treg signature across four (DN) TIL clusters (CD56 − IKZF2 + , CD56 − IKZF2 - , CD56 + IKZF2 - , and CD56 + IKZF2 + (DN) TILs). A dashed line indicates the median of the signature score of the CD56 − IKZF2 + (DN) TIL. Median and interquartile were shown in the boxplots. Kruskal-Wallis test was used to determine the significance. (G) Kaplan-Meier survival curves for PFS (top) and OS (bottom) in NPC patients stratified according to the abundance (high abundance vs. low abundance) of CD56 − IKZF2 + (DN) TILs inferred from (sc)RNA sequencing data ( n = 26). p values were determined based on the two-sided log rank test. See also .

Article Snippet: To evaluate the suppression mechanism of DN TILs, neutralizing antibodies targeting TGF-β (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHB215) and IL-10 (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHA915) were added, along with a Fas-FasL signaling antagonist (10 μM; MedChemExpress, HY- P10102 ) and GITR inhibitor (20 nM; KKL Med Inc., KM2426) at an effector-to-target (E:T) ratio of 1:1.

Techniques: Expressing, Labeling, Protein-Protein interactions, RNA Sequencing

Journal: Cell Reports Medicine

Article Title: Tumor-infiltrated double-negative regulatory T cells predict outcome of T cell-based immunotherapy in nasopharyngeal carcinoma

doi: 10.1016/j.xcrm.2025.102096

Figure Lengend Snippet: Associations of CD8 + TIL subsets with (DN) TILs and clinical outcome (A) Dot color represents the communication probability of the specific ligand-receptor pairs, including TGF-β, IL-10, and Fas-FasL signaling between the indicated sender cluster and receiver clusters. (B) Representative histograms and statistical graph showing carboxyfluorescein succinimidyl ester (CFSE) dilution (left) and proliferation inhibition rates (right) of (DN) TILs with CD4 + and CD8 + naive T cells at a ratio of 4:1, 2:1, 1:1, and 1:2. The data are presented as mean ± SD, and three biological replicates were included. (C) Representative histograms and statistical graph showing CFSE dilution (left) and proliferation inhibition rates (right) of (DN) TILs with CD4 + and CD8 + naive T cells at a 1:1 ratio with neutralizing antibodies against TGF-β and IL-10, as well as the presence or absence of a Fas-FasL signaling antagonist. The data are presented as mean ± SD, and three biological replicates were included. p values were evaluated by t test (two-sided). ∗∗ p < 0.01; ∗∗∗ p < 0.001. (D) Bubble plot showing the correlation between the frequency of (DN) TILs and other TIL subsets in TIL infusion products ( n = 26). p values were determined by Spearman correlation analysis. ∗ p < 0.05. (E) Dot plot showing the correlation between the frequency of (DN) TILs and CD8 + TILs in TIL infusion products ( n = 26). p values were determined by Spearman correlation analysis. (F) Representative histograms and statistical graph showing CFSE dilution (left) and proliferation inhibition rates (right) of (DN) TILs with autologous expanded CD8 + TILs at ratios of 4:1, 2:1, and 1:1. Conventional CD4 + (i)Tregs were included as a control. The data are presented as mean ± SD, and three biological replicates were included. (G–K) Kaplan-Meier survival curves of PFS and OS in NPC patients stratified according to the abundance (high vs. low) of total CD8 + TILs (G), C2_proliferation_CD8_T_cells-PCNA (H), C4_MHC_II_CD8_T_cells (I), C6_proliferation_CD8_T_cells-TOP2A (J), and C12_tissue_resident_memory_CD8_T_cells (K) inferred from (sc)RNA sequencing data ( n = 26). p values were determined based on the two-sided log rank test. See also and .

Article Snippet: To evaluate the suppression mechanism of DN TILs, neutralizing antibodies targeting TGF-β (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHB215) and IL-10 (5 μg/mL; Sanyou Biopharmaceuticals Co. Ltd, CHA915) were added, along with a Fas-FasL signaling antagonist (10 μM; MedChemExpress, HY- P10102 ) and GITR inhibitor (20 nM; KKL Med Inc., KM2426) at an effector-to-target (E:T) ratio of 1:1.

Techniques: Inhibition, Control, RNA Sequencing