Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition

doi: 10.1136/jitc-2023-006677

Figure Lengend Snippet: 8B and 9G glioblastoma cells differently affected macrophages (Mφs). (A) One thousand 8B or 9G cells were transplanted into C57BL/6 mouse brain (8B (red solid line) n=30, 9G (blue solid line) n=27) or NOD/SCID mouse brain (8B (red dotted line) n=5, 9G (blue dotted line) n=5); then, mouse survival was observed. Statistical analyses were performed using the log-rank test with Bonferroni correction. The p values<0.05/6=0.0083 (Bonferroni correction) were considered as statistically significant. NS, not significant. (B) Proliferation of immune cells in co-culture with 8B or 9G cells. Whole splenocytes from C57BL/6 mice were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and then co-cultured with 8B or 9G for 5 days. The resulting cells were stained with the individual immune cell marker indicated in the plots; CFSE reduction in each immune cell was flow cytometrically measured. The proliferation immune cell in 8B-co-cultured cells is shown in red histograms; 9G-co-cultured cells are shown in blue histograms. The percentage of proliferated immune cell in 8B-co-cultured cells is shown in red numbers; corresponding result of 9G-co-cultured cells are shown in blue numbers. (C) 8B and 9G cells were co-cultured with whole splenic cells for 4 days. The percentage of Ki67-positive cells among CD11b, F4/80, B220, and CD3 positive cells was determined by flow cytometry analysis. The bars indicate the mean (±SD). Each dot represents the Ki-67 positive percentage in each sample. *p<0.05, Student’s t-test. (D) Phase contrast images of generated Mφs by co-culture of splenic F4/80 + cells and 8B or 9G supernatant. Bars; 50 µm. (E) Whole splenocytes were cultured in 8B supernatant or 9G supernatant for 11 days. Generated cells were analyzed by flow cytometry. Forward scatter and side scatter of generated CD11b + cells are shown. Red lines show 8B-cultured cells; blue lines show 9G-cultured cells. (F and G) Morphological analysis of generated Mφs that proliferated in response to 8B (F) or 9G (G) supernatants. CFSE-labeled splenocytes were co-cultured with 8B or 9G for 4 days. Quiescent or proliferated CD11b + cells were sorted based on CFSE fluorescence reduction. Proliferated CD11b + cells were further subdivided into Ly6c-positive or Ly6c-negative fractions. Each fractionalized cell type was stained using a Diff-quick stain. Bars; 10 µm.

Article Snippet: Whole splenocytes from C57BL/6 mice were stained with 5-(and −6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Dojindo, Kumamoto, Japan) and then co-cultured with 8B or 9G for 4 days.

Techniques: Co-Culture Assay, Staining, Cell Culture, Marker, Flow Cytometry, Generated, Labeling, Fluorescence, Diff-Quik

Journal: Journal for Immunotherapy of Cancer

Article Title: Tumor cell-induced macrophage senescence plays a pivotal role in tumor initiation followed by stable growth in immunocompetent condition

doi: 10.1136/jitc-2023-006677

Figure Lengend Snippet: Supplementation of nicotinamide mononucleotide (NMN) inhibited macrophages (Mφs) to go into a senescence-like state, induced expression of an immunosuppressive phenotype and improved survival of immunocompetent mice after lethal inoculation of tumor cells. We screened expressed surface proteins on 8B-sup cultured senescence-like Mφs. (A) 8B-supernatant-cultured or 9G-supernatant-cultured Mφs were stained with anti-CD38 monoclonal antibody (mAb). (B) Mouse brains that were given 8B or 9G tumor cells 7 days earlier were stained with F4/80, CD38, and DAPI. The bar graph shows the proportion of CD38 positive area in the F4/80 positive area. Ten independent regions were analyzed, and the means were compared using unpaired Student’s t-test. ***p<0.001. Each dot represents the percentage of CD38 positive area in F4/80 + area in each sample. Bars; 100 µm. (C) Splenic F4/80 + cells were cultured in 8B-supernatant with/without NMN for 14 days followed by a senescence-associated β-galactosidase (SA-β-Gal) assay. The left image is a phase-contrast microscopy view, while the right is a bright-field view. Black arrowheads indicate SA-β-Gal + cells. SA-β-Gal positive and negative cells in 10 different views were counted; the proportion of positive cells was calculated from the total number. The bars indicate the mean (±SD) of percentage SA-β-Gal positive cells from three independent cultures. Each dot represents the percentage β-Galactosidase + cells in each independent culture. **p<0.01, Student’s t-test. (D) Arginase-1 expression of the generated cells in B. The bars indicate the mean (±SD) from three independent flow cytometry analyses. Each dot represents the percentage of Arginase-1 + cells in each independent culture. **p<0.01, Student’s t-test. (E) Assessment of NMN treatment on the immune activating capacity of 8B-Mφs. The allogeneic T cell activating capacity of Mφs were evaluated by carboxyfluorescein diacetate succinimidyl ester (CFSE) assay. 8B-Mφs were induced with/without NMN. Macrophage colony stimulating factor (M-CSF) induced Mφ was used as a positive control. The Mφs and CFSE-labeled Thy1 + T cells from BALB/c were co-cultured for 4 days; then stained with CD4 and CD8, their proliferation was analyzed by CFSE reduction. The bars indicate the mean (±SD) from four independent flow cytometry analyses. Each dot represents the percentage of proliferated cells in each culture. **p<0.01, NS: not significant, Tukey-Kramer honest significant difference test. (F) 8B cells (1×10 4 ) were transplanted into the C57BL/6 mouse brain. NMN or saline was inoculated twice per week. Mouse survival was observed. The dark blue line shows NMN-treated mice (n=6); the gray line shows saline-treated mice (n=6). *p<0.05, log-rank test.

Article Snippet: Whole splenocytes from C57BL/6 mice were stained with 5-(and −6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Dojindo, Kumamoto, Japan) and then co-cultured with 8B or 9G for 4 days.

Techniques: Expressing, Cell Culture, Staining, Microscopy, Generated, Flow Cytometry, CFSE Assay, Positive Control, Labeling, Saline

Journal: Scientific Reports

Article Title: A lncRNA landscape in breast cancer reveals a potential role for AC009283.1 in proliferation and apoptosis in HER2-enriched subtype

doi: 10.1038/s41598-020-69905-z

Figure Lengend Snippet: ( A ) Real-time qPCR illustrating gene silencing by shRNA against AC009283.1 in SKBR3, a HER2-enriched cell line model. ( B ) Supervised hierarchical cluster analysis shows 158 differentially expressed genes after AC009283.1 knockdown (Foldchange < − 1.2, > 1.2 p-value < 0.05); we observed 94 up-regulated and 64 down-regulated genes in shRNA-2 vs NC and ( C ). Their distribution across the genome. ( D ) Ingenuity Pathway Analysis of 158 genes differentially expressed after knockdown of AC009283.1 in SKBR3 cells. ( E ) Enrichment pathway analysis of genes differently expressed in samples of HER2-enriched tumors from TCGA with high vs low expression of AC009283.1.

Article Snippet: Human BRCA cell lines included in the analyses (MCF7, ZR-75-1, MDA-MB-361, SKBR3, MDA-MB-468, HCC1187, HS578T, MDA-MB-231, MDA-MB-453) and non-tumorigenic epithelial cell line MCF10A, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: shRNA, Knockdown, Expressing

Journal: Scientific Reports

Article Title: A lncRNA landscape in breast cancer reveals a potential role for AC009283.1 in proliferation and apoptosis in HER2-enriched subtype

doi: 10.1038/s41598-020-69905-z

Figure Lengend Snippet: ( A ) Proliferation assay performed following knockdown of AC009283.1 with shRNA2, shRNA NC as negative control, and wildtype cell line. Cells were counted every day for four days. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing with shRNA NC for each condition via Student’s t test. ( B ) Proliferation index using CFSE assay in SKBR3 cells with shRNA2 AC009283.1 and shRNA NC. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing with shRNA NC for each condition via Student’s t test. ( C ) Representative CFSE flow-cytometry histograms showing the effect on in vitro SKBR-3 cell proliferation with shRNA NC and shRNA2 (AC009283.1 knock-down). ( D ) Cell cycle assay with flow-cytometry using (propidium iodide) PI on in vitro SKBR-3 cells, AC009283.1 knockdown causes accumulation of cells at S phase. *p < 0.05, **p < 0.005, ***p < 0.0005, comparing to shRNA NC for each condition via Student’s t test.

Article Snippet: Human BRCA cell lines included in the analyses (MCF7, ZR-75-1, MDA-MB-361, SKBR3, MDA-MB-468, HCC1187, HS578T, MDA-MB-231, MDA-MB-453) and non-tumorigenic epithelial cell line MCF10A, were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Proliferation Assay, Knockdown, shRNA, Negative Control, CFSE Assay, Flow Cytometry, In Vitro, Cell Cycle Assay

Journal:

Article Title: Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity

doi: 10.1111/j.1365-2567.2004.01821.x

Figure Lengend Snippet: (a) In vitro proliferation of carboxy fluorescein diacetate succinimidyl ester (CFSE)-labelled F5 T cells in response to cross-presentation of Δ.2-Rag−/−, 48-Rag−/− and Rag−/− pituitary extracts by bone marrow-derived H-2b dendritic cells (DCs). (b) In vitro proliferation of CFSE-labelled F5 T cells incubated with DCs and 1 nm ASNENMDAM peptide. (c) Semi-quantitative PCR analysis of influenza nucleoprotein A/NT/60/68 (NP) transgene expression. RNA was extracted from the pituitaries of 48-Rag−/− and D.2-Rag−/− mice and reverse transcribed, as described. Dilutions of cDNA were amplified with primers specific to NP and growth hormone (GH). The GH semiquantitative PCR was used to control for the concentration of cDNA in each sample.

Article Snippet: F5 lymph node T cells were incubated at 37° for 10 min at a cell density of 10 7 /ml with 1 μ m carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in phosphate-buffered saline (PBS) and washed thoroughly.

Techniques: In Vitro, Derivative Assay, Incubation, Real-time Polymerase Chain Reaction, Expressing, Reverse Transcription, Amplification, Control, Concentration Assay

Journal:

Article Title: Factors affecting the susceptibility of the mouse pituitary gland to CD8 T-cell-mediated autoimmunity

doi: 10.1111/j.1365-2567.2004.01821.x

Figure Lengend Snippet: Characteristics of double-transgenic Δ.2-Rag−/− F5 mice. (a) Growth of male Δ.2-Rag−/− F5 (•) mice compared with F5 (○) and 48-Rag−/− F5 (×) mice. Growth data for the 48-Rag−/− F5 mice have been previously published20 and are included here for comparison only. (b) Pituitary GH levels in individual 16-week-old male Δ.2-Rag−/− F5 (•; n = 4) and Rag−/− F5 (○; n = 6) mice. The mean value for each group is indicated by a horizontal line. (c) High CD44 expression in pituitary-infiltrating CD8+ T-cell receptor (TCR)+ cells isolated from a 12-week-old Δ.2-Rag−/− F5 mouse. (d) In vivo proliferation of transferred, CFSE-labelled F5 T cells in the cervical lymph nodes of B10, 48-B10 and Δ.2-B10 mice of various ages. B10 mice (n = 5), 48-B10 (13 days old; n = 8) and Δ.2-B10 mice (14 days old, n = 7; 23 days old, n = 5; and > 35 days old, n = 10) were injected intravenously (i.v.) with 10 × 106 T cells. The percentage of transferred T cells that had undergone one or more divisions was determined by fluorescence-activated cell sorter (FACS) analysis, 4 days after cell transfer. Numbers indicate the proportion of mice in each group in which the F5 T-cell proliferation was observed.

Article Snippet: F5 lymph node T cells were incubated at 37° for 10 min at a cell density of 10 7 /ml with 1 μ m carboxy fluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes, Eugene, OR) in phosphate-buffered saline (PBS) and washed thoroughly.

Techniques: Transgenic Assay, Comparison, Expressing, Isolation, In Vivo, Injection, Fluorescence

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 3. Fn facilitates anti-PD-1 efficacy by systematically activating CD8+ TILs in germ-free immunohumanized mice bearing subcutane- ous MSS CRC xenografts and SPF mice bearing orthotopic MSS CRC allografts (A) Model design for germ-free, CD34+-humanized mice bearing subcutaneous HT29 xenografts and experimental groups (n = 6/group). Mice received Fn, Streptococcus mutans UA159 (UA159), or PBS by gavage 3 times a week. Related results can be found in B–F. (B) Engraftment of human immune cells in CD34+-humanized mice were evaluated 14 weeks post implantation (n = 6/group). (C) Representative tumor morphology, weight, and volume (n = 6/group). Red circles indicate the location of subcutaneous tumors. (D) Ki-67, PCNA, and TUNEL staining of HT29 xenografts (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti-PCNA, or TUNEL kit. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. (E) CD8 and PD-1 co-immunofluorescence staining (n = 4/group) and PD-1 IHC in HT29 xenografts (n = 6/group). Tumor tissues were stained using DAPI, Alexa Fluor 647 anti-human CD8, anti-mouse PD-1, and Alexa Fluor 488 anti-Rabbit IgG H&L antibodies for immunofluorescence staining, and anti-PD-1 for IHC. For immunofluorescence staining, red: CD8+ cells, green: PD-1+ cells, blue: DAPI+ cells, yellow: PD-1+ CD8+ cells. For PD-1 IHC staining, blue: hematoxylin+ cells, brown: PD-1+ cells. (F) Flow cytometry analyses of infiltration of CD8+ T cells in HT29 xenografts, and PD-1, IFN-g, TNF-a, and GZMB expression in CD8+ TILs (n = 6/group). (G) Model design for SPF BALB/c mice bearing orthotopic CT26 allografts and experimental groups. Mice were gavaged with Fn conditioned medium (CM), UA159 CM (UACM), or broth Ctrl daily, with anti-PD-1 mAb (aPD-1) or IgG twice a week (n = 8–12/group). Red circles indicate the location of orthotopic tumors. Related results can be found in (H) and (I). (H) Representative tumor morphology, weight, and volume of CT26 allografts (n = 8–12/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: TUNEL Assay, Staining, Immunohistochemistry, Flow Cytometry, Expressing

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 4. Fn decreases PD-1 expression in CD8+ T cell through its small-molecule metabolites (A) Effects of Fn CM fractions on decreasing PD-1 expression in CD8+ T cell after co-culture for 18 h, as determined by flow cytometry (n = 6/group). Fn CM PK: Fn CM treated with proteinase K; >100 kDa: Fn CM > 100 kDa fraction; 10–100 kDa: Fn CM 10–100 kDa fraction; 3–10 kDa: Fn CM 3–10 kDa fraction; <3 kDa: Fn CM < 3 kDa fraction. (B) Workflow to define candidate metabolites from Fn culture medium that suppress PD-1 expression in CD8+ T and promote CD8+ T cell activation in vitro. (C) Orthogonal partial least squares discriminant analysis (OPLS-DA) score plot based on non-targeted metabolomic analysis of Fn CM < 3 kDa vs. control (left) and Fn gavage vs. PBS gavage germ-free mouse feces (right) (n = 6/group). (D) Venn diagram of enriched metabolites in Fn CM < 3 kDa vs. control CM, and Fn gavage vs. PBS gavage in germ-free mouse feces, and metabolomic heatmap of metabolites enriched after Fn gavage vs. PBS gavage germ-free mouse feces as determined by untargeted metabolomics (n = 6/group). (E) Targeted metabolomic analysis of short-chain fatty acids (SFCAs) in Fn- and PBS-gavaged germ-free mouse feces (left). Butyric acid quantification in CM and feces from germ-free mice (right) (n = 6/group). (F) Effect of sodium butyrate (NaB) on the expression of PD-1 and GZMB in splenic CD8+ T cells from tumor-bearing mice (n = 6/group).

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Expressing, Co-Culture Assay, Cytometry, Activation Assay, In Vitro, Control

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 5. Fn boosts response to anti-PD-1 therapy through its metabolite butyric acid (A) Workflow for the construction of FN0271 mutant Fn strain deficient in butyric acid biosynthesis. PCR products of FN0271 were analyzed by agarose gel electrophoresis to confirm the insertion mutant of Fn (left). GC-MS was conducted to detect butyric acid levels in Fn CM and FN0271 mutant (Mut) Fn CM (right). (B) Effect of Fn CM, Mut Fn CM, and NaB on PD-1 and GZMB expression in splenic CD8+ T cells (n = 6/group). (C) Evaluation of T cell proliferation by carboxyfluorescein succinimidyl ester (CFSE) assay and flow cytometry (n = 6/group). (D) Workflow to assess Fn CM and Mut Fn CM on anti-PD-1 mAb efficacy in vitro using CD8+ TILs from CT26 tumor-bearing mice. (E) Effect of Fn CM and Mut Fn CM in combination with anti-PD-1 mAb (aPD-1) on PD-1, GZMB, and IFN-g expression in CD8+ TILs (n = 6/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S6.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Mutagenesis, Agarose Gel Electrophoresis, Gas Chromatography-Mass Spectrometry, Expressing, CFSE Assay, Cytometry, In Vitro

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 6. Fn boosts anti-PD-1 mAb tumor-killing effects through its metabolite butyric acid in autologous co-culture system of PBMCs with tumor organoids from patients with MSS CRC (A) Schematic diagram for the evaluation of Fn CM and metabolites on anti-PD-1 therapy-mediated killing of CRC organoids with autologous peripheral blood mononuclear cell (PBMC)-derived CD8+ T cells. (B) Representative images of anti-PD-1 (aPD-1) therapy combined with Fn CM, FN0271 Mut Fn CM, or NaB on tumor killing efficiency. MSS CRC organoids (red) were labeled with CellTrace Far Red and apoptotic cells were labeled with green caspase-3/7 probe (n = 6/group). Black circle: organoid, small dot: T cell, red: CellTrace far red+ cells, green: caspase-3/7+ cells. (C) Apoptosis in CRC organoids, as determined by flow cytometry (n = 6/group). (D) Flow cytometry of PD-1, GZMB, and IFN-g in autologous CD8+ T cells (n = 3/group). All results are presented as mean ± SD. Each data point in bar plots represents one subject. Statistical significance was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. See also Figure S7 and Table S2.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Co-Culture Assay, Derivative Assay, Labeling, Cytometry, Flow Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 7. Fn-derived butyrate facilitates anti-PD-1 therapy in MSS CRC in a CD8+ T cell-dependent manner (A) CT26 subcutaneous allografts tumor-bearing BALB/c mice received NaB in drinking water, together with anti-PD-1 mAb (aPD-1), anti-CD8a mAb, or IgG twice a week (n = 6–10/group). Related results can be found in B–H. (B) Representative tumor gross morphology, tumor weight, and tumor volume. NaB potentiated with anti-PD-1 to suppress tumor growth, an effect abolished by the depletion of CD8+ T cells by anti-CD8a (n = 6–10/group). Red circles indicate the location of subcutaneous tumors. (C) Tumoral butyrate among different groups (n = 6/group). (D) TUNEL, (E) Ki-67, and PCNA IHC in CT26 allografts to analyze cell apoptosis and proliferation (n = 6/group). Tumor tissues were stained using anti-Ki-67, anti- PCNA, or TUNEL kit. For TUNEL staining, green: methyl green+ cells, brown: apoptotic cells. For Ki-67 and PCNA IHC staining, blue: hematoxylin+ cells, brown: Ki-67+ or PCNA+ cells. (F) Infiltration of CD8+ TILs in CT26 allografts, as determined by flow cytometry and (G) IHC (n = 6/group). Tumor tissues were stained using anti-CD8. Blue: hematoxylin+ cells, brown: CD8+ cells.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Derivative Assay, TUNEL Assay, Staining, Immunohistochemistry, Cytometry

Journal: Cancer cell

Article Title: Fusobacterium nucleatum facilitates anti-PD-1 therapy in microsatellite stable colorectal cancer.

doi: 10.1016/j.ccell.2024.08.019

Figure Lengend Snippet: Figure 8. Fn metabolite butyric acid regulates CD8+ T cell through an epigenetic mechanism leading to upregulation of TBX21 (T-bet) (A) CD8+ T cells treated with Fn CM, FN0271 Mut Fn CM, and control CM were analyzed by RNA sequencing. Principal component analysis (PCA) and heatmap analysis of differentially expressed genes, and validation by qPCR (n = 3/group). qPCR data were normalized using Actb as an internal control.

Article Snippet: For CD8+ T cell depletion experiment, 200 mg of InVivoMAb anti-mouse CD8a (BE0004-1, clone: 53-6.7, Bio X Cell, USA) or isotype control IgG (BE0089, clone: 2A3, Bio X Cell, USA) was injected intraperitoneally twice a week.

Techniques: Control, RNA Sequencing, Biomarker Discovery

Journal: Matrix Biology Plus

Article Title: Temporal expression and spatial distribution of the proteoglycan versican during cardiac fibrosis development

doi: 10.1016/j.mbplus.2023.100135

Figure Lengend Snippet: Versican ( VCAN ) expression is regulated by cytokines and mechanical stretch in cardiac cells. (A) mRNA expression of versican was upregulated by TGF-β1, and downregulated by TNF-α, and IL-1β in cardiac fibroblasts, and (B) in addition by IL-4 in cardiomyocytes compared to control (Control, n = 3). (C, D) Versican and α-SMA ( ACTA2 ) mRNA expression were increased after biaxial strain of 10% (stretch) for 24 h at 1 Hz frequency in fibroblasts compared to non-stretched cells (Control, n = 9). Gene expression is presented as values relative to Control. Bar graphs represent mean ± SD. One-way ANOVA with Dunnett’s multiple comparisons test (A, B) and Student’s t -test (C, D) were used for statistical analysis. P values < 0.05 were considered statistically significant. *P < 0.05 Control vs. cytokine treatment or stretch.

Article Snippet: Fetal human cardiac fibroblasts (CFs) (Cell Applications, CA, #306-05F) and primary human cardiomyocytes (CMs) (PromoCell, Germany, #C-12810) were used for in vitro experiments.

Techniques: Expressing, Control, Gene Expression

Journal: Blood

Article Title: Patients with CD3G mutations reveal a role for human CD3γ in T reg diversity and suppressive function

doi: 10.1182/blood-2018-02-835561

Figure Lengend Snippet: T-cell activation and cell division following mitogen stimulation. (A) Representative plots and (B) cumulative percentage of (top) CD25 expression, (middle) Ki67 expression, and (bottom) CFSE staining in CD4+ T cells from controls (solid gray) or CD3G patients (red) in unstimulated samples or following activation with PHA, anti-CD3, or anti-CD3/CD28. (C) Representative plots and (D) cumulative data for (top) CD25 expression, (middle) Ki67 expression, and (bottom) CFSE staining in CD8+ T cells from controls (solid gray) or patients (red) among the indicated groups. (A-D) Response to anti-CD3+anti-CD28 was studied in 5 patients (P1-P5) because not enough cells were available from P6. Dose titration curve of CFSE dilution among (E) CD4+ and (F) CD8+ T cells following stimulation with PHA (top) or anti-CD3 (bottom) for patient P6 (red line) and control sample (blue line). Unstim, unstimulated.

Article Snippet: PBMC were isolated to form a single cell suspension and then stained with carboxyfluorescein succinimidyl ester (CFSE) (Thermo Fisher, Carlsbad, CA) at a concentration of 5 μM/mL for 10 minutes at 37°C in phosphate-buffered saline.

Techniques: Activation Assay, Expressing, Staining, Titration, Control

Journal: Signal Transduction and Targeted Therapy

Article Title: Intranasal prime-boost RNA vaccination elicits potent T cell response for lung cancer therapy

doi: 10.1038/s41392-025-02191-1

Figure Lengend Snippet: AMs and cDC1s are mainly responsible for boosting antigen-specific T cells in lung tissues after intranasal vaccination. a Timeline of the experiment to evaluate the role CD11c+ APCs on boosting antigen-specific T cells. WT, Batf3 −/− or CD11c-DTR mice were pre-treated with cyclophosphamide (CTX) and diphtheria toxin (DT) for lymphodepletion in all mice and CD11c+ cells depletion in CD11c-DTR mice. Activated OT-1 cells were labeled with CFSE and then transferred to mice immunized with intranasal circRNA vaccine. T cell proliferation at lung tissues was analyzed at day 3. b , c Representative flow cytometry histograms ( b ) and statistical results ( c ) of T cell proliferation with different treatments. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. n = 3 for mock vac. group; n = 4 for other groups. For LNP uptake analysis, mice received intranasal DiD-labeled LNP and lung tissues were analyzed 4 h later. d , e Representative flow cytometry histograms ( d ) and statistical results ( e ) of LNP uptake among different cell types. f Scheme of the experiment to test the potential APCs that can boost antigen-specific T cells. Mice were immunized with intranasal LNP without RNA (mock) or circRNA vaccine and 24 h later, lung tissues were collected and APCs were sorted and co-cultured with OT-1 cells for another 36 h in IFN-γ Elispot wells. ( n = 3 for mock group; n = 5 for circRNA vaccine group, repeated for three times) Created with BioRender.com. g , h Representative images and statistical results of IFN-γ Elispot assay. Data were analyzed by Student’s t test. i Timeline of the experiment to explore whether vaccine can boost T cells at lung tissues. Mice were treated with FTY720 during the prime-boost process or only the boosting process or normal saline as the control group. The number of antigen-specific T cells at lung tissues was analyzed. j , k Representative plots ( j ) and statistical results ( k ) of the antigen-specific T cells with different treatments. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. All data are represented as mean ± SEM

Article Snippet: Activated OT-1 cells were labeled with 5 μM CFSE (Selleck) for 15 min at 37 °C and washed with PBS two times.

Techniques: Labeling, Flow Cytometry, Cell Culture, Enzyme-linked Immunospot, Saline, Control

Journal: PLoS ONE

Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

doi: 10.1371/journal.pone.0074363

Figure Lengend Snippet: A. sCAR-PPAb did not enhance the phagocytosis of K562 cells by macrophages. K562 cells stained with CFSE and pretreated with sCAR-PPAb or PBS were injected into the abdominal cavity in ICR mice. After 5h, cells in the abdominal cavity were then collected and stained with a primary antibody against F4/80 or IgG isotype followed by a goat anti-rat IgG-PE antibody. Cells were then analyzed by flow cytometry. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p>0.05, n=3). B. sCAR-PPAb enhanced the phagocytosis of K562/ADR by macrophages. Data were reported as the percent of macrophages undergoing phagocytosis, calculated by CFSE/PE double positive cells divided by all PE positive cells. Values are shown as mean ± SEM (p<0.05, n=3). C. The cytotoxicity of sCAR-PPAb on K562 and K562/ADR was analyzed by MTT assay. D. Mouse macrophages from the abdominal cavity were isolated and cultured in 96-well plates. The cells were then treated with 5μg of sCAR-PPAb for 5 hours. PBS treatment served as the control. The cell viability was analyzed by MTT assay.

Article Snippet: K562/ADR cells were stained with carboxyl fluorescein succinimidyl ester (CFSE), and 1 x 10 6 cells were mixed with 10μg of a mouse anti-SLMAP mAb (Santa Cruz Biotechnology) or a mouse IgG (Sigma-Aldrich, Co., St. Louis, MO, USA) followed by adding into wells pre-cultured with macrophages.

Techniques: Staining, Injection, Flow Cytometry, MTT Assay, Isolation, Cell Culture, Control

Journal: PLoS ONE

Article Title: Pinellia pedatisecta Agglutinin Targets Drug Resistant K562/ADR Leukemia Cells through Binding with Sarcolemmal Membrane Associated Protein and Enhancing Macrophage Phagocytosis

doi: 10.1371/journal.pone.0074363

Figure Lengend Snippet: A. sCAR-PPAb combined with a CAR antibody was used in immunoprecipitation. PBS combined with CAR antibody served as the control. Precipitates were analyzed by Western blot for SLMAP. Whole cell lysis (WCL) was monitored for Actin levels. B. SLMAP levels on K562 and K562/ADR were determined by Western blot. Actin served as a loading control. C. A SLMAP antibody enhanced the phagocytosis of K562/ADR by macrophages in vitro. An IgG isotype served as the control. The portion of macrophages ingesting CFSE stained K562/ADR cells was observed under a fluorescence microscope (200 x). A significant enhancement of phagocytosis upon SLMAP antibody treatment was determined (p<0.05).

Article Snippet: K562/ADR cells were stained with carboxyl fluorescein succinimidyl ester (CFSE), and 1 x 10 6 cells were mixed with 10μg of a mouse anti-SLMAP mAb (Santa Cruz Biotechnology) or a mouse IgG (Sigma-Aldrich, Co., St. Louis, MO, USA) followed by adding into wells pre-cultured with macrophages.

Techniques: Immunoprecipitation, Control, Western Blot, Lysis, In Vitro, Staining, Fluorescence, Microscopy

Journal: bioRxiv

Article Title: SARS-CoV-2 variant B.1.1.7 caused HLA-A2 + CD8 + T cell epitope mutations for impaired cellular immune response

doi: 10.1101/2021.03.28.437363

Figure Lengend Snippet: A: The schematic of mutation sites of the SARS-CoV-2 variant B.1.1.7. B: List of the predicted epitopes for following experiments. The mutated amino acids were highlighted as red in varian B.1.1.7t. a The antigenic vaule threshold was > 0.4000 C - D: Comparison of ancestral and mutant epitope binding affinity to HLA-A2 in T2A2 cells. ancestral and mutated epitopes were synthesized and incubated with T2A2 cells. The binding of the peptide on T2A2 was measured with anti-HLA-A2 staining with flow cytometry. Binding capacity was presented as mean fluorescence intensity (MFI) of HLA-A2 staining. C was the representative plot of D. Each symbol represents an independent experiment. Ancestral: Wuhan strain epitope; Mutant: varian B.1.1.7t epitope. E: Evaluation of epitope binding to HLA-A2 with ELISA assay. Peptide exchanged assay was performed with coated UV-sensitive peptide/MHC complex and given peptides. The binding capability was measured with pMHC ELISA assay. Data shown are mean plus standard error of the mean (SEM). Threshold for pMHC formation positivity was set as above the average OD value of the negative-control cohort. Blank: no peptides; Neg ctrl: negative control, Zika virus peptide GLQRLGYVL; Pos ctrl: positive control, influenza A M1 peptide GILGFVFTL.

Article Snippet: 0.5×10 6 CD8 + T cells isolated from health donors were co-cultured with 0.5 × 10 6 peptide-loaded T2A2 cells stained with 5 µmol/L CFSE (TargetMol), and stimulated with 1 µg/mL anti-human CD28 antibodies (BioLegend) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2( 125 Ala) Injection).

Techniques: Mutagenesis, Variant Assay, Binding Assay, Synthesized, Incubation, Staining, Flow Cytometry, Fluorescence, Enzyme-linked Immunosorbent Assay, Negative Control, Positive Control

Journal: bioRxiv

Article Title: SARS-CoV-2 variant B.1.1.7 caused HLA-A2 + CD8 + T cell epitope mutations for impaired cellular immune response

doi: 10.1101/2021.03.28.437363

Figure Lengend Snippet: Mitomycin pretreated T2A2 cells were loaded with mixed peptides from ancestral or mutant, and incubated with CD8 + T cell from health donors at 1:1 ratio, respectively. Epitope specific CD8 + T cells were generated after 7 day stimulation. A - B: The CD69 expression level of activated CD8 + T cell was evaluated with flow cytometry after 16 hour stimulation. A was the representative plot of B. n=4 per group. Day 0 ctrl: staining before stimulation; T2A2 ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. C - E: Epitope specific CD8 + T cell measurement before (C) and after (D) 7 day stimulation. The cells were stained with corresponding ancestral or mutated tetramer, and compared before and after stimulation (E). Four and five repeats were performed for decreased and nonchanged comparison, respectively. Please also see Supplementary Figure 1 A-C. F-I: Cross-detection of epitope specific CD8 + T cells with tetramers based on ancestral and corresponding mutant peptides. F-G: ancestral or mutant epitopes stimulated CD8 + T cells were stained with ancestral peptide-based tetramer. H-I: mutant or ancestral epitopes stimulated CD8 + T cells were stained with mutant peptide-based tetramer. n=3 per group. Symbols in G and I represented individual person. The p values were calculated by paired-samples T test. ** p < 0.01. Neg ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. J - M: Epitope specific CD8 + T cell mediated cytotoxicity was evaluated after 7 day culture (J&K). The remained CFSE labeled T2A2 cells were calculated as survived target cells. J was the representative plot of K. Apoptosis of T2A2 cells at day 7 after culture. The proportion of CFSE + AnnexinV + cells was calculated as indicator for epitope stimulated T cell mediated T2A2 apoptosis (L&M). L was the representative plot of M. n=4 per group. N - O: The expression of IFN-γ after epitope stimulation for 7 days. IFN-γ was measured with intracellular stained flow cytometry. N was the representative plot of O. n=4 per group. P - Q: The expression of Granzyme B after epitope stimulation for 7 days. Granzyme B was measured with intracellular stained flow cytometry. P was the representative plot of Q. n=4 per group. Day 0 ctrl: staining before stimulation; T2A2 ctrl: T2A2 without peptide loading; Pos ctrl: T2A2 loaded with influenza A M1 peptide GILGFVFTL. The p values were calculated by paired-samples T test, *** p < 0.001.

Article Snippet: 0.5×10 6 CD8 + T cells isolated from health donors were co-cultured with 0.5 × 10 6 peptide-loaded T2A2 cells stained with 5 µmol/L CFSE (TargetMol), and stimulated with 1 µg/mL anti-human CD28 antibodies (BioLegend) and 50 IU/mL IL-2 (SL PHARM, Recombinant Human Interleukin-2( 125 Ala) Injection).

Techniques: Mutagenesis, Incubation, Generated, Expressing, Flow Cytometry, Staining, Labeling