rabbit anti human cd8  (Bioss)

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) UMAP plot for the different subclusters of CD8 + T cells in human HCC tissues. ( B ) UMAP plots show marker genes in different CD8 + T clusters. ( C ) Heatmap shows cell markers in each CD8 + T subtype. ( D ) Volcano plot for differential expression analysis of T EX and T EFF clusters. P adj , adjusted P value; n.s., not significant. ( E ) Venn plot for differential genes between T EX and T EFF clusters in three scRNA-seq datasets and one bulk RNA-seq dataset. ( F ) Dot plot shows specific function markers enriched in different CD8 + T subclusters in Zhongshan cohort. ( G ) UMAP plot for the different subclusters of CD8 + T cells in GSE149614 dataset. T CM , central memory T cells. ( H ) Relative expression of HMGB2 in CD8 + T cells at different tumor stages. ( I ) Relative expression of HMGB2 in CD8 + T cells in the normal liver tissues and tumor tissues. ( J ) UMAP plot for the different subclusters of CD8 + T cells in GSE140228 dataset. ( K ) UMAP plot shows the expression of HMGB2 in different CD8 + T cell subclusters. ( L ) Relative expression of HMGB2 in CD8 + T cells in the normal liver tissues and tumor tissues. ( M ) UMAP plot for the different subclusters of CD8 + T cells in mouse HCC tissues. ( N ) UMAP plot shows Hmgb2 expression of CD8 + T cells in mouse HCC tissues. ( O ) Dot plot shows specific function markers enriched in CD8 + T subclusters of mouse HCC tissues. Wilcoxon test.

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Marker, Quantitative Proteomics, RNA Sequencing, Expressing

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Gene Ontology (GO) analysis reveals changes in Hmgb2 -cKO CD8 + T cells. FDR, false discovery rate. ( B ) Gene set enrichment analysis (GSEA) shows top pathway enriched in Hmgb2 -cKO CD8 + T cells. ( C ) mRNA levels of electron transport chain genes in NC and Hmgb2 -cKO CD8 + T cells. ( D ) Heatmap for energy metabolites in isolated NC and Hmgb2 -cKO CD8 + T cells detected by liquid chromatography–MS (LC-MS) analysis. cAMP, adenosine 3′,5′-monophosphate; GTP, guanosine 5′-triphosphate; NAD, nicotinamide adenine dinucleotide; NADH, reduced form of NAD + ; NADPH, reduced form of nicotinamide adenine dinucleotide phosphate; CoA, coenzyme A; AMP, adenosine 5′-monophosphate; UDP, uridine 5′-diphosphate; ADP, adenosine 5′-diphosphate; GDP, guanosine diphosphate; NADP, beta-nicotinamide adenine dinucleotide phosphoric acid. ( E ) Intensity of ATP, NAD + , and fumaric acid as in (D) ( n = 3). ( F ) KEGG analysis shows top metabolic pathway changes as in (D). ( G ) Seahorse extracellular flux analysis of OCR in isolated NC and Hmgb2 -cKO CD8 + T cells. FCCP, carbonyl cyanide p -trifluoromethoxyphenylhydrazone. ( H ) Quantification of OCR as in (G) ( n = 32). ( I ) Immunofluorescence micrographs of NC and Hmgb2 -cKO OT-I CD8 + T cells stained with MitoTracker (red) and 4′,6-diamidino-2-phenylindole (DAPI) (blue) after coculture with Hepa1-6–OVA cells. Scale bar, 10 μm. ( J ) Comparison of fluorescence of stained MitoTracker as in (I) ( n = 5). ( K ) Transmission electron microscope images of mitochondria in activated NC and Hmgb2 -cKO OT-I CD8 + T cells after coculture with Hepa1-6–OVA cells. The density of mitochondrial cristae is compared ( n = 7). ( L ) Flow cytometry analysis of CD44 hi CD62L lo effector CD8 + T cells in NC and Hmgb2 -cKO OT-I CD8 + T cells after coculture with Hepa1-6–OVA cells ( n = 4). ( M ) Flow cytometry analysis of GranB + IFN-γ + CD8 + T cells as in (L). ( N ) Flow cytometry analysis of TNF-α + CD8 + T cells as in (L). ( O ) Flow cytometry analysis of PD1 + LAG-3 + CD8 + T cells as in (L). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Student’s t test for (C), (E), and (J) to (O). Two-way analysis of variance (ANOVA) test for (H).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Isolation, Liquid Chromatography, Liquid Chromatography with Mass Spectroscopy, Immunofluorescence, Staining, Comparison, Fluorescence, Transmission Assay, Microscopy, Flow Cytometry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Location of differential accessible ATAC-seq peaks in NC and Hmgb2 -cKO CD8 + T cells. 3′UTR, 3′ untranslated region; 5′UTR, 5′ untranslated region. ( B ) Chromatin accessibility changes of genes associated with effector function and mitochondrial transcription factors. ( C ) The correlation between Hmgb2 , Tfam , and Tfb1m expression in CD8 + T cells from murine scRNA-seq data. ( D ) mRNA levels of Keap1 and Nfe2l2 in NC and Hmgb2 -cKO CD8 + T cells ( n = 6). ( E ) mRNA levels of ARE genes in NC and Hmgb2 -cKO CD8 + T cells ( n = 3). ( F ) Protein changes of KEAP1 and NRF2 after Hmgb2 knockout. ( G ) The ubiquitination of NRF2 after Hmgb2 knockout. IB, immunoblot. ( H ) Protein changes of KEAP1 and NRF2 after stimulation of mouse recombinant HMGB2 protein and IN-1. NC CD8 + T cells were treated with recombinant HMGB2 protein (500 ng/ml) and/or IN-1 (10 μM) for 48 hours. ( I ) The ubiquitination of NRF2 after cell treatment as in (H). ( J ) Immunofluorescence staining of spontaneous HCC tissues in NC and Hmgb2 -cKO mice. NRF2 + KEAP1 − CD8 + T cells were labeled. Scale bars, 20 μm (left) and 10 μm (right). HP, high power field. ( K ) Seahorse extracellular flux analysis of OCR in different CD8 + T cell groups ( n = 6 to 8) after cell treatment as in (H). ( L ) Quantification of seahorse extracellular flux analysis of OCR of different CD8 + T cells as in (K). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Pearson test for (C) Student’s t test for (D), (E), and (J). Two-way ANOVA test for (L).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Expressing, Knock-Out, Ubiquitin Proteomics, Western Blot, Recombinant, Immunofluorescence, Staining, Labeling

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) UMAP plot for the different subclusters of CD8 + T cells in human HCC tissues scheduled to neoadjuvant anti–PD-1 monotherapy. ( B ) Histogram plot shows the proportions of CD8 + T cells as in (A). ( C ) UMAP plot for the HMGB2 expression of CD8 + T cells in nonresponse and PR HCC tissues. ( D ) Relative expression of HMGB2 in CD8 + T cells as in (C). ( E ) Comparison of HMGB2 expression in CD8 + T cells in pretreatment and postoperative tissues. ( F ) The schematic diagram shows the medication regimen in vivo. ( G ) Representative images of HCC spontaneous models in different treatment groups. Scale bar, 1 cm. ( H ) Tumor numbers of HCC spontaneous models as in (G). ( I ) Survival time of HCC spontaneous models as in (G). ( J ) Flow cytometry of intratumoral IFN-γ + CD8 + T cells and GranB + CD8 + T cells from spontaneous HCC tissues. ( K ) Quantification of intratumoral IFN-γ + CD8 + T cells ( n = 5). ( L ) Quantification of intratumoral GranB + CD8 + T cells ( n = 5). ( M ) Immunofluorescence staining of effector markers in spontaneous HCC tissues as in (G). Scale bar, 50 μm. Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Wilcoxon test for (D) and (E). One-way ANOVA test for (H), (K), and (L). The icon in (F) is cited from BioRender: W. Qu (2025; https://BioRender.com/m63n001 ).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Expressing, Comparison, In Vivo, Flow Cytometry, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Representative images of harvested Hepa1-6 subcutaneous HCC tumors. Scale bar, 1 cm ( B ) Tumor growth curves of Hepa1-6 subcutaneous tumors in BALB/C nude mice and C57BL/6J mice. ( C ) Tumor weights of subcutaneous Hepa1-6 tumors in BALB/C nude mice and C57BL/6J mice ( n = 6). ( D ) Difference of tumor volumes between shCtrl and shHmgb2 subcutaneous tumors in BALB/C nude mice and C57BL/6J mice ( n = 6). ( E ) Differential GO pathways in Hepa1-6 shHmgb2 cells. JAK, Janus kinase. ( F ) GSEA analysis shows top pathway enriched in Hepa1-6 shHmgb2 cells. ( G ) Western blotting experiment shows STAT1 pathway changes in Hepa1-6 cells and Huh7 cells. Cells were treated with IFN-γ (10 ng/ml) or fludarabine (10 μM) for 24 hours. ( H ) Annexin V apoptosis analysis for Hepa1-6 cells treated with vehicle and IFN-γ (10 or 20 ng/ml). PI, propidium iodide. ( I ) Quantification for proportions of apoptotic cells after treatment of IFN-γ. ( J ) T cell killing assay with Hepa1-6 shCtrl and shHmgb2 cells and wild-type (WT) CD8 + T cells ( n = 5). RLU, relative light unit. ( K ) Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining and quantification of Hepa1-6 subcutaneous tumors ( n = 4). Scale bar, 25 μm. HPF, high power field. ( L ) Representative immunohistochemistry images of CD8, IFN-γ, and CXCL10 staining in Hepa1-6 subcutaneous tumors. Scale bars, 20 μm. ( M ) Quantification of CD8, IFN-γ, and CXCL10 staining in Hepa1-6 subcutaneous tumors ( n = 4). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001. Two-way ANOVA test for (B) Student’s t test for (C), (D), (I), (J), (K), and (M).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Western Blot, End Labeling, TUNEL Assay, Staining, Immunohistochemistry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) ChIP assay shows the interaction of HMGB2 with Stat1 chromatin ( n = 3). ( B ) Colocalization of HMGB2, STAT1 and TRIM 24 in HCC subcutaneous tumor tissue. Scale bar, 10 μm. ( C ) CoIP assay shows the interaction of HMGB2 and TRIM24. ( D ) ChIP-PCR shows that TRIM24 modulates the transcriptional level of Stat1 ( n = 3). ( E ) Luciferase reporter assay shows that the Trim24 / Hmgb2 signal modulates the transcriptional level of Stat1 ( n = 3). OE, overexpression. ( F ) Representative images of the orthotopic HCC models. ( G ) Tumor weights of different groups from the orthotopic HCC models ( n = 6). ( H ) Overall survival of different treatment groups from the orthotopic HCC models. ( I ) Flow cytometry of intratumoral CD3 + CD8 + T cells as in (G). ( J ) Flow cytometry of intratumoral effector markers as in (G). ( K ) Quantification of intratumoral IFN-γ + CD8 + T cells and GranB + CD8 + T cells in different treatment groups ( n = 5). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Student’s t test for (A) and (D). One-way ANOVA test for (E), (G), and (K).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Co-Immunoprecipitation Assay, Luciferase, Reporter Assay, Over Expression, Flow Cytometry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: ( A ) Proliferative inhibition curve for tannic acid on Hepa1-6 cells. IC 50 = 21.23 μM. ( B ) Representative images of orthotopic HCC models. ( C ) Tumor volumes of different groups from the orthotopic HCC model ( n = 6). ( D ) Representative images of colorectal subcutaneous tumors constructed by MC38 cell injection ( n = 6). ( E ) Tumor growth curves of subcutaneous tumors constructed by MC38 cell injection ( n = 6). ( F ) Immunohistochemistry staining images of CD8 in the subcutaneous tumors constructed by MC38 cell injection. Scale bar, 50 μm. ( G ) Representative images of spontaneous HCC model. ( H ) Tumor weights of different treatment groups in the spontaneous HCC model ( n = 6). ( I ) Overall survival of different treatment groups in the spontaneous HCC model ( n = 6). ( J ) Flow cytometry and quantification of intratumoral CD3 + CD8 + T cells as in (H). ( K ) Quantification of flow cytometry analysis on intratumoral effector CD8 + T cells and exhausted CD8 + T cells as in (H). Data are presented as the means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. One-way ANOVA test for (C), (H), (J), and (K). Two-way ANOVA test for (E).

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Inhibition, Construct, Injection, Immunohistochemistry, Staining, Flow Cytometry

Journal: Science Advances

Article Title: Targeting HMGB2 acts as dual immunomodulator by bolstering CD8 + T cell function and inhibiting tumor growth in hepatocellular carcinoma

doi: 10.1126/sciadv.ads8597

Figure Lengend Snippet: Markers and article numbers of antibodies. PE, phycoerythrin; FITC, fluorescein isothiocyanate; APC, antigen-presenting cell; HRP, horseradish peroxidase; mAb, monoclonal antibody.

Article Snippet: Anti-human/mouse CD8 , 85336 , Cell Signaling Technology, USA.

Techniques: Ubiquitin Proteomics, Purification, In Vivo

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: The Sd/Gv-based CAR effectively redirected T cells to eliminate target cells. A Schematic representation of the design of modular CAR by Sd/Gv platform. Sd: SDCatcher, Gv: GVoptiTag. B and C Coomassie blue staining and western blotting analysis of Gv-VRC01 scFv and Gv-CD5-CD30 scFvs. Anti-His antibody was used to confirm the expression and purity of each protein. D The Sd-28BBZ3-P2A-tCD19 elements were transduced into HEK293T cells, and the expression of CD19 indirectly indicated the expression of Sd-28BBZ3. Meanwhile, Gv-scFvs (VRC01 scFv or CD5-CD30 scFvs) were expressed as fusion proteins with a 6-Histidine tag. The cells expressing Sd-28BBZ3-P2A-tCD19 were incubated with Gv-scFv proteins, allowing for indirect detection of Gv-scFV binding to Sd on the cell surface through the presence of His tag. Immunofluorescence staining analysis of His-tagged Gv-scFv proteins conjugating to Sd-28BBZ3-P2A-tCD19 on HEK293T cells with anti-CD19 (red) and anti-His (green) antibodies. DAPI was used to stain the nuclei (blue). Scale bars represented 10 μm. E The Sd-28BBZ3-P2A-tCD19 elements were transduced into HEK293T cells. After 12 h, the culture medium was removed and the cells were cultured with fresh medium supplemented with 100 nM Gv-scFVs (VRC01 scFv or CD5-CD30 scFvs) proteins. Following an additional 12 h, the membrane proteins were extracted and the covalent binding of Sd-Gv was detected using western blot with anti-Gv/Sd antibodies. F and G Direct killing of target cell lines was performed utilizing the Cyto-Tox nonradioactive cytotoxicity kit with Jurkat gp160 ( F ) or Karpas 299 ( G ) cells as targets. The effector cells included the following cells. Control CD8 + T: CD8 + T cells transduced with empty vector. Sd-28BBZ3 CD8 + T: CD8 + T cells transduced with Sd-28BBZ3. Sd-28BBZ3 + Gv-scFv: CD8 + T cells transduced with Sd-28BBZ3 and incubated with proteins of Gv-scFvs (VRC01 scFv or CD5-CD30 scFvs). VRC01-CAR: CD8 + T cells transduced with conventional VRC01-CAR. CD5-CD30-CAR: CD8 + T cells transduced with conventional CD5-CD30-CAR. Different ratios of effector cells and target cells were co-incubated. Data represented as mean ± SD. N = 3 independent biological replicates. Statistical analysis was performed by two-way ANOVA followed by Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: Staining, Western Blot, Expressing, Incubation, Binding Assay, Immunofluorescence, Cell Culture, Membrane, Control, Transduction, Plasmid Preparation

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: CRISPR/Cas9-mediated TCR and HLA-I knockout did not affect the function of T cells and reduced alloreactivity. A The expression of CD5-CD30-CAR on non-disrupted and TCR − /HLA-I − CD8 + T cells. tCD19 was in-frame expressed with CD5-CD30-CAR to indicate the expression of CAR molecules. B The killing results of Karpas 299 target cells under different effector/target ratios. Data represented as mean ± SD. N = 3 independent biological replicates. C CD5-CD30-CAR-transduced CD8 + T cells derived from non-disrupted and TCR − /HLA-I − cells were co-cultured with Karpas 299 at a ratio of 4:1 respectively. IFN-γ secretion within each group was analyzed by ELISPOT assay. Data represented as mean ± SD. N = 3 independent biological replicates. D and E The inhibition of alloreactivity upon TCR/HLA-I disruption was determined utilizing the IFN-γ ELISPOT assay. Allogenic PBMCs were co-cultured with irradiated gene-edited T cells ( D ). Irradiated allogenic PBMCs were also co-cultured with gene-edited T cells ( E ). Data represented as mean ± SD. N = 7 independent biological replicates. Statistical analysis was performed by Student’s t -test. **** p < 0.0001

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: CRISPR, Knock-Out, Expressing, Derivative Assay, Cell Culture, Enzyme-linked Immunospot, Inhibition, Disruption, Irradiation

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: Candidate T cells were screened and obtained utilizing modified culture conditions. A Phenotypic analysis of T cells was performed based on the expression of CD45RA, CCR7, CD122 and CD95 after exposure to either IL-2 or a combination of IL-7 and IL-15. The percentage of T SCM (CD45RA + CCR7 + CD122 + CD95 + ) cells was shown. Data represented as mean ± SD. N = 9 independent biological replicates. B T cells which were induced by either IL-2 or the combination of IL-7 and IL-15 were mixed with Karpas 299 target cells to conduct the cytotoxicity assay. Data represented as mean ± SD. N = 3 independent biological replicates. C Expansion of CD8 + T cells derived from 20 healthy donors within OptiVitro T-SFM medium containing IL-7 and IL-15. D and E The viability and expansion of five dominant CD8 + T cells, along with one suboptimal CD8 + T cells, following cryopreservation and thawing cycles. F and G TCR − /HLA-I − CD8 + T cells derived from five dominant donors were transduced with CD5-CD30 CAR and incubated with Karpas 299 cells for 18 to 24 h. IFN-γ secretion within each group was analyzed utilizing ELISPOT assays. Data in ( A ) were analyzed by Student’s t -test. Data in ( B ) were analyzed by two-way ANOVA with Sidak’s multiple comparisons test. Data in ( G ) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.00

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: Modification, Expressing, Cytotoxicity Assay, Derivative Assay, Transduction, Incubation, Enzyme-linked Immunospot

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: Generation and functional validation of MU-CAR-T cells. A Flowchart illustrating the protocol for generating MU-CAR-T cells. Briefly, α-CD3/α-CD28-activated CD8 + T cells were electroporated with Cas9 mRNA and sgRNAs targeting B2M and TRAC on Day 3 and Day 4. On Day 6, TCR − /HLA-I − T cells were enriched. Then, enriched T cells were transduced with SdΔN17-28BBZ3-tCD19 through lentiviral infection on Day 7. CD19-positive selection was performed on Day 9. After incubation with the specific Gv-scFv, the resulting products were identified as MU-CAR-T cells. B and C The cytotoxicity of MU-CAR-T cells against Jurkat gp160 and Karpas 299 target cells was observed upon being conjugated with VRC01 scFv ( B ) and CD5-CD30 scFvs ( C ) respectively. Data represented as mean ± SD. N = 3 independent biological replicates. D Control CD8 + T, SdΔN17-28BBZ3-expressing CD8 + T, conventional CAR-T and MU-CAR-T cells were co-cultured with target cells at a 4:1 ratio respectively. IFN-γ secretion was detected utilizing ELISPOT assay. PHA-stimulated effector cells were treated as the positive control (PC), and effector cells-only group was treated as the negative control (NC). Data represented as mean ± SD. N = 3 independent biological replicates. ( E , F and G ) The production of cytokines including Granzyme B ( E ), IFN-γ ( F ) and TNFα ( G ) was assessed upon co-culturing of control CD8 + T cells and CAR-T cells with different target cells at the ratio of 4:1. Data represented as mean ± SD. N = 3 independent biological replicates. Data in ( B ) and ( C ) were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Data in ( D - G ) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: Functional Assay, Biomarker Discovery, Transduction, Infection, Selection, Incubation, Control, Expressing, Cell Culture, Enzyme-linked Immunospot, Positive Control, Negative Control

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: MU-CAR-T cells efficiently suppressed HIV-1 rebound after the withdrawal of antiviral treatment in vitro. A Flow chart of the experimental design. PHA-activated CD4 + T cells were infected with wild-type HIV-1 NL4 − 3 . On Day 6, inhibitory drugs including AZT and Lopinavir were added to inhibit viral replication. On Day 14, anti-HIV-1 drugs were withdrawn, and infected CD4 + T cells were co-cultured with various groups of T cells including control CD8 + T cells, SdΔN17-28BBZ3-expressing CD8 + T cells, conventional CAR-T and VRC01-MU-CAR-T cells. On Day 26, infected CD4 + T cells were harvested to detect viral RNAs. HIV-1 p24 proteins within the supernatant were monitored every 2 days. B The expression of activation markers including CD25 and HLA-DR was analyzed on unstimulated and activated CD4 + T cells, as well as CD4 + T cells on Day 14 post infection. The percentage of cells was indicated in each quadrant. C Culture supernatants were tested for the presence of p24 utilizing ELISA assay every 2 days, which represented the expression and rebound of HIV-1. Data represented as mean ± SD. N = 3 independent biological replicates. D Cells from each group were harvested on Day 26, and cell-associated HIV-1 RNAs were quantified utilizing real-time RT-qPCR. Data represented as mean ± SD. N = 3 independent biological replicates. Data in ( C ) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. Data in ( D ) were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. **** p < 0.0001

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: In Vitro, Infection, Cell Culture, Control, Expressing, Activation Assay, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Molecular Cancer

Article Title: The construction of modular universal chimeric antigen receptor T (MU-CAR-T) cells by covalent linkage of allogeneic T cells and various antibody fragments

doi: 10.1186/s12943-024-01938-8

Figure Lengend Snippet: The effectiveness of MU-CAR-T cells within tumor-bearing mice in vivo . A Flow chart of the experimental design. NCG mice were subcutaneously injected with Karpas 299 lymphoma cells. On Day 9, when the tumor volume reached approximately 100 mm 3 , control CD8 + T, SdΔN17-28BBZ3-expressing CD8 + T, conventional CAR-T and CD5-CD30-MU-CAR-T cells were intravenously injected into mice respectively, followed by the intraperitoneal injection of IL-7 and IL-15 every 3 days. On Day 24, mice were euthanized and their tumors were collected for subsequent experiments. B Mice from each group were monitored for tumor growth every 3 days. These mice were euthanized at two weeks after infusion of CAR-T cells, and their tumors were collected. Scale bar represented 10 mm. C , D , and E ) Mononuclear cells were isolated from collected tumors. Subsequently, the proportion of infiltrating T cells ( C ) as well as their capabilities of Granzyme B ( D ) and IFN-γ (E) secretion were evaluated utilizing flow cytometry analysis. F IHC staining results of CD8 + T cells, Granzyme B cytotoxic molecules and IFN-γ cytokines were acquired within tumors derived from various groups. Scale bars represented 50 μm. Data represented as mean ± SD. N = 5 independent biological replicates. Data in ( B ) were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. Data in ( C - E ) were analyzed by one-way ANOVA with Tukey’s multiple comparisons test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Article Snippet: The nonspecific binding site was blocked with BSA for 30 minutes at room temperature, and then samples were incubated overnight at 4 °C with rabbit anti-human CD8, IFN-γ and Granzyme B (Proteintech, USA) antibodies at a dilution of 1:200 respectively.

Techniques: In Vivo, Injection, Control, Expressing, Isolation, Flow Cytometry, Immunohistochemistry, Derivative Assay