primary cd8 t cells  (Thermo Fisher)


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    Thermo Fisher primary cd8 t cells
    Primary Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    anti human cd8  (Thermo Fisher)


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    Thermo Fisher anti human cd8
    Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, <t>CD8,</t> neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005
    Anti Human Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Lung Inflammation in alpha-1-antitrypsin deficient individuals with normal lung function"

    Article Title: Lung Inflammation in alpha-1-antitrypsin deficient individuals with normal lung function

    Journal: Respiratory Research

    doi: 10.1186/s12931-023-02343-3

    Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, CD8, neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005
    Figure Legend Snippet: Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, CD8, neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005

    Techniques Used: Incubation

    anti cd8  (Thermo Fisher)


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    Thermo Fisher anti cd8
    HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of <t>CD8</t> + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented
    Anti Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway"

    Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    doi: 10.1186/s13046-023-02609-0

    HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented
    Figure Legend Snippet: HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Techniques Used: Western Blot, Flow Cytometry, Expressing

    HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA ( C ) or PTP1B-mcherry ( D ) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( J ). K IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented
    Figure Legend Snippet: HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA ( C ) or PTP1B-mcherry ( D ) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( J ). K IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Techniques Used: SDS Page, Silver Staining, Immunoprecipitation, Transfection, Magnetic Beads, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Cell Culture, Flow Cytometry

    HERC2 promoted hepatic STAT3 activation and facilitated HCC tumorigenesis in vivo. A Diagram for mouse primary HCC induction. 15-day-old male mice were intraperitoneally injected with 25 μg/g DEN. Two weeks later, the mice were intraperitoneally injected with 0.5 μl/g CCl4 once a week for consecutive 22 weeks. B Tumor formation in liver tissues was observed, and the red arrow indicates typical tumor nodes. C The statistical diagram for tumor numbers is presented. D The statistical diagram for the liver index is presented. ( E ) H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. F The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 100 μm. G Western blot analysis was performed to detect JAK2/STAT3 signaling activation. H RT-qPCR analysis was conducted to evaluate the levels of cancer stem cell-related genes. I Western blot analysis was performed to detect PD-L1 expression in liver tissues. J PD-L1 expression was evaluated by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. K The expression of CD8 was determined by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. L-N A total of 2 × 10 6 or 2 × 10 5 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. L A diagram of mouse orthotopically implanted HCC is presented. M Tumor formation in liver tissues is presented. N A statistical diagram for liver tumor volume is presented. O-Q A total of 2 × 10 6 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. O H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. P The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 250 μm. Q CD133 expression in liver tissues was determined by immunohistochemical assay. Top scale bars = 100 μm, and bottom scale bars = 25 μm. * p < 0.05, ** p < 0.01. Data from one representative experiment of three independent experiments are presented
    Figure Legend Snippet: HERC2 promoted hepatic STAT3 activation and facilitated HCC tumorigenesis in vivo. A Diagram for mouse primary HCC induction. 15-day-old male mice were intraperitoneally injected with 25 μg/g DEN. Two weeks later, the mice were intraperitoneally injected with 0.5 μl/g CCl4 once a week for consecutive 22 weeks. B Tumor formation in liver tissues was observed, and the red arrow indicates typical tumor nodes. C The statistical diagram for tumor numbers is presented. D The statistical diagram for the liver index is presented. ( E ) H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. F The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 100 μm. G Western blot analysis was performed to detect JAK2/STAT3 signaling activation. H RT-qPCR analysis was conducted to evaluate the levels of cancer stem cell-related genes. I Western blot analysis was performed to detect PD-L1 expression in liver tissues. J PD-L1 expression was evaluated by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. K The expression of CD8 was determined by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. L-N A total of 2 × 10 6 or 2 × 10 5 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. L A diagram of mouse orthotopically implanted HCC is presented. M Tumor formation in liver tissues is presented. N A statistical diagram for liver tumor volume is presented. O-Q A total of 2 × 10 6 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. O H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. P The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 250 μm. Q CD133 expression in liver tissues was determined by immunohistochemical assay. Top scale bars = 100 μm, and bottom scale bars = 25 μm. * p < 0.05, ** p < 0.01. Data from one representative experiment of three independent experiments are presented

    Techniques Used: Activation Assay, In Vivo, Injection, Staining, Expressing, Immunohistochemical staining, Western Blot, Quantitative RT-PCR

    cd8  (Thermo Fisher)


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    Thermo Fisher cd8
    TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + <t>CD8</t> neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.
    Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV"

    Article Title: Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV

    Journal: Nature Communications

    doi: 10.1038/s41467-023-36163-2

    TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + CD8 neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.
    Figure Legend Snippet: TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + CD8 neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.

    Techniques Used: Infection, Blocking Assay, Expressing, Derivative Assay, Two Tailed Test

    A HTOC were TCR-activated and infected with HIV. ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) and ARI for 6 days as described in methods ( n = 5 independent experiments). Representative flow cytometric data showing ODC-1 expression in CD3 + CD8 negative cells (CD4+ T cells) (top) and statistical analyses of MFI from five independent experiments (bottom) are shown. B Key intermediate steps in polyamine synthesis. Cells were stimulated and processed for flow cytometry as in A for determining the expression of EIF5A ( C ) and hypusinated EIF5A in CD4 + T cells ( D ). Light and dark shaded gray histograms represent staining controls for EIF5A ( C ) and hypusinated EIF5A ( D ) respectively. Mean values +/− SEM; **** P < 0.0001; *** P < 0.0002; Two-tailed; Unpaired t test in A – D . E Fluorometric polyamine estimation in cellular lysates from cells stimulated as in A for 4 or 7 days ( n = 3 independent experiments). Polyamine concentrations were normalized to cell numbers. Mean values +/− SD; *** P = 0.0002; Two-tailed; Welch’s t test. Circles within box plots represent each replicate.
    Figure Legend Snippet: A HTOC were TCR-activated and infected with HIV. ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) and ARI for 6 days as described in methods ( n = 5 independent experiments). Representative flow cytometric data showing ODC-1 expression in CD3 + CD8 negative cells (CD4+ T cells) (top) and statistical analyses of MFI from five independent experiments (bottom) are shown. B Key intermediate steps in polyamine synthesis. Cells were stimulated and processed for flow cytometry as in A for determining the expression of EIF5A ( C ) and hypusinated EIF5A in CD4 + T cells ( D ). Light and dark shaded gray histograms represent staining controls for EIF5A ( C ) and hypusinated EIF5A ( D ) respectively. Mean values +/− SEM; **** P < 0.0001; *** P < 0.0002; Two-tailed; Unpaired t test in A – D . E Fluorometric polyamine estimation in cellular lysates from cells stimulated as in A for 4 or 7 days ( n = 3 independent experiments). Polyamine concentrations were normalized to cell numbers. Mean values +/− SD; *** P = 0.0002; Two-tailed; Welch’s t test. Circles within box plots represent each replicate.

    Techniques Used: Infection, Blocking Assay, Expressing, Flow Cytometry, Staining, Two Tailed Test

    cd8 t cells  (Thermo Fisher)


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    Thermo Fisher cd8 t cells
    ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the <t>CD8</t> + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.
    Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy"

    Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

    Journal: Nature Communications

    doi: 10.1038/s41467-023-36045-7

    ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the CD8 + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.
    Figure Legend Snippet: ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the CD8 + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.

    Techniques Used: Injection, Expressing, Modification

    a Expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at −20, −4, or 37 °C for 10 min ( n = 3 independent experiments). b A schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8 + cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). c , d Typical flow cytometry plots ( c ) and quantitative data ( d ) on maturing bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for 24 h ( n = 3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at −20 °C for 10 min. e , f Quantitative data ( e ) and typical flow cytometry plots ( f ) of CD11c + Kb-SIINFEKL + BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells ( n = 3 independent experiments). g , h Typical flow cytometry histograms ( g ) and quantitative data ( h ) of CD8 + T cell proliferation after incubating them with the BMDCs matured by the EO771-OVA cells treated with aforementioned formulations ( n = 3 independent experiments). i, j Typical flow cytometry plots ( i ) and quantitative data ( j ) of activated CD8 + T cells following co-culture with the aforementioned BMDCs ( n = 3 independent experiments). k Representative images showing the process of activated CD8 + T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. l , m Typical flow cytometry plots ( l ) and quantitative data ( m ) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations ( n = 3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey’s multiple comparisons and correction. Data are presented as mean ± SD ( a , d , e , h , j , m ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a Expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at −20, −4, or 37 °C for 10 min ( n = 3 independent experiments). b A schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8 + cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). c , d Typical flow cytometry plots ( c ) and quantitative data ( d ) on maturing bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for 24 h ( n = 3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at −20 °C for 10 min. e , f Quantitative data ( e ) and typical flow cytometry plots ( f ) of CD11c + Kb-SIINFEKL + BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells ( n = 3 independent experiments). g , h Typical flow cytometry histograms ( g ) and quantitative data ( h ) of CD8 + T cell proliferation after incubating them with the BMDCs matured by the EO771-OVA cells treated with aforementioned formulations ( n = 3 independent experiments). i, j Typical flow cytometry plots ( i ) and quantitative data ( j ) of activated CD8 + T cells following co-culture with the aforementioned BMDCs ( n = 3 independent experiments). k Representative images showing the process of activated CD8 + T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. l , m Typical flow cytometry plots ( l ) and quantitative data ( m ) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations ( n = 3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey’s multiple comparisons and correction. Data are presented as mean ± SD ( a , d , e , h , j , m ). Source data are provided as a Source Data file.

    Techniques Used: Expressing, Flow Cytometry, Co-Culture Assay, Labeling, Cell Culture

    a – d Quantitative flow cytometry data of matured DCs (CD11c + CD86 + ) in inguinal lymph nodes (LNs, a ), and percentage of infiltrated CD8 + T cells ( b ), ratios of CD8 + /Treg cells ( c ), and percentage of cytotoxic T cells (CTLs, CD8 + GZMB + , d ) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery ( n = 3 mice). e Representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery ( n = 3 mice). f Representative flow cytometry plots showing the percentage of effector memory T cell (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. g Quantitative flow cytometry data of T EM in blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). h CD8 + /Treg ratios in the blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). i Representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. j Quantitative flow cytometry data of CD8 + /Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. k Quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. l Representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations ( n = 3 mice). Statistical analyses were done using two-way ANOVA with Sidak’s post-test and correction for multiple comparisons. The experiments for e and I were repeated three times independently ( n = 3 mice) with similar results. Data are presented as mean ± SD ( a – d , g , h , j , k ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a – d Quantitative flow cytometry data of matured DCs (CD11c + CD86 + ) in inguinal lymph nodes (LNs, a ), and percentage of infiltrated CD8 + T cells ( b ), ratios of CD8 + /Treg cells ( c ), and percentage of cytotoxic T cells (CTLs, CD8 + GZMB + , d ) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery ( n = 3 mice). e Representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery ( n = 3 mice). f Representative flow cytometry plots showing the percentage of effector memory T cell (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. g Quantitative flow cytometry data of T EM in blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). h CD8 + /Treg ratios in the blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). i Representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. j Quantitative flow cytometry data of CD8 + /Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. k Quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. l Representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations ( n = 3 mice). Statistical analyses were done using two-way ANOVA with Sidak’s post-test and correction for multiple comparisons. The experiments for e and I were repeated three times independently ( n = 3 mice) with similar results. Data are presented as mean ± SD ( a – d , g , h , j , k ). Source data are provided as a Source Data file.

    Techniques Used: Flow Cytometry, Immunofluorescence, Injection

    a A schematic illustration of the in vivo experimental design. Mice received an intravenous injection of various formulations every 3 days. Cryosurgery was performed on the orthotopic tumor (OT) at 8 h after the first injection of the various formulations. The lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day 10 after the first injection of the different formulations. b Weight of lungs collected from mice at the end of the study, suggesting inhibition of lung metastasis by ICIE ( n = 6 mice). c Representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. d Quantification of lung metastasis nodes per mouse from mice at the end of the study ( n = 6 mice). e Representative H&E staining images of lung tissues collected at the end of the study, showing the decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 µm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently ( n = 3 mice) with similar results. f The percentage of effector memory T cells (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood collected from mice with the various treatments ( n = 3 mice). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test and correction. Data are presented as mean ± SD ( b , d , f ). Source data are provided as a Source Data file.
    Figure Legend Snippet: a A schematic illustration of the in vivo experimental design. Mice received an intravenous injection of various formulations every 3 days. Cryosurgery was performed on the orthotopic tumor (OT) at 8 h after the first injection of the various formulations. The lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day 10 after the first injection of the different formulations. b Weight of lungs collected from mice at the end of the study, suggesting inhibition of lung metastasis by ICIE ( n = 6 mice). c Representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. d Quantification of lung metastasis nodes per mouse from mice at the end of the study ( n = 6 mice). e Representative H&E staining images of lung tissues collected at the end of the study, showing the decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 µm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently ( n = 3 mice) with similar results. f The percentage of effector memory T cells (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood collected from mice with the various treatments ( n = 3 mice). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test and correction. Data are presented as mean ± SD ( b , d , f ). Source data are provided as a Source Data file.

    Techniques Used: In Vivo, Injection, Inhibition, Isolation, Staining

    human cd3 cd4 cd8 mixture cd3 cd4 cd8  (Thermo Fisher)


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    Thermo Fisher human cd3 cd4 cd8 mixture cd3 cd4 cd8
    Comparison of lymphocyte subsets by acne severity
    Human Cd3 Cd4 Cd8 Mixture Cd3 Cd4 Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment"

    Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

    Journal: Indian Journal of Pharmacology

    doi: 10.4103/ijp.ijp_695_21

    Comparison of lymphocyte subsets by acne severity
    Figure Legend Snippet: Comparison of lymphocyte subsets by acne severity

    Techniques Used:

    Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment
    Figure Legend Snippet: Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment

    Techniques Used:

    cd8 t cells  (Thermo Fisher)


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    Thermo Fisher cd8 t cells
    (A – D) BM cells collected from tumor-free C57Bl/6J mice were cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor GM-CSF (20 ng/mL) and E0771 conditioned media (CM) (50% V/V) for 4 days in the presence of Veh or PEP to generate MDSCs. (A) Experimental outline and gating scheme. (B) Representative flow plot of CD11b + Gr-1 + cells in Veh- or PEP-treated BM cultures. (C) Frequencies and (D) absolute cell numbers of CD11b + Gr-1 + cells (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (*P < 0.05, ***P < 0.005). (E) Ccr2 gene expression in M-MDSC isolated from the spleens (sM-MDSC) of E0771 tumor-bearing mice and treated ex vivo with PEP (20 nM) or Veh for 72h as analyzed by qRT-PCR (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (****P < 0.001). (F–I) Splenic CD11b + Gr-1 + MDSCs (sMDSCs) were isolated from E0771 tumor-bearing mice and co-cultured with CellTrace Violet (CTV)-labeled <t>CD8</t> + T cells isolated from the spleens of tumor-free mice at a 1:2 T cell/MDSC ratio. sMDSCs were pre-treated with PEP (20 nM) or Veh for 3h prior to the addition of T cells and Dynabeads (anti-CD28/CD3). MDSC suppressive properties were assessed by CD8 + T cell activation (IFNγ expression) and proliferation (CTV dilution). (F) Representative contour plots and (G) IFNγ expression in CD8 + T cells (n = 3 mice). One-way ANOVA with multiple comparisons with Tukey correction (***P < 0.0001). (H–I) sPMN-MDSCs and sM-MDSCs were isolated from the spleens of tumor-bearing mice as previously described. Percent of CD8 + T cell proliferation after coculture with Veh- or PEP (20 nM)-pre-treated (H) sPMN-MDSCs and (I) sM-MDSCs at the indicated T cell:MDSC ratios (n = 3 mice). Shown are Means ± SEM. (**P < 0.01, ***P < 0.005).
    Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8 + T cells in breast cancer"

    Article Title: PKC agonism restricts innate immune suppression, promotes antigen cross-presentation and synergizes with agonistic CD40 antibody therapy to activate CD8 + T cells in breast cancer

    Journal: Cancer letters

    doi: 10.1016/j.canlet.2022.01.017

    (A – D) BM cells collected from tumor-free C57Bl/6J mice were cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor GM-CSF (20 ng/mL) and E0771 conditioned media (CM) (50% V/V) for 4 days in the presence of Veh or PEP to generate MDSCs. (A) Experimental outline and gating scheme. (B) Representative flow plot of CD11b + Gr-1 + cells in Veh- or PEP-treated BM cultures. (C) Frequencies and (D) absolute cell numbers of CD11b + Gr-1 + cells (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (*P < 0.05, ***P < 0.005). (E) Ccr2 gene expression in M-MDSC isolated from the spleens (sM-MDSC) of E0771 tumor-bearing mice and treated ex vivo with PEP (20 nM) or Veh for 72h as analyzed by qRT-PCR (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (****P < 0.001). (F–I) Splenic CD11b + Gr-1 + MDSCs (sMDSCs) were isolated from E0771 tumor-bearing mice and co-cultured with CellTrace Violet (CTV)-labeled CD8 + T cells isolated from the spleens of tumor-free mice at a 1:2 T cell/MDSC ratio. sMDSCs were pre-treated with PEP (20 nM) or Veh for 3h prior to the addition of T cells and Dynabeads (anti-CD28/CD3). MDSC suppressive properties were assessed by CD8 + T cell activation (IFNγ expression) and proliferation (CTV dilution). (F) Representative contour plots and (G) IFNγ expression in CD8 + T cells (n = 3 mice). One-way ANOVA with multiple comparisons with Tukey correction (***P < 0.0001). (H–I) sPMN-MDSCs and sM-MDSCs were isolated from the spleens of tumor-bearing mice as previously described. Percent of CD8 + T cell proliferation after coculture with Veh- or PEP (20 nM)-pre-treated (H) sPMN-MDSCs and (I) sM-MDSCs at the indicated T cell:MDSC ratios (n = 3 mice). Shown are Means ± SEM. (**P < 0.01, ***P < 0.005).
    Figure Legend Snippet: (A – D) BM cells collected from tumor-free C57Bl/6J mice were cultured in vitro in the presence of granulocyte-macrophage colony stimulating factor GM-CSF (20 ng/mL) and E0771 conditioned media (CM) (50% V/V) for 4 days in the presence of Veh or PEP to generate MDSCs. (A) Experimental outline and gating scheme. (B) Representative flow plot of CD11b + Gr-1 + cells in Veh- or PEP-treated BM cultures. (C) Frequencies and (D) absolute cell numbers of CD11b + Gr-1 + cells (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (*P < 0.05, ***P < 0.005). (E) Ccr2 gene expression in M-MDSC isolated from the spleens (sM-MDSC) of E0771 tumor-bearing mice and treated ex vivo with PEP (20 nM) or Veh for 72h as analyzed by qRT-PCR (n = 4 mice). Shown are Means ± SEM. Unpaired t-test (****P < 0.001). (F–I) Splenic CD11b + Gr-1 + MDSCs (sMDSCs) were isolated from E0771 tumor-bearing mice and co-cultured with CellTrace Violet (CTV)-labeled CD8 + T cells isolated from the spleens of tumor-free mice at a 1:2 T cell/MDSC ratio. sMDSCs were pre-treated with PEP (20 nM) or Veh for 3h prior to the addition of T cells and Dynabeads (anti-CD28/CD3). MDSC suppressive properties were assessed by CD8 + T cell activation (IFNγ expression) and proliferation (CTV dilution). (F) Representative contour plots and (G) IFNγ expression in CD8 + T cells (n = 3 mice). One-way ANOVA with multiple comparisons with Tukey correction (***P < 0.0001). (H–I) sPMN-MDSCs and sM-MDSCs were isolated from the spleens of tumor-bearing mice as previously described. Percent of CD8 + T cell proliferation after coculture with Veh- or PEP (20 nM)-pre-treated (H) sPMN-MDSCs and (I) sM-MDSCs at the indicated T cell:MDSC ratios (n = 3 mice). Shown are Means ± SEM. (**P < 0.01, ***P < 0.005).

    Techniques Used: Cell Culture, In Vitro, Expressing, Isolation, Ex Vivo, Quantitative RT-PCR, Labeling, Activation Assay

    (A – C) sMDSCs were pretreated with Veh or PEP (20 nM) overnight, then loaded with Alexa 647-conjugated ovalbumin (Alexa 647-OVA) for 3h before co-culture with CTV-labeled CD8 + T cells isolated from the spleens of OT-I mice for an additional 72h. OT-I CD8 + T cell proliferation and activation were assessed by CTV dilution via flow and IFNγ concentration in supernatants via ELISA, respectively. (A) MDSC OVA uptake was evaluated by Alexa 647-OVA MFI upon pretreatment with Veh or PEP (20 nM). Peak in gray represents OVA uptake by MDSCs loaded with Alexa 647-OVA for 3h on ice (negative control). Shown is a representative histogram (left) and Means ± SEM (right). (n = 5 mice). Unpaired t -test (***P < 0.005). (B) CTV dilution assessing OT-I CD8 + T cell proliferation. Shown is a representative histogram (n = 5 mice). (C) Bar graph shows IFNγ concentration in co-culture supernatants (n = 5 mice). One-way ANOVA with multiple comparisons with Tukey correction (****P < 0.001). (D – G) 1×10 6 CD45.2 + CD8 + T cells were isolated from the spleens of OT-I mice (donor), labeled with CTV, and adoptively transferred into CD45.1 mice (recipient) via tail vein injection on day −1. On day 1, sMDSCs that were pretreated with Veh or PEP (20 nM overnight) were loaded with OVA for 3h, then adoptively transferred into the same recipient mice via tail vein injection. Negative control consisted of adoptively transferred MDSCs without OVA. OT-I CD8 + T cells were analyzed via flow. (D) Experimental outline. (E) Representative flow plots of CD45.2 + OT-I CD8 + T cells in the spleens of recipient mice. (F) OT-I CD8 + T cell frequencies (left) and absolute cell numbers (right) in the spleens of recipient mice. (G) CTV dilution within gated CD45.2 + OT-I CD8 + T cells. Shown is a representative histogram (n = 3–4 mice). Shown are Means ± SEM. One-way ANOVA with multiple comparisons with Tukey correction (*P < 0.05, **P < 0.01).
    Figure Legend Snippet: (A – C) sMDSCs were pretreated with Veh or PEP (20 nM) overnight, then loaded with Alexa 647-conjugated ovalbumin (Alexa 647-OVA) for 3h before co-culture with CTV-labeled CD8 + T cells isolated from the spleens of OT-I mice for an additional 72h. OT-I CD8 + T cell proliferation and activation were assessed by CTV dilution via flow and IFNγ concentration in supernatants via ELISA, respectively. (A) MDSC OVA uptake was evaluated by Alexa 647-OVA MFI upon pretreatment with Veh or PEP (20 nM). Peak in gray represents OVA uptake by MDSCs loaded with Alexa 647-OVA for 3h on ice (negative control). Shown is a representative histogram (left) and Means ± SEM (right). (n = 5 mice). Unpaired t -test (***P < 0.005). (B) CTV dilution assessing OT-I CD8 + T cell proliferation. Shown is a representative histogram (n = 5 mice). (C) Bar graph shows IFNγ concentration in co-culture supernatants (n = 5 mice). One-way ANOVA with multiple comparisons with Tukey correction (****P < 0.001). (D – G) 1×10 6 CD45.2 + CD8 + T cells were isolated from the spleens of OT-I mice (donor), labeled with CTV, and adoptively transferred into CD45.1 mice (recipient) via tail vein injection on day −1. On day 1, sMDSCs that were pretreated with Veh or PEP (20 nM overnight) were loaded with OVA for 3h, then adoptively transferred into the same recipient mice via tail vein injection. Negative control consisted of adoptively transferred MDSCs without OVA. OT-I CD8 + T cells were analyzed via flow. (D) Experimental outline. (E) Representative flow plots of CD45.2 + OT-I CD8 + T cells in the spleens of recipient mice. (F) OT-I CD8 + T cell frequencies (left) and absolute cell numbers (right) in the spleens of recipient mice. (G) CTV dilution within gated CD45.2 + OT-I CD8 + T cells. Shown is a representative histogram (n = 3–4 mice). Shown are Means ± SEM. One-way ANOVA with multiple comparisons with Tukey correction (*P < 0.05, **P < 0.01).

    Techniques Used: Co-Culture Assay, Labeling, Isolation, Activation Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Negative Control, Injection

    E0771 tumor-bearing mice were treated with Veh, PEP (1 μg/mouse-IT), CD40 agonist (100μg/mouse-i.p.) or PEP-CD40 agonist combination on days indicated in A. Mice were sacrificed on day 19 and spleens and tumors were harvested for flow analysis. (A) Experimental outline. Tumor burden was assessed by (B) tumor volume as a function of time and (C) tumor weight at endpoint. (D) Percent change in body weights reported between days 6 (before treatment) and 19 (endpoint) (n = 5 mice). (E) Representative flow plots showing tumor IFNγ + TNFα + and CD103 + CD69 + CD8 + T cells. Frequencies of tumor (F) IFNγ + TNFα + CD8 + T cells and (G) CD103+ CD69 + CD8 + T cells (n = 3–5 mice). Shown are Means ± SEM. One-way ANOVA with multiple comparisons with Tukey correction (*P < 0.05, **P < 0.01).
    Figure Legend Snippet: E0771 tumor-bearing mice were treated with Veh, PEP (1 μg/mouse-IT), CD40 agonist (100μg/mouse-i.p.) or PEP-CD40 agonist combination on days indicated in A. Mice were sacrificed on day 19 and spleens and tumors were harvested for flow analysis. (A) Experimental outline. Tumor burden was assessed by (B) tumor volume as a function of time and (C) tumor weight at endpoint. (D) Percent change in body weights reported between days 6 (before treatment) and 19 (endpoint) (n = 5 mice). (E) Representative flow plots showing tumor IFNγ + TNFα + and CD103 + CD69 + CD8 + T cells. Frequencies of tumor (F) IFNγ + TNFα + CD8 + T cells and (G) CD103+ CD69 + CD8 + T cells (n = 3–5 mice). Shown are Means ± SEM. One-way ANOVA with multiple comparisons with Tukey correction (*P < 0.05, **P < 0.01).

    Techniques Used:

    cd8 t cells  (Thermo Fisher)


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    Thermo Fisher cd8 t cells
    a Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or <t>CD8</t> + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. b Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. * p < 0.05. One-way ANOVA using transformed data with Tukey’s post-hoc test. c Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. d Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. **** p < 0.0001. One-way ANOVA using transformed data with Tukey’s post-hoc test.
    Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4 + memory T cells"

    Article Title: The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4 + memory T cells

    Journal: Communications Biology

    doi: 10.1038/s42003-023-04420-1

    a Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. b Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. * p < 0.05. One-way ANOVA using transformed data with Tukey’s post-hoc test. c Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. d Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. **** p < 0.0001. One-way ANOVA using transformed data with Tukey’s post-hoc test.
    Figure Legend Snippet: a Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. b Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEA, SEB, SEC3, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. * p < 0.05. One-way ANOVA using transformed data with Tukey’s post-hoc test. c Frequency of CD69 + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. d Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEE, SpeC, or TSST-1. Each dot represents one donor. Horizontal bars indicate median values. **** p < 0.0001. One-way ANOVA using transformed data with Tukey’s post-hoc test.

    Techniques Used: Cell Culture, Transformation Assay

    a , b Frequency of CD107a + (blue filled circles), IFN-γ + (green filled circles), TNF-α + (red filled circles), or IL-2 + cells (black filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEB, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. c , d Functional profiles of TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) from human PBMCs cultured in medium alone (unstimulated) or stimulated for 24 h with SEB, SpeC, or TSST-1. Pie chart segments from concatenated data ( n = 4) represent the fractions of cells displaying the indicated numbers of functions (key). Arcs denote individual functions (key). The functional profiles elicited by SpeC and TSST-1 were significantly different from the corresponding functional profiles elicited by SEB ( p < 0.05; permutation test).
    Figure Legend Snippet: a , b Frequency of CD107a + (blue filled circles), IFN-γ + (green filled circles), TNF-α + (red filled circles), or IL-2 + cells (black filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEB, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. c , d Functional profiles of TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) from human PBMCs cultured in medium alone (unstimulated) or stimulated for 24 h with SEB, SpeC, or TSST-1. Pie chart segments from concatenated data ( n = 4) represent the fractions of cells displaying the indicated numbers of functions (key). Arcs denote individual functions (key). The functional profiles elicited by SpeC and TSST-1 were significantly different from the corresponding functional profiles elicited by SEB ( p < 0.05; permutation test).

    Techniques Used: Cell Culture, Functional Assay

    a , b Frequency of PD-1 + (mauve filled circles), LAG-3 + (aqua filled circles), or TIM-3 + cells (pink filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEB, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. c , d Expression profiles of coIRs (PD-1, LAG-3, and TIM-3) among TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) from human PBMCs stimulated for 24 h with SEB, SEE, SpeC, or TSST-1. Baseline expression was negligible. Pie chart segments from concatenated data ( n = 6) represent the fractions of cells expressing the indicated numbers of coIRs (key). The expression profiles elicited by SEE, SpeC, and TSST-1 were significantly different from the corresponding expression profiles elicited by SEB ( p < 0.05; permutation test). e , f Frequency of PD-1 + LAG-3 + (teal filled circles), PD-1 + TIM-3 + (gray filled circles), or PD-1 + LAG-3 + TIM-3 + cells (orange filled circles) among TRBV12-3/12-4 + CD4 + ( e ) or CD8 + T cells ( f ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h or 48 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. g Frequency of TIGIT + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h or 48 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05. One-way ANOVA with Tukey’s post-hoc test.
    Figure Legend Snippet: a , b Frequency of PD-1 + (mauve filled circles), LAG-3 + (aqua filled circles), or TIM-3 + cells (pink filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h with SEB, SpeC, or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. c , d Expression profiles of coIRs (PD-1, LAG-3, and TIM-3) among TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) from human PBMCs stimulated for 24 h with SEB, SEE, SpeC, or TSST-1. Baseline expression was negligible. Pie chart segments from concatenated data ( n = 6) represent the fractions of cells expressing the indicated numbers of coIRs (key). The expression profiles elicited by SEE, SpeC, and TSST-1 were significantly different from the corresponding expression profiles elicited by SEB ( p < 0.05; permutation test). e , f Frequency of PD-1 + LAG-3 + (teal filled circles), PD-1 + TIM-3 + (gray filled circles), or PD-1 + LAG-3 + TIM-3 + cells (orange filled circles) among TRBV12-3/12-4 + CD4 + ( e ) or CD8 + T cells ( f ) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h or 48 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. g Frequency of TIGIT + cells among TRBV12-3/12-4 + CD4 + (gray filled circles) or CD8 + T cells (green filled circles) from human PBMCs cultured in medium alone (unstim) or stimulated for 24 h or 48 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05. One-way ANOVA with Tukey’s post-hoc test.

    Techniques Used: Cell Culture, Expressing

    a , b Heatmaps showing the frequency (key) of TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) expressing CD69, IFN-γ, TNF-α, IL-2, CD107a, PD-1, LAG-3, TIM-3, or TIGIT across the T N , T SCM , T CM , T EM , and T EMRA subsets from human PBMCs stimulated for 24 h with SpeC or TSST-1. Concatenated data are shown ( n = 4). c , d Stacked bar charts showing the frequency of TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) across the T N , T SCM , T CM , T EM , and T EMRA subsets (key) from human PBMCs cultured in medium alone (unstimulated) or stimulated for 24 h or 48 h with SpeC or TSST-1 ( n = 4). Data are shown as mean ± SEM. e Frequency of Ki67 + cells among TRBV12-3/12-4 + CD4 + (gray filled symbols) or CD8 + T cells (green filled symbols) from human PBMCs cultured in medium alone (0) or stimulated for 24 h, 48 h, or 120 h with SpeC (circles) or TSST-1 (squares) ( n = 4). Data are shown as mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. f Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled symbols) or CD8 + T cells (green filled symbols) from human PBMCs cultured in medium alone (0) or stimulated for 24 h, 48 h, or 120 h with SpeC (circles) or TSST-1 (squares) ( n = 4). Data are shown as mean ± SEM.
    Figure Legend Snippet: a , b Heatmaps showing the frequency (key) of TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) expressing CD69, IFN-γ, TNF-α, IL-2, CD107a, PD-1, LAG-3, TIM-3, or TIGIT across the T N , T SCM , T CM , T EM , and T EMRA subsets from human PBMCs stimulated for 24 h with SpeC or TSST-1. Concatenated data are shown ( n = 4). c , d Stacked bar charts showing the frequency of TRBV12-3/12-4 + CD4 + ( c ) or CD8 + T cells ( d ) across the T N , T SCM , T CM , T EM , and T EMRA subsets (key) from human PBMCs cultured in medium alone (unstimulated) or stimulated for 24 h or 48 h with SpeC or TSST-1 ( n = 4). Data are shown as mean ± SEM. e Frequency of Ki67 + cells among TRBV12-3/12-4 + CD4 + (gray filled symbols) or CD8 + T cells (green filled symbols) from human PBMCs cultured in medium alone (0) or stimulated for 24 h, 48 h, or 120 h with SpeC (circles) or TSST-1 (squares) ( n = 4). Data are shown as mean ± SEM. * p < 0.05, *** p < 0.001, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test. f Frequency of TRBV12-3/12-4 + cells among CD4 + (gray filled symbols) or CD8 + T cells (green filled symbols) from human PBMCs cultured in medium alone (0) or stimulated for 24 h, 48 h, or 120 h with SpeC (circles) or TSST-1 (squares) ( n = 4). Data are shown as mean ± SEM.

    Techniques Used: Expressing, Cell Culture

    a , b Frequency of CD69 + (gray filled circles), TNF-α + (red filled circles), IL-2 + (black filled circles), or IFN-γ + cells (green filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs pretreated with anti-human HLA-DR or an isotype control antibody (IgG 2a ) and then stimulated for 24 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01. One-way ANOVA with Tukey’s post-hoc test.
    Figure Legend Snippet: a , b Frequency of CD69 + (gray filled circles), TNF-α + (red filled circles), IL-2 + (black filled circles), or IFN-γ + cells (green filled circles) among TRBV12-3/12-4 + CD4 + ( a ) or CD8 + T cells ( b ) from human PBMCs pretreated with anti-human HLA-DR or an isotype control antibody (IgG 2a ) and then stimulated for 24 h with SpeC or TSST-1. Each dot represents one donor. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01. One-way ANOVA with Tukey’s post-hoc test.

    Techniques Used:

    a Representative flow cytometry plots showing the frequency of SEE or TSST-1 tetramer + events among clonal TRBV12-3/12-4 + CD8 + T cells cultured at a 1:1 ratio with the MR B-LCL. Plots are gated on live singlets (left) or doublets (right). b , c Frequency of SEE or TSST-1 tetramer + events among clonal TRBV12-3/12-4 + CD8 + T cells gated as singlets ( b ) or doublets ( c ) in the absence (dark gray filled circles) or presence of the MR B-LCL (green filled circles). Each dot represents one experiment. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test.
    Figure Legend Snippet: a Representative flow cytometry plots showing the frequency of SEE or TSST-1 tetramer + events among clonal TRBV12-3/12-4 + CD8 + T cells cultured at a 1:1 ratio with the MR B-LCL. Plots are gated on live singlets (left) or doublets (right). b , c Frequency of SEE or TSST-1 tetramer + events among clonal TRBV12-3/12-4 + CD8 + T cells gated as singlets ( b ) or doublets ( c ) in the absence (dark gray filled circles) or presence of the MR B-LCL (green filled circles). Each dot represents one experiment. Data are shown as mean ± SEM. * p < 0.05, ** p < 0.01, **** p < 0.0001. One-way ANOVA with Tukey’s post-hoc test.

    Techniques Used: Flow Cytometry, Cell Culture

    anti cd8 antibody  (Thermo Fisher)


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    Thermo Fisher anti cd8 antibody
    Anti Cd8 Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cd8a bv786  (Thermo Fisher)


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    Thermo Fisher cd8a bv786
    A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of <t>CD8</t> T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
    Cd8a Bv786, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/cd8a bv786/product/Thermo Fisher
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cd8a bv786 - by Bioz Stars, 2023-02
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    1) Product Images from "FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies"

    Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

    Journal: bioRxiv

    doi: 10.1101/2023.01.19.522856

    A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
    Figure Legend Snippet: A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Techniques Used: In Vivo, Flow Cytometry, Expressing, Fluorescence, SPR Assay, Binding Assay, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Variant Assay

    A) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 (200 ug alone or in combination with 50 ug intratumoral 2B6 blockade prior on days 0 and 3) comapred to 2B6 only control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). Blockade of the inhibitory FcyR (FcyRIIB) leads to significantly less Tregs when coadministered with 10D1-IgG1 compared to 10D1-IgG2. B) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants (e.g. IgG1 or IgG2) were given alone or in combination with 2B6 antibody blockade (50 ug given intratumoral) on days 0, 3, and 6. The combination of the IgG1-subclass variant led to significantly improved tumor control when compared to anti-CTLA-4 antibody alone or the anti-CTLA-4 IgG2 subclass antibody with 2B6. C) Table of the 10D1 IgG subclass variants generated for these studies, along with their relative binding affinities for activating vs inhibitory FcyRs. D) ELISA-based quantification of the relative affinity of the various subclass variants tested (in E and F). E) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of anti-CTLA-4 antibody variants (200 ug on days 0 and 3) comapred to to IgG control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). F) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to control, Fc-null (N297A), or IgG1. G) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1 or 1121) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced GAALIE variants led to significantly improved tumor control, even when placed on the 1121 (tremelimumab) backbone. n = 4-7 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
    Figure Legend Snippet: A) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 (200 ug alone or in combination with 50 ug intratumoral 2B6 blockade prior on days 0 and 3) comapred to 2B6 only control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). Blockade of the inhibitory FcyR (FcyRIIB) leads to significantly less Tregs when coadministered with 10D1-IgG1 compared to 10D1-IgG2. B) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants (e.g. IgG1 or IgG2) were given alone or in combination with 2B6 antibody blockade (50 ug given intratumoral) on days 0, 3, and 6. The combination of the IgG1-subclass variant led to significantly improved tumor control when compared to anti-CTLA-4 antibody alone or the anti-CTLA-4 IgG2 subclass antibody with 2B6. C) Table of the 10D1 IgG subclass variants generated for these studies, along with their relative binding affinities for activating vs inhibitory FcyRs. D) ELISA-based quantification of the relative affinity of the various subclass variants tested (in E and F). E) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of anti-CTLA-4 antibody variants (200 ug on days 0 and 3) comapred to to IgG control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). F) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to control, Fc-null (N297A), or IgG1. G) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1 or 1121) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced GAALIE variants led to significantly improved tumor control, even when placed on the 1121 (tremelimumab) backbone. n = 4-7 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Techniques Used: In Vivo, Blocking Assay, Flow Cytometry, Variant Assay, Generated, Binding Assay, Enzyme-linked Immunosorbent Assay

    A) In vivo evaluation of the Fc-optimized anti-CCR8 antibody (GAALIE variant with decreased FcyRIIB binding) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma and MB49 bladder tumor microenvironment following systemic (i.p.) administration of 200 ug of CCR8-IgG1, CCR8-GRLR, or CCR8-GAALIE on days 0 and 3 following randomization. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). B) In vivo evaluation of the effect of Fc-enhanced anti-CCR8 antibodies on tumor control in the MC38, MB49, and B16 tumor models. Here, 200 ug of anti-CCR8 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variant GAALIE led to significantly improved tumor control when compared to Fc-null (GRLR), or IgG1. C) In vivo comparison of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) versus anti-CCR8 antibodies on tumor control in the MC38 tumor model. Here, 200 ug of each subclass variant was given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to IgG1 or afucosylated versions, with anti-CCR8 GAALIE demonstrating improved activity over anti-CTLA-4 GAALIE. n = 5-10 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
    Figure Legend Snippet: A) In vivo evaluation of the Fc-optimized anti-CCR8 antibody (GAALIE variant with decreased FcyRIIB binding) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma and MB49 bladder tumor microenvironment following systemic (i.p.) administration of 200 ug of CCR8-IgG1, CCR8-GRLR, or CCR8-GAALIE on days 0 and 3 following randomization. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). B) In vivo evaluation of the effect of Fc-enhanced anti-CCR8 antibodies on tumor control in the MC38, MB49, and B16 tumor models. Here, 200 ug of anti-CCR8 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variant GAALIE led to significantly improved tumor control when compared to Fc-null (GRLR), or IgG1. C) In vivo comparison of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) versus anti-CCR8 antibodies on tumor control in the MC38 tumor model. Here, 200 ug of each subclass variant was given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to IgG1 or afucosylated versions, with anti-CCR8 GAALIE demonstrating improved activity over anti-CTLA-4 GAALIE. n = 5-10 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Techniques Used: In Vivo, Variant Assay, Binding Assay, Flow Cytometry, Activity Assay

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    Thermo Fisher primary cd8 t cells
    Primary Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher anti human cd8
    Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, <t>CD8,</t> neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005
    Anti Human Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of <t>CD8</t> + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented
    Anti Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + <t>CD8</t> neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.
    Cd8, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher cd8 t cells
    ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the <t>CD8</t> + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.
    Cd8 T Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Comparison of lymphocyte subsets by acne severity
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    Comparison of lymphocyte subsets by acne severity
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    A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of <t>CD8</t> T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.
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    Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, CD8, neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005

    Journal: Respiratory Research

    Article Title: Lung Inflammation in alpha-1-antitrypsin deficient individuals with normal lung function

    doi: 10.1186/s12931-023-02343-3

    Figure Lengend Snippet: Large airway inflammatory cells in normal and alpha1- antitrypsin deficient Individuals. A Bronchial biopsies from normal and alpha1- antitrypsin deficient individuals were incubated with primary antibodies against CD68, CD45, CD8, neutrophil elastase (NE), and AA1; HRP-conjugated secondary antibody and HRP-DAB developer as described in the methods. B The cells were counted and expressed as the number of cells per mm2 of basement membrane in the section. * P < 0.05, ** P < 0.005

    Article Snippet: Cat # MA5-32166) (T-helper cells), anti-human CD8 (ThermoFischer Scientifics, Waltham, MA.

    Techniques: Incubation

    HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

    doi: 10.1186/s13046-023-02609-0

    Figure Lengend Snippet: HERC2 participated in the immune evasion of HCC cells. A HERC2-deficient Huh7 cells and HERC2-overexpressing HCC-97 h cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. B and C Activated PBMCs were cocultured with Huh7 cells or HERC2-overexpressing HCC-97 h cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( B ). C IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. D The correlation between HERC2 expression and levels of PD-L1 based on TCGA datasets ( n = 369). E The correlation between HERC2 expression and liver immune cell recruitment based on TCGA datasets ( n = 369). ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

    Techniques: Western Blot, Flow Cytometry, Expressing

    HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA ( C ) or PTP1B-mcherry ( D ) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( J ). K IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

    doi: 10.1186/s13046-023-02609-0

    Figure Lengend Snippet: HERC2 regulated JAK2/STAT3 signaling through direct interaction with PTP1B. A Huh7 cells were treated with 50 ng/ml IL-6 for 20 min. The extracted protein was precipitated with an antibody against HERC2 and separated by SDS-PAGE followed by silver staining. On the other hand, the band was analyzed with 4-D label-free quantitative proteomics. B Huh7 cells were treated with 50 ng/ml IL-6 for 20 min, and the interaction between HERC2 and PTP1B was validated by an immunoprecipitation assay. C-D HEK293T cells were co-transfected with HERC2-Flag and PTP1B-HA ( C ) or PTP1B-mcherry ( D ) plasmids. C Immunoprecipitation was performed using an anti-FLAG magnetic beads. The presence of coprecipitated HERC2 was determined by immunoblotting with the anti-HA antibody. D The colocalization of HERC2 and PTP1B in the cells was analyzed by immunofluorescence assay. E HERC2 and PTP1B double-deficient Huh7 cell lines were established. The cells were treated with 50 ng/ml IL-6 for 20 min, and the phosphorylation levels of JAK2 and STAT3 were detected by western blot analysis. F Cells were treated with 50 ng/ml IL-6 for 24 h. The RT-qPCR assay was used to detect the mRNA expression of CSC-related genes. G Cells were cultured with medium containing 100 × N2, 50 × B27, 20 ng/ml EGF, 10 nmol FGF, 5 μg/ml insulin, and 0.4% BSA for 7 days, scale bars = 100 μm. H The cells were treated with 20 μM sorafenib for 24 h. A flow cytometry assay was used to determine the percentage of apoptotic cells. I Cells were treated with 50 ng/ml IL-6 for 24 h and then subjected to western blot assay for PD-L1 detection. J and K Activated PBMCs were cocultured with HCC cells at the ratio of 4:1 for 24 h. Apoptosis of HCC cells was detected by flow cytometry assay ( J ). K IFN-γ levels of CD8 + T cells were determined by flow cytometry analysis. NS: not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. Data from one representative experiment of three independent experiments are presented

    Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

    Techniques: SDS Page, Silver Staining, Immunoprecipitation, Transfection, Magnetic Beads, Western Blot, Immunofluorescence, Quantitative RT-PCR, Expressing, Cell Culture, Flow Cytometry

    HERC2 promoted hepatic STAT3 activation and facilitated HCC tumorigenesis in vivo. A Diagram for mouse primary HCC induction. 15-day-old male mice were intraperitoneally injected with 25 μg/g DEN. Two weeks later, the mice were intraperitoneally injected with 0.5 μl/g CCl4 once a week for consecutive 22 weeks. B Tumor formation in liver tissues was observed, and the red arrow indicates typical tumor nodes. C The statistical diagram for tumor numbers is presented. D The statistical diagram for the liver index is presented. ( E ) H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. F The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 100 μm. G Western blot analysis was performed to detect JAK2/STAT3 signaling activation. H RT-qPCR analysis was conducted to evaluate the levels of cancer stem cell-related genes. I Western blot analysis was performed to detect PD-L1 expression in liver tissues. J PD-L1 expression was evaluated by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. K The expression of CD8 was determined by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. L-N A total of 2 × 10 6 or 2 × 10 5 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. L A diagram of mouse orthotopically implanted HCC is presented. M Tumor formation in liver tissues is presented. N A statistical diagram for liver tumor volume is presented. O-Q A total of 2 × 10 6 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. O H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. P The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 250 μm. Q CD133 expression in liver tissues was determined by immunohistochemical assay. Top scale bars = 100 μm, and bottom scale bars = 25 μm. * p < 0.05, ** p < 0.01. Data from one representative experiment of three independent experiments are presented

    Journal: Journal of Experimental & Clinical Cancer Research : CR

    Article Title: HERC2 promotes inflammation-driven cancer stemness and immune evasion in hepatocellular carcinoma by activating STAT3 pathway

    doi: 10.1186/s13046-023-02609-0

    Figure Lengend Snippet: HERC2 promoted hepatic STAT3 activation and facilitated HCC tumorigenesis in vivo. A Diagram for mouse primary HCC induction. 15-day-old male mice were intraperitoneally injected with 25 μg/g DEN. Two weeks later, the mice were intraperitoneally injected with 0.5 μl/g CCl4 once a week for consecutive 22 weeks. B Tumor formation in liver tissues was observed, and the red arrow indicates typical tumor nodes. C The statistical diagram for tumor numbers is presented. D The statistical diagram for the liver index is presented. ( E ) H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. F The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 100 μm. G Western blot analysis was performed to detect JAK2/STAT3 signaling activation. H RT-qPCR analysis was conducted to evaluate the levels of cancer stem cell-related genes. I Western blot analysis was performed to detect PD-L1 expression in liver tissues. J PD-L1 expression was evaluated by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. K The expression of CD8 was determined by immunohistochemical assay. Top scale bars = 250 μm, and bottom scale bars = 50 μm. L-N A total of 2 × 10 6 or 2 × 10 5 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. L A diagram of mouse orthotopically implanted HCC is presented. M Tumor formation in liver tissues is presented. N A statistical diagram for liver tumor volume is presented. O-Q A total of 2 × 10 6 HERC2-overexpressing Hepa1-6 cells or control cells were orthotopically injected into mouse livers. O H&E staining was performed to examine the pathological changes in liver tissues. Top scale bars = 250 μm, and bottom scale bars = 25 μm. P The expression of Ki67 and PCNA in liver tissues was determined by immunohistochemical assay. Scale bars = 250 μm. Q CD133 expression in liver tissues was determined by immunohistochemical assay. Top scale bars = 100 μm, and bottom scale bars = 25 μm. * p < 0.05, ** p < 0.01. Data from one representative experiment of three independent experiments are presented

    Article Snippet: Cells were then stained with anti-CD8 (17–0086-42, Thermo Fisher Scientific), anti-CD4 (12–0049-42, Thermo Fisher Scientific), or anti-CD56 (11–0566-42, Thermo Fisher Scientific) antibodies at 4 °C in the dark for 30 min.

    Techniques: Activation Assay, In Vivo, Injection, Staining, Expressing, Immunohistochemical staining, Western Blot, Quantitative RT-PCR

    TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + CD8 neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.

    Journal: Nature Communications

    Article Title: Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV

    doi: 10.1038/s41467-023-36163-2

    Figure Lengend Snippet: TCR-stimulated HTOC was infected with HIV. Control cultures were not infected (Uninfected). In some cultures, ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) for 6 days. A FOXP3 and CD25 expression (top) and PD-1 and IFN-γ expression (bottom) in CD4 + (CD3 + CD8 neg ) FOXP3 + cells, showing the frequency of T regs and T regDys . Expression of IL-17A and ROR-γt ( B ), and IL-17A and CCR6 ( C ) in CD4 + cells, showing the frequency of Th17 cells. Representative contour plots (left), statistical analyses of proportions of the cells from 5 independent experiments showing the frequency of these subsets ( A , B , right). D Statistical analysis of T regDys /Th17 ratio based on the results in A and B . Results derived from five independent experiments are presented as mean values +/− SEM. A – D **** P < 0.0001; *** P < 0.0002; * P < 0.02; Two-tailed; Unpaired t test. Circles within box plots represent each replicate.

    Article Snippet: Flow cytometry antibodies for ROR-γt (AFKJS-9), IL-1β (CRM56), CCR6 (R6H1), IL-17A(eBio64DEC17), CD25 (M-A251), Ki-67 (SolA15), HIF-1α (Mgc3), CD4 (OKT4), CD45(HI30), CD8 (RPA-T8), IFN-γ (4S.B3), FOXP3(236A/E7), AREG (AREG559), HLADR, and phospho-caspase 1 (Ser376) were all purchased from Invitrogen/Thermofisher Scientific.

    Techniques: Infection, Blocking Assay, Expressing, Derivative Assay, Two Tailed Test

    A HTOC were TCR-activated and infected with HIV. ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) and ARI for 6 days as described in methods ( n = 5 independent experiments). Representative flow cytometric data showing ODC-1 expression in CD3 + CD8 negative cells (CD4+ T cells) (top) and statistical analyses of MFI from five independent experiments (bottom) are shown. B Key intermediate steps in polyamine synthesis. Cells were stimulated and processed for flow cytometry as in A for determining the expression of EIF5A ( C ) and hypusinated EIF5A in CD4 + T cells ( D ). Light and dark shaded gray histograms represent staining controls for EIF5A ( C ) and hypusinated EIF5A ( D ) respectively. Mean values +/− SEM; **** P < 0.0001; *** P < 0.0002; Two-tailed; Unpaired t test in A – D . E Fluorometric polyamine estimation in cellular lysates from cells stimulated as in A for 4 or 7 days ( n = 3 independent experiments). Polyamine concentrations were normalized to cell numbers. Mean values +/− SD; *** P = 0.0002; Two-tailed; Welch’s t test. Circles within box plots represent each replicate.

    Journal: Nature Communications

    Article Title: Polyamine metabolism impacts T cell dysfunction in the oral mucosa of people living with HIV

    doi: 10.1038/s41467-023-36163-2

    Figure Lengend Snippet: A HTOC were TCR-activated and infected with HIV. ARI was added the next day to block subsequent rounds of infection. Cells were allowed to expand in the presence of TGF-β1 (10 ng/ml) and IL-2 (100 U/ml) and ARI for 6 days as described in methods ( n = 5 independent experiments). Representative flow cytometric data showing ODC-1 expression in CD3 + CD8 negative cells (CD4+ T cells) (top) and statistical analyses of MFI from five independent experiments (bottom) are shown. B Key intermediate steps in polyamine synthesis. Cells were stimulated and processed for flow cytometry as in A for determining the expression of EIF5A ( C ) and hypusinated EIF5A in CD4 + T cells ( D ). Light and dark shaded gray histograms represent staining controls for EIF5A ( C ) and hypusinated EIF5A ( D ) respectively. Mean values +/− SEM; **** P < 0.0001; *** P < 0.0002; Two-tailed; Unpaired t test in A – D . E Fluorometric polyamine estimation in cellular lysates from cells stimulated as in A for 4 or 7 days ( n = 3 independent experiments). Polyamine concentrations were normalized to cell numbers. Mean values +/− SD; *** P = 0.0002; Two-tailed; Welch’s t test. Circles within box plots represent each replicate.

    Article Snippet: Flow cytometry antibodies for ROR-γt (AFKJS-9), IL-1β (CRM56), CCR6 (R6H1), IL-17A(eBio64DEC17), CD25 (M-A251), Ki-67 (SolA15), HIF-1α (Mgc3), CD4 (OKT4), CD45(HI30), CD8 (RPA-T8), IFN-γ (4S.B3), FOXP3(236A/E7), AREG (AREG559), HLADR, and phospho-caspase 1 (Ser376) were all purchased from Invitrogen/Thermofisher Scientific.

    Techniques: Infection, Blocking Assay, Expressing, Flow Cytometry, Staining, Two Tailed Test

    ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the CD8 + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.

    Journal: Nature Communications

    Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

    doi: 10.1038/s41467-023-36045-7

    Figure Lengend Snippet: ICIE combines cryosurgery with intravenously injected (i.v. ) irinotecan (or camptothecin/CPT) and PD-L1 silencing siRNA (siR)-laden cold-responsive nanoparticles (CPT&siR CRNPs) to engineer the tumor microenvironment (TME), via promoting immunogenic cell death (ICD) and reducing the expression of programmed death Ligand 1 (PD-L1) in cancer cells. This turns the TME from immunologically “cold” (i.e., immunosuppressive) into immunologically “hot” (i.e., immuno-active). As a result, the CD8 + T cells can be activated to exert tumor eradication both locally and systemically. The CPT&siR CRNPs are prepared by a double-emulsion method detailed in the Methods section using CPT, siR, Poly (D, L-lactide-co-glycolide) (PLGA), poly (N-isopropylacrylamide copolymerized butyl acrylate) (pNIPAAm-BA), chitosan-modified PF-127 (CS-PF-127), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and sodium chloride (NaCl). HMGB1: high mobility group box protein 1, CRT: calreticulin, HSP-70: heat shock protein-70, HSP-90: heat shock protein-90, DC: dendritic cell, and T EM : effective memory T cell.

    Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

    Techniques: Injection, Expressing, Modification

    a Expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at −20, −4, or 37 °C for 10 min ( n = 3 independent experiments). b A schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8 + cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). c , d Typical flow cytometry plots ( c ) and quantitative data ( d ) on maturing bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for 24 h ( n = 3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at −20 °C for 10 min. e , f Quantitative data ( e ) and typical flow cytometry plots ( f ) of CD11c + Kb-SIINFEKL + BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells ( n = 3 independent experiments). g , h Typical flow cytometry histograms ( g ) and quantitative data ( h ) of CD8 + T cell proliferation after incubating them with the BMDCs matured by the EO771-OVA cells treated with aforementioned formulations ( n = 3 independent experiments). i, j Typical flow cytometry plots ( i ) and quantitative data ( j ) of activated CD8 + T cells following co-culture with the aforementioned BMDCs ( n = 3 independent experiments). k Representative images showing the process of activated CD8 + T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. l , m Typical flow cytometry plots ( l ) and quantitative data ( m ) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations ( n = 3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey’s multiple comparisons and correction. Data are presented as mean ± SD ( a , d , e , h , j , m ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

    doi: 10.1038/s41467-023-36045-7

    Figure Lengend Snippet: a Expression of HMGB1, CRT, HSP-70, and HSP-90 in EO771 cells after incubating them with various formulations at −20, −4, or 37 °C for 10 min ( n = 3 independent experiments). b A schematic illustration of ICD production following treatment of CPT&siR CRNPs in combination with cryosurgery to mature DCs, present the tumor-specific antigen to T cells by the matured DCs, and activate CD8 + cytotoxic T lymphocytes (CTLs) to attack cancer cells with reduced expression of PD-L1 via the release of granzyme B (GZMB). c , d Typical flow cytometry plots ( c ) and quantitative data ( d ) on maturing bone marrow dendritic cells (BMDCs) after co-culturing them with EO771-OVA cells for 24 h ( n = 3 independent experiments). The EO771-OVA cells were pretreated with the indicated various formulations. “+C” represents cold treatment at −20 °C for 10 min. e , f Quantitative data ( e ) and typical flow cytometry plots ( f ) of CD11c + Kb-SIINFEKL + BMDC percentage following co-culture with the aforementioned, pretreated EO771-OVA cells ( n = 3 independent experiments). g , h Typical flow cytometry histograms ( g ) and quantitative data ( h ) of CD8 + T cell proliferation after incubating them with the BMDCs matured by the EO771-OVA cells treated with aforementioned formulations ( n = 3 independent experiments). i, j Typical flow cytometry plots ( i ) and quantitative data ( j ) of activated CD8 + T cells following co-culture with the aforementioned BMDCs ( n = 3 independent experiments). k Representative images showing the process of activated CD8 + T cells attacking EO771-OVA cells labeled with CellTracker™ Green CMFDA Dye. The T cells were activated by the BMDCs co-cultured with EO771-OVA cells that received CPT&siR CRNPs+C treatment. The experiment was repeated three times independently with similar results. l , m Typical flow cytometry plots ( l ) and quantitative data ( m ) of dead EO771-OVA cells after T cell attacking. T cells were activated by BMDCs cocultured with EO771-OVA cells with different formulations ( n = 3 independent experiments). Statistical analyses were done using one-way ANOVA with Tukey’s multiple comparisons and correction. Data are presented as mean ± SD ( a , d , e , h , j , m ). Source data are provided as a Source Data file.

    Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

    Techniques: Expressing, Flow Cytometry, Co-Culture Assay, Labeling, Cell Culture

    a – d Quantitative flow cytometry data of matured DCs (CD11c + CD86 + ) in inguinal lymph nodes (LNs, a ), and percentage of infiltrated CD8 + T cells ( b ), ratios of CD8 + /Treg cells ( c ), and percentage of cytotoxic T cells (CTLs, CD8 + GZMB + , d ) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery ( n = 3 mice). e Representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery ( n = 3 mice). f Representative flow cytometry plots showing the percentage of effector memory T cell (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. g Quantitative flow cytometry data of T EM in blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). h CD8 + /Treg ratios in the blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). i Representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. j Quantitative flow cytometry data of CD8 + /Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. k Quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. l Representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations ( n = 3 mice). Statistical analyses were done using two-way ANOVA with Sidak’s post-test and correction for multiple comparisons. The experiments for e and I were repeated three times independently ( n = 3 mice) with similar results. Data are presented as mean ± SD ( a – d , g , h , j , k ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

    doi: 10.1038/s41467-023-36045-7

    Figure Lengend Snippet: a – d Quantitative flow cytometry data of matured DCs (CD11c + CD86 + ) in inguinal lymph nodes (LNs, a ), and percentage of infiltrated CD8 + T cells ( b ), ratios of CD8 + /Treg cells ( c ), and percentage of cytotoxic T cells (CTLs, CD8 + GZMB + , d ) in primary (Prim) tumors of mice with cryosurgery and L (left side) tumors of mice with no cryosurgery ( n = 3 mice). e Representative immunofluorescence images of infiltrated CTLs in primary (Prim) tumors from mice with cryosurgery ( n = 3 mice). f Representative flow cytometry plots showing the percentage of effector memory T cell (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood (BL) collected from mice with cryosurgery and injection of one of the various formulations. g Quantitative flow cytometry data of T EM in blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). h CD8 + /Treg ratios in the blood of mice with/without cryosurgery and injection of one of the different formulations ( n = 3 mice). i Representative flow cytometry plots of Treg cells in blood collected from mice with cryosurgery and injection of one of the different formulations. j Quantitative flow cytometry data of CD8 + /Treg ratios in distant (Dist) tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. k Quantitative flow cytometry data of the percentage of infiltrated CTLs in Dist tumors of mice with cryosurgery and R (right side) tumors of mice without cryosurgery ( n = 3 mice). All mice were injected with one of the indicated formulations. l Representative immunofluorescence images of infiltrated CTLs in Dist tumors from mice with cryosurgery on Prim tumors and injection of one of the different formulations ( n = 3 mice). Statistical analyses were done using two-way ANOVA with Sidak’s post-test and correction for multiple comparisons. The experiments for e and I were repeated three times independently ( n = 3 mice) with similar results. Data are presented as mean ± SD ( a – d , g , h , j , k ). Source data are provided as a Source Data file.

    Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

    Techniques: Flow Cytometry, Immunofluorescence, Injection

    a A schematic illustration of the in vivo experimental design. Mice received an intravenous injection of various formulations every 3 days. Cryosurgery was performed on the orthotopic tumor (OT) at 8 h after the first injection of the various formulations. The lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day 10 after the first injection of the different formulations. b Weight of lungs collected from mice at the end of the study, suggesting inhibition of lung metastasis by ICIE ( n = 6 mice). c Representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. d Quantification of lung metastasis nodes per mouse from mice at the end of the study ( n = 6 mice). e Representative H&E staining images of lung tissues collected at the end of the study, showing the decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 µm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently ( n = 3 mice) with similar results. f The percentage of effector memory T cells (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood collected from mice with the various treatments ( n = 3 mice). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test and correction. Data are presented as mean ± SD ( b , d , f ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: In-situ cryo-immune engineering of tumor microenvironment with cold-responsive nanotechnology for cancer immunotherapy

    doi: 10.1038/s41467-023-36045-7

    Figure Lengend Snippet: a A schematic illustration of the in vivo experimental design. Mice received an intravenous injection of various formulations every 3 days. Cryosurgery was performed on the orthotopic tumor (OT) at 8 h after the first injection of the various formulations. The lung metastasis model was induced by intravenous injection of 4T1 cancer cells on day 10 after the first injection of the different formulations. b Weight of lungs collected from mice at the end of the study, suggesting inhibition of lung metastasis by ICIE ( n = 6 mice). c Representative photographs of lung tissues isolated at the end of the study, showing inhibition of lung metastasis by the ICIE treatment. Scale bar: 1 mm. d Quantification of lung metastasis nodes per mouse from mice at the end of the study ( n = 6 mice). e Representative H&E staining images of lung tissues collected at the end of the study, showing the decreased formation of metastasis in the lungs of mice with the ICIE treatment. Scale bar: 1 mm for low-magnification images and 200 µm for zoom-in images. The areas circled by blue dashed lines are metastases in the lungs. The experiments were repeated three times independently ( n = 3 mice) with similar results. f The percentage of effector memory T cells (T EM , CD3 + CD8 + CD44 + CD62L − ) in blood collected from mice with the various treatments ( n = 3 mice). Statistical analyses were performed using one-way ANOVA with Tukey’s multiple comparisons test and correction. Data are presented as mean ± SD ( b , d , f ). Source data are provided as a Source Data file.

    Article Snippet: To determine T cell proliferation, OT-I CD8 + T cells (1 × 10 5 ) that could recognize the OVA antigen were labeled with CellTrace™ Violet Cell Proliferation Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction and co-cultured for 3 days with BMDCs (1 × 10 5 ) pre-incubated with EO771-OVA cells with the various treatments for 24 h given above in 96-well plate.

    Techniques: In Vivo, Injection, Inhibition, Isolation, Staining

    Comparison of lymphocyte subsets by acne severity

    Journal: Indian Journal of Pharmacology

    Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

    doi: 10.4103/ijp.ijp_695_21

    Figure Lengend Snippet: Comparison of lymphocyte subsets by acne severity

    Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

    Techniques:

    Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment

    Journal: Indian Journal of Pharmacology

    Article Title: Evaluation of peripheral lymphocyte subsets in acne vulgaris patients before and after systemic isotretinoin treatment

    doi: 10.4103/ijp.ijp_695_21

    Figure Lengend Snippet: Comparison of lymphocyte subsets in patients with acne vulgaris before and after systemic isotretinoin treatment

    Article Snippet: CD3/CD16/CD56 monoclonal antibody (M2AB, C5.9, J5511), fluorescein isothiocyanate, PE (catalog number: MA1–12207), MS anti-HU CD45 PERCP (catalog number: MHCD4531), anti-human CD19 APC SJ25C1 (catalog number: 17-0198-41), human CD3/CD4/CD8 mixture CD3/CD4/CD8 (catalog number: CD348A), anti-human CD38 APC-eFluor® 780 (catalog number: 47-0389-41), anti-human HLA-DR APC (LN3) (catalog number: 17-9956-41), anti-human CD45RA APC HI100 (catalog number: 17-0458-41), and anti-human CD45RO APC-eFluor® 780 (catalog number: 47-0457-41) were obtained from Thermo Fisher Scientific (Meridian Road Rockford, IL 61105 USA).

    Techniques:

    A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Journal: bioRxiv

    Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

    doi: 10.1101/2023.01.19.522856

    Figure Lengend Snippet: A) Cartoon illustration of humanized CTLA-4 mice crossed to hFcyR mice to generate a hCTLA-4/hFcyR colony for the in vivo evaluation of fully human anti-CTLA-4 antibody variants. B) Flow cytometry evaluation of endogenous murine CTLA-4 cell surface expression in WT C57BL/6 mice compared to hCTLA-4/hFcyR mice, expressed as mean fluorescence intensity (MFI). C) Ipilimumab is a human IgG1 antibody whereas tremelimumab is a human IgG2 subclass. D) Surface plasmon resonance characterization of binding to human CTLA-4 by recombinant variants of ipilimumab and tremelimumab and their respective KDs. E) Evaluation of the ability of 10D1 and 1121 Fabs on IgG2 backbone to block interaction of hCTLA-4 with B7.1 as measured by ELISA. F) In vivo evaluation of the MC38 colon carcinoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). G) Evaluation of the in vivo activity of anti-CTLA-4 subclass variant on MC38 tumor growth. Ipilimumab and tremelimumab were tested as both IgG1 and IgG2 Fc subclass variants. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0, 3 and 6. H) Ipilimumab and tremelimumab were both placed on an IgG2 Fc backbone and tested for antitumor activity in the MC38 tumor model. I) In vivo evaluation of the B16 melanoma tumor microenvironment of subcutaneous tumors following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 when compared to IgG1 isotype control. Tumor bearing hCTLA-4/hFcγR mice were treated with 200 µg of anti-CTLA-4 mAb variants on days 0 and 3 following randomization, and sacrificed 24 hours later for flow cytometry analysis of CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). J) Ipilimumab in either and IgG1 or IgG2 format was tested for antitumor activity in the B16 tumor model. n = 4-8 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

    Techniques: In Vivo, Flow Cytometry, Expressing, Fluorescence, SPR Assay, Binding Assay, Recombinant, Blocking Assay, Enzyme-linked Immunosorbent Assay, Activity Assay, Variant Assay

    A) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 (200 ug alone or in combination with 50 ug intratumoral 2B6 blockade prior on days 0 and 3) comapred to 2B6 only control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). Blockade of the inhibitory FcyR (FcyRIIB) leads to significantly less Tregs when coadministered with 10D1-IgG1 compared to 10D1-IgG2. B) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants (e.g. IgG1 or IgG2) were given alone or in combination with 2B6 antibody blockade (50 ug given intratumoral) on days 0, 3, and 6. The combination of the IgG1-subclass variant led to significantly improved tumor control when compared to anti-CTLA-4 antibody alone or the anti-CTLA-4 IgG2 subclass antibody with 2B6. C) Table of the 10D1 IgG subclass variants generated for these studies, along with their relative binding affinities for activating vs inhibitory FcyRs. D) ELISA-based quantification of the relative affinity of the various subclass variants tested (in E and F). E) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of anti-CTLA-4 antibody variants (200 ug on days 0 and 3) comapred to to IgG control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). F) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to control, Fc-null (N297A), or IgG1. G) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1 or 1121) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced GAALIE variants led to significantly improved tumor control, even when placed on the 1121 (tremelimumab) backbone. n = 4-7 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Journal: bioRxiv

    Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

    doi: 10.1101/2023.01.19.522856

    Figure Lengend Snippet: A) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of 10D1-IgG1 or 10D1-IgG2 (200 ug alone or in combination with 50 ug intratumoral 2B6 blockade prior on days 0 and 3) comapred to 2B6 only control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). Blockade of the inhibitory FcyR (FcyRIIB) leads to significantly less Tregs when coadministered with 10D1-IgG1 compared to 10D1-IgG2. B) In vivo evaluation of the effect of FcyRIIB blocking antibody (clone 2B6) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants (e.g. IgG1 or IgG2) were given alone or in combination with 2B6 antibody blockade (50 ug given intratumoral) on days 0, 3, and 6. The combination of the IgG1-subclass variant led to significantly improved tumor control when compared to anti-CTLA-4 antibody alone or the anti-CTLA-4 IgG2 subclass antibody with 2B6. C) Table of the 10D1 IgG subclass variants generated for these studies, along with their relative binding affinities for activating vs inhibitory FcyRs. D) ELISA-based quantification of the relative affinity of the various subclass variants tested (in E and F). E) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma tumor microenvironment following systemic (i.p.) administration of anti-CTLA-4 antibody variants (200 ug on days 0 and 3) comapred to to IgG control. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). F) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to control, Fc-null (N297A), or IgG1. G) In vivo evaluation of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1 or 1121) on tumor control in the MC38 tumor model. Here, 200 ug of 10D1 subclass variants were given on days 0, 3, and 6. The Fc-enhanced GAALIE variants led to significantly improved tumor control, even when placed on the 1121 (tremelimumab) backbone. n = 4-7 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

    Techniques: In Vivo, Blocking Assay, Flow Cytometry, Variant Assay, Generated, Binding Assay, Enzyme-linked Immunosorbent Assay

    A) In vivo evaluation of the Fc-optimized anti-CCR8 antibody (GAALIE variant with decreased FcyRIIB binding) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma and MB49 bladder tumor microenvironment following systemic (i.p.) administration of 200 ug of CCR8-IgG1, CCR8-GRLR, or CCR8-GAALIE on days 0 and 3 following randomization. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). B) In vivo evaluation of the effect of Fc-enhanced anti-CCR8 antibodies on tumor control in the MC38, MB49, and B16 tumor models. Here, 200 ug of anti-CCR8 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variant GAALIE led to significantly improved tumor control when compared to Fc-null (GRLR), or IgG1. C) In vivo comparison of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) versus anti-CCR8 antibodies on tumor control in the MC38 tumor model. Here, 200 ug of each subclass variant was given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to IgG1 or afucosylated versions, with anti-CCR8 GAALIE demonstrating improved activity over anti-CTLA-4 GAALIE. n = 5-10 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Journal: bioRxiv

    Article Title: FcyRIIB is a novel immune checkpoint in the tumor microenvironment limiting activity of Treg-targeting antibodies

    doi: 10.1101/2023.01.19.522856

    Figure Lengend Snippet: A) In vivo evaluation of the Fc-optimized anti-CCR8 antibody (GAALIE variant with decreased FcyRIIB binding) on Treg depletion. Flow cytometric evaluation of the MC38 colon carcinoma and MB49 bladder tumor microenvironment following systemic (i.p.) administration of 200 ug of CCR8-IgG1, CCR8-GRLR, or CCR8-GAALIE on days 0 and 3 following randomization. Mice were sacrificed 24 hours later and flow cytometry analysis was performed for CD8 T cells, CD4 effector T cells, and T regulatory cells (Tregs). B) In vivo evaluation of the effect of Fc-enhanced anti-CCR8 antibodies on tumor control in the MC38, MB49, and B16 tumor models. Here, 200 ug of anti-CCR8 subclass variants were given on days 0, 3, and 6. The Fc-enhanced variant GAALIE led to significantly improved tumor control when compared to Fc-null (GRLR), or IgG1. C) In vivo comparison of the effect of Fc-enhanced anti-CTLA-4 antibodies (clone 10D1) versus anti-CCR8 antibodies on tumor control in the MC38 tumor model. Here, 200 ug of each subclass variant was given on days 0, 3, and 6. The Fc-enhanced variants led to significantly improved tumor control when compared to IgG1 or afucosylated versions, with anti-CCR8 GAALIE demonstrating improved activity over anti-CTLA-4 GAALIE. n = 5-10 mice per group; bars represent SEM, and *p < 0.05, **p < 0.01, ***p < 0.001. ****p < 0.0001.

    Article Snippet: For cell surface staining, cells were incubated for 30 minutes at 4C with the following anti-mouse antibodies from Biolegend CD4-FITC, clone GK1.5, CD8a-BV786, clone 53-6.7; CD3e-Percp-Cy5.5, clone 17A2; and ThermoFisher CD45-AlexaFluor 700, clone 30-F11.

    Techniques: In Vivo, Variant Assay, Binding Assay, Flow Cytometry, Activity Assay