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Becton Dickinson igg2a pe
( A ): Surface expression of α and β integrins on PC3 cells. Solid line: specific fluorescence, dashed line: IgG1-PE or <t>IgG2a-PE.</t> The abscissa shows the relative logarithmic distribution of the relative fluorescence intensity of α2, α3, α5, α6, β1, and β4. The ordinate shows cell number. 10,000 cells were counted. The figure is representative for n = 6. Scatter plots are shown in Supplement S1 . ( B ): Influence of sE-cadherin (5 µg/mL) on the integrin expression profile of PC3 cells. The untreated control is set to 100%. Values are means ± SD, n = 4; * indicates significant difference to controls. ( C ): Western blot of α and β integrins, ILK, FAK, pFAK, and pAkt in PC3 depending on the influence of sE-cadherin (5 µg/mL). Protein levels were measured 24 h after treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample. ( D ): Adhesion to collagen and chemotaxis of PC3 cells after blockade of integrins α3 or β1. The untreated control is set to 100%. 5 separate fields of 0.25 mm 2 were counted at 200× magnification (means ± SD, n = 3); * indicates significant difference to controls. ( E ): PC3 cell growth after blockade of integrins α3 or β1. Cell number evaluated after 24, 48, and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05. ( F ): Western blot of cytoskeleton-related proteins in PC3 following sE-cadherin exposure (0.5, 5 µg/mL). Protein levels were measured after 24 h of treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample.
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1) Product Images from "Deciphering the Molecular Machinery—Influence of sE-Cadherin on Tumorigenic Traits of Prostate Cancer Cells"

Article Title: Deciphering the Molecular Machinery—Influence of sE-Cadherin on Tumorigenic Traits of Prostate Cancer Cells

Journal: Biology

doi: 10.3390/biology10101007

( A ): Surface expression of α and β integrins on PC3 cells. Solid line: specific fluorescence, dashed line: IgG1-PE or IgG2a-PE. The abscissa shows the relative logarithmic distribution of the relative fluorescence intensity of α2, α3, α5, α6, β1, and β4. The ordinate shows cell number. 10,000 cells were counted. The figure is representative for n = 6. Scatter plots are shown in Supplement S1 . ( B ): Influence of sE-cadherin (5 µg/mL) on the integrin expression profile of PC3 cells. The untreated control is set to 100%. Values are means ± SD, n = 4; * indicates significant difference to controls. ( C ): Western blot of α and β integrins, ILK, FAK, pFAK, and pAkt in PC3 depending on the influence of sE-cadherin (5 µg/mL). Protein levels were measured 24 h after treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample. ( D ): Adhesion to collagen and chemotaxis of PC3 cells after blockade of integrins α3 or β1. The untreated control is set to 100%. 5 separate fields of 0.25 mm 2 were counted at 200× magnification (means ± SD, n = 3); * indicates significant difference to controls. ( E ): PC3 cell growth after blockade of integrins α3 or β1. Cell number evaluated after 24, 48, and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05. ( F ): Western blot of cytoskeleton-related proteins in PC3 following sE-cadherin exposure (0.5, 5 µg/mL). Protein levels were measured after 24 h of treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample.
Figure Legend Snippet: ( A ): Surface expression of α and β integrins on PC3 cells. Solid line: specific fluorescence, dashed line: IgG1-PE or IgG2a-PE. The abscissa shows the relative logarithmic distribution of the relative fluorescence intensity of α2, α3, α5, α6, β1, and β4. The ordinate shows cell number. 10,000 cells were counted. The figure is representative for n = 6. Scatter plots are shown in Supplement S1 . ( B ): Influence of sE-cadherin (5 µg/mL) on the integrin expression profile of PC3 cells. The untreated control is set to 100%. Values are means ± SD, n = 4; * indicates significant difference to controls. ( C ): Western blot of α and β integrins, ILK, FAK, pFAK, and pAkt in PC3 depending on the influence of sE-cadherin (5 µg/mL). Protein levels were measured 24 h after treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample. ( D ): Adhesion to collagen and chemotaxis of PC3 cells after blockade of integrins α3 or β1. The untreated control is set to 100%. 5 separate fields of 0.25 mm 2 were counted at 200× magnification (means ± SD, n = 3); * indicates significant difference to controls. ( E ): PC3 cell growth after blockade of integrins α3 or β1. Cell number evaluated after 24, 48, and 72 h by MTT assay. Error bars indicate standard deviation, * = p ≤ 0.05. ( F ): Western blot of cytoskeleton-related proteins in PC3 following sE-cadherin exposure (0.5, 5 µg/mL). Protein levels were measured after 24 h of treatment. All bands are representative of n = 3. β-actin served as loading control and is representatively shown once. 50 µg were used per sample.

Techniques Used: Expressing, Fluorescence, Western Blot, Chemotaxis Assay, MTT Assay, Standard Deviation

2) Product Images from "Co-inhibition of TIGIT, PD1, and Tim3 reverses dysfunction of Wilms tumor protein-1 (WT1)-specific CD8+ T lymphocytes after dendritic cell vaccination in gastric cancer"

Article Title: Co-inhibition of TIGIT, PD1, and Tim3 reverses dysfunction of Wilms tumor protein-1 (WT1)-specific CD8+ T lymphocytes after dendritic cell vaccination in gastric cancer

Journal: American Journal of Cancer Research

doi:

TIGIT inhibition synergizes with PD1 and Tim3 inhibition to promote the frequencies of cytokine-producing WT1-specific CD8+ T cells. A. Dot plots from one representative gastric cancer patient showing the proportions of IFN-γ-, TNF- and IL-2-producing A2/WT1 235-243 tet+ CD8+ T cells among total CD8+ T cells. PBMCs from gastric cancer patients were incubated for 6 days with WT1 235-243 peptide and blocking mAbs against TIGIT (αTIGIT) and/or PD1 (αPD1) and/or Tim3 (αTim3) or isotype control mAbs (IgG), prior to evaluating intracellular cytokine production of A2/WT1 235-243 tet+ CD8+ T cells in response to the cognate peptide. B. Fold changes of the frequencies of IFN-γ-, TNF- and IL-2-producing A2/WT1 235-243 tet+ CD8+ T cells after a 6-day in vitro stimulation with cognate peptide and the indicated mAb (n = 10). Each data-point represents the proportion of WT1-specific CTLs for each patient. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: TIGIT inhibition synergizes with PD1 and Tim3 inhibition to promote the frequencies of cytokine-producing WT1-specific CD8+ T cells. A. Dot plots from one representative gastric cancer patient showing the proportions of IFN-γ-, TNF- and IL-2-producing A2/WT1 235-243 tet+ CD8+ T cells among total CD8+ T cells. PBMCs from gastric cancer patients were incubated for 6 days with WT1 235-243 peptide and blocking mAbs against TIGIT (αTIGIT) and/or PD1 (αPD1) and/or Tim3 (αTim3) or isotype control mAbs (IgG), prior to evaluating intracellular cytokine production of A2/WT1 235-243 tet+ CD8+ T cells in response to the cognate peptide. B. Fold changes of the frequencies of IFN-γ-, TNF- and IL-2-producing A2/WT1 235-243 tet+ CD8+ T cells after a 6-day in vitro stimulation with cognate peptide and the indicated mAb (n = 10). Each data-point represents the proportion of WT1-specific CTLs for each patient. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Inhibition, Incubation, Blocking Assay, In Vitro

3) Product Images from "Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages"

Article Title: Dopamine Receptor Activation Increases HIV Entry into Primary Human Macrophages

Journal: PLoS ONE

doi: 10.1371/journal.pone.0108232

CCR5 is necessary for dopamine-mediated increase in HIV entry. Macrophages were infected with HIV BaL in the presence of 10 9 M or 10 6 M dopamine and analyzed by flow cytometry for CCR5. (A) A representative histogram of CCR5 surface expression in a single donor in response to HIV alone (IgG – dashed grey lines, CCR5 – solid grey lines) is shown. (B) The mean MFI of CCR5 on the surface of macrophages from seven donors infected with HIV alone (white), HIV + 10 9 M DA (light gray) or HIV + 10 6 M DA (dark gray) relative HIV alone. Neither concentration of dopamine-induced significant changes in the expression of this protein on the cell surface relative to the macrophages infected with HIV alone. (C) Macrophages were pretreated with 2×10 7 M TAK779, and then an MOI of 0.01 β-lac HIV was added in the presence or absence of 10 8 M dopamine. As controls, both macrophages pretreated with TAK779 and macrophages not pretreated were infected with HIV alone. Dopamine significantly increased HIV entry (n = 6, p = 0.0194 *), and pretreatment with 2×10 7 M of the CCR5 inhibitor TAK779 blocked HIV entry into macrophages infected in both the presence and absence of dopamine (HIV + TAK779, p = 0.0001 **, HIV + TAK779 + DA, p = 0.0003 ***).
Figure Legend Snippet: CCR5 is necessary for dopamine-mediated increase in HIV entry. Macrophages were infected with HIV BaL in the presence of 10 9 M or 10 6 M dopamine and analyzed by flow cytometry for CCR5. (A) A representative histogram of CCR5 surface expression in a single donor in response to HIV alone (IgG – dashed grey lines, CCR5 – solid grey lines) is shown. (B) The mean MFI of CCR5 on the surface of macrophages from seven donors infected with HIV alone (white), HIV + 10 9 M DA (light gray) or HIV + 10 6 M DA (dark gray) relative HIV alone. Neither concentration of dopamine-induced significant changes in the expression of this protein on the cell surface relative to the macrophages infected with HIV alone. (C) Macrophages were pretreated with 2×10 7 M TAK779, and then an MOI of 0.01 β-lac HIV was added in the presence or absence of 10 8 M dopamine. As controls, both macrophages pretreated with TAK779 and macrophages not pretreated were infected with HIV alone. Dopamine significantly increased HIV entry (n = 6, p = 0.0194 *), and pretreatment with 2×10 7 M of the CCR5 inhibitor TAK779 blocked HIV entry into macrophages infected in both the presence and absence of dopamine (HIV + TAK779, p = 0.0001 **, HIV + TAK779 + DA, p = 0.0003 ***).

Techniques Used: Infection, Flow Cytometry, Cytometry, Expressing, Concentration Assay

4) Product Images from "Resistance to the mTOR Inhibitor Temsirolimus Alters Adhesion and Migration Behavior of Renal Cell Carcinoma Cells through an Integrin α5– and Integrin β3–Dependent Mechanism"

Article Title: Resistance to the mTOR Inhibitor Temsirolimus Alters Adhesion and Migration Behavior of Renal Cell Carcinoma Cells through an Integrin α5– and Integrin β3–Dependent Mechanism

Journal: Neoplasia (New York, N.Y.)

doi: 10.1016/j.neo.2014.03.011

Integrin α and β expression in KTC par and KTC res . To evaluate background staining of PE-conjugated antibodies, goat anti-mouse IgG1-PE or IgG2a-PE was used. Fluorescence was measured using a FACScan flow cytometer. * indicates significant difference between the resistant and the sensitive tumor subline. (B) Modification of intracellular integrin protein level. Tumor cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted on the membrane incubated with appropriate mAbs. β-Actin served as the internal control. The figure shows one representative from three separate experiments.
Figure Legend Snippet: Integrin α and β expression in KTC par and KTC res . To evaluate background staining of PE-conjugated antibodies, goat anti-mouse IgG1-PE or IgG2a-PE was used. Fluorescence was measured using a FACScan flow cytometer. * indicates significant difference between the resistant and the sensitive tumor subline. (B) Modification of intracellular integrin protein level. Tumor cell lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and blotted on the membrane incubated with appropriate mAbs. β-Actin served as the internal control. The figure shows one representative from three separate experiments.

Techniques Used: Expressing, Staining, Fluorescence, Flow Cytometry, Cytometry, Modification, Polyacrylamide Gel Electrophoresis, Incubation

5) Product Images from "Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and β1-triggered motility, migration and invasion of prostate cancer cells"

Article Title: Resistance to the mTOR-inhibitor RAD001 elevates integrin α2- and β1-triggered motility, migration and invasion of prostate cancer cells

Journal: British Journal of Cancer

doi: 10.1038/bjc.2012.313

FACS analysis of integrin α and β subtype expression on PC3 par versus PC3 res cells. Cells were washed in blocking solution and then stained with specific monoclonal antibodies as listed in Materials and Methods. To evaluate background staining of PE-conjugated antibodies, goat anti-mouse IgG1-PE or IgG2a-PE was used (dotted lines). Fluorescence was analysed using a FACScan flow cytometer. Mean fluorescence values are given below the histograms. One from three independent experiments.
Figure Legend Snippet: FACS analysis of integrin α and β subtype expression on PC3 par versus PC3 res cells. Cells were washed in blocking solution and then stained with specific monoclonal antibodies as listed in Materials and Methods. To evaluate background staining of PE-conjugated antibodies, goat anti-mouse IgG1-PE or IgG2a-PE was used (dotted lines). Fluorescence was analysed using a FACScan flow cytometer. Mean fluorescence values are given below the histograms. One from three independent experiments.

Techniques Used: FACS, Expressing, Blocking Assay, Staining, Fluorescence, Flow Cytometry, Cytometry

6) Product Images from "CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1"

Article Title: CD8+ T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1

Journal: Cancer Research

doi: 10.1158/0008-5472.CAN-11-2637

BTLA blockade increases the frequencies of proliferating and total NY-ESO-1-specific CD8+ T cells and adds to PD-1 and Tim-3 blockades. ( A ) Representative flow cytometry analysis from two melanoma patients showing percentages of CFSE lo A2/NY-ESO-1 157-165 tet + CD8+ T cells among total CD8+ T cells. CFSE-labeled PBMCs were incubated for 6 days with NY-ESO-1 157-165 peptide or HIVpol 476-484 peptide and blocking mAbs against BTLA ( aBTLA ) and/or PD-1 ( aPD-1 ) and/or Tim-3 (aTim-3) or an isotype control antibody ( IgG ). Fold changes of the frequencies of CFSE lo ( B ) and total ( C ) A2/NY-ESO-1 157-165 tet + CD8+ T cells after 6-day IVS with cognate peptide and blocking anti-BTLA and/or anti-PD-1 and/or anti-Tim-3 mAbs ( n = 10). The ratio of the percentages of CFSE lo and total A2/NY-ESO-1 157-165 tet + CD8+ T cells in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: BTLA blockade increases the frequencies of proliferating and total NY-ESO-1-specific CD8+ T cells and adds to PD-1 and Tim-3 blockades. ( A ) Representative flow cytometry analysis from two melanoma patients showing percentages of CFSE lo A2/NY-ESO-1 157-165 tet + CD8+ T cells among total CD8+ T cells. CFSE-labeled PBMCs were incubated for 6 days with NY-ESO-1 157-165 peptide or HIVpol 476-484 peptide and blocking mAbs against BTLA ( aBTLA ) and/or PD-1 ( aPD-1 ) and/or Tim-3 (aTim-3) or an isotype control antibody ( IgG ). Fold changes of the frequencies of CFSE lo ( B ) and total ( C ) A2/NY-ESO-1 157-165 tet + CD8+ T cells after 6-day IVS with cognate peptide and blocking anti-BTLA and/or anti-PD-1 and/or anti-Tim-3 mAbs ( n = 10). The ratio of the percentages of CFSE lo and total A2/NY-ESO-1 157-165 tet + CD8+ T cells in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Flow Cytometry, Cytometry, Labeling, Incubation, Blocking Assay

BTLA blockade increases the frequencies of cytokine-producing NY-ESO-1-specific CD8+ T cells and adds to PD-1 and Tim-3 blockades. ( A ) Representative flow cytometry analysis from one melanoma patient showing percentages of IFN-γ-, TNF and IL-2-producing A2/NY-ESO-1 157-165 tet + CD8+ T cells among total CD8+ T cells. PBMCs were incubated for 6 days with NY-ESO-1 157-165 peptide or with HIVpol 476-484 peptide and blocking mAbs against BTLA ( aBTLA ) and/or PD-1 ( aPD-1 ) and/or Tim-3 ( aTim-3 ) or an isotype control antibody ( IgG ), prior to evaluating intracellular cytokine production of A2/NY-ESO-1 157-165 tet + CD8+ T cells in response to cognate peptide. Fold changes of the frequencies of IFN-γ- ( B ), TNF- ( C ) and IL-2-producing ( D ) A2/NY-ESO-1 157-165 tet + CD8+ T cells after 6-day IVS with cognate peptide and blocking anti-BTLA and/or anti-PD-1 and/or anti-Tim-3 mAbs. The ratio of the frequency of cytokine-producing tet + CD8+ T cells from melanoma patients ( n = 10) in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: BTLA blockade increases the frequencies of cytokine-producing NY-ESO-1-specific CD8+ T cells and adds to PD-1 and Tim-3 blockades. ( A ) Representative flow cytometry analysis from one melanoma patient showing percentages of IFN-γ-, TNF and IL-2-producing A2/NY-ESO-1 157-165 tet + CD8+ T cells among total CD8+ T cells. PBMCs were incubated for 6 days with NY-ESO-1 157-165 peptide or with HIVpol 476-484 peptide and blocking mAbs against BTLA ( aBTLA ) and/or PD-1 ( aPD-1 ) and/or Tim-3 ( aTim-3 ) or an isotype control antibody ( IgG ), prior to evaluating intracellular cytokine production of A2/NY-ESO-1 157-165 tet + CD8+ T cells in response to cognate peptide. Fold changes of the frequencies of IFN-γ- ( B ), TNF- ( C ) and IL-2-producing ( D ) A2/NY-ESO-1 157-165 tet + CD8+ T cells after 6-day IVS with cognate peptide and blocking anti-BTLA and/or anti-PD-1 and/or anti-Tim-3 mAbs. The ratio of the frequency of cytokine-producing tet + CD8+ T cells from melanoma patients ( n = 10) in the presence of indicated antibody treatment and isotype control antibody is shown. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Blocking Assay

7) Product Images from "Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis"

Article Title: Molecular targeting of prostate cancer cells by a triple drug combination down-regulates integrin driven adhesion processes, delays cell cycle progression and interferes with the cdk-cyclin axis

Journal: BMC Cancer

doi: 10.1186/1471-2407-11-375

Analysis of integrin surface expression (left) and intracellular integrin protein level (right) . PC-3 or LNCaP cells were treated either with 1 μM AEE788, 1 mM VPA or 1 nM RAD001, or with all compounds simultaneously (triple). Non-treated cells served as the controls. To explore integrin surface expression, cells were washed in blocking solution and stained with specific monoclonal antibodies as listed in materials and methods. A mouse IgG1-PE or IgG2a-PE was used as the isotype control. Fluorescence was analysed using a FACScan flow cytometer, and a histogram plot was generated to show PE-fluorescence. The mean fluorescence units are given in percentage difference to the controls. One of three independent experiments is shown here. *indicates significant difference to controls, #indicates significant difference to single drug treatment. To carry out western blotting, cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies (including anti-ILK, anti-FAK and anti-pFAK). β-actin served as the internal control. The figure shows one representative from three separate experiments.
Figure Legend Snippet: Analysis of integrin surface expression (left) and intracellular integrin protein level (right) . PC-3 or LNCaP cells were treated either with 1 μM AEE788, 1 mM VPA or 1 nM RAD001, or with all compounds simultaneously (triple). Non-treated cells served as the controls. To explore integrin surface expression, cells were washed in blocking solution and stained with specific monoclonal antibodies as listed in materials and methods. A mouse IgG1-PE or IgG2a-PE was used as the isotype control. Fluorescence was analysed using a FACScan flow cytometer, and a histogram plot was generated to show PE-fluorescence. The mean fluorescence units are given in percentage difference to the controls. One of three independent experiments is shown here. *indicates significant difference to controls, #indicates significant difference to single drug treatment. To carry out western blotting, cell lysates were subjected to SDS-PAGE and blotted on the membrane incubated with the respective monoclonal antibodies (including anti-ILK, anti-FAK and anti-pFAK). β-actin served as the internal control. The figure shows one representative from three separate experiments.

Techniques Used: Expressing, Blocking Assay, Staining, Fluorescence, Flow Cytometry, Cytometry, Generated, Western Blot, SDS Page, Incubation

8) Product Images from "Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients"

Article Title: Upregulation of Tim-3 and PD-1 expression is associated with tumor antigen-specific CD8+ T cell dysfunction in melanoma patients

Journal: The Journal of Experimental Medicine

doi: 10.1084/jem.20100637

Blockade of the Tim-3–Tim-3L pathway alone or in combination PD-1–PD-L1 blockade with prolonged antigen stimulation increases the frequency of proliferating and total NY-ESO-1–specific CD8 + T cells. Representative flow cytometry analysis from two melanoma patients showing percentages of CFSE lo A2/NY-ESO-1 157–165 tet + CD8 + T cells among total CD8 + T cells (A) and pooled data from melanoma patients ( n = 9) showing the variation in the numbers of CFSE lo (B) and total (C) A2/NY-ESO-1 157–165 tet + cells for 10 6 CD8 + T cells. CFSE-labeled PBMCs were incubated for 6 d with NY-ESO-1 157–165 peptide or HIVpol 476–484 peptide and blocking anti–Tim-3 (aTim-3), and/or anti–PD-L1 (aPD-L1) mAbs or an isotype control antibody (IgG). (D and E) Fold change of the frequencies of CFSE lo (D) and total (E) A2/NY-ESO-1 157–165 tet + CD8 + T cells after 6-d IVS with cognate peptide and blocking anti–Tim-3 (aTim-3) and/or anti–PD-L1 (aPD-L1) mAbs ( n = 9). The ratio of the percentages of CFSE lo and total A2/NY-ESO-1 157–165 tet + CD8 + T cells in the presence of indicated antibody treatment and isotype control antibody is shown. The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: Blockade of the Tim-3–Tim-3L pathway alone or in combination PD-1–PD-L1 blockade with prolonged antigen stimulation increases the frequency of proliferating and total NY-ESO-1–specific CD8 + T cells. Representative flow cytometry analysis from two melanoma patients showing percentages of CFSE lo A2/NY-ESO-1 157–165 tet + CD8 + T cells among total CD8 + T cells (A) and pooled data from melanoma patients ( n = 9) showing the variation in the numbers of CFSE lo (B) and total (C) A2/NY-ESO-1 157–165 tet + cells for 10 6 CD8 + T cells. CFSE-labeled PBMCs were incubated for 6 d with NY-ESO-1 157–165 peptide or HIVpol 476–484 peptide and blocking anti–Tim-3 (aTim-3), and/or anti–PD-L1 (aPD-L1) mAbs or an isotype control antibody (IgG). (D and E) Fold change of the frequencies of CFSE lo (D) and total (E) A2/NY-ESO-1 157–165 tet + CD8 + T cells after 6-d IVS with cognate peptide and blocking anti–Tim-3 (aTim-3) and/or anti–PD-L1 (aPD-L1) mAbs ( n = 9). The ratio of the percentages of CFSE lo and total A2/NY-ESO-1 157–165 tet + CD8 + T cells in the presence of indicated antibody treatment and isotype control antibody is shown. The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Flow Cytometry, Cytometry, Labeling, Incubation, Blocking Assay

Blockade of the Tim-3–Tim-3L pathway alone or in combination with PD-1–PD-L1 blockade with prolonged antigen stimulation increases the frequency of cytokine-producing NY-ESO-1–specific CD8 + T cells. (A and B) Representative flow cytometry analysis from one melanoma patient showing percentages of IFN-γ-, TNF-, and IL-2–producing A2/NY-ESO-1 157–165 tet + CD8 + T cells among total CD8 + T cells (A) and pooled data from melanoma patients ( n = 9) showing the variation in the frequencies of IFN-γ–, TNF-, and IL-2–producing NY-ESO-1 tet + cells for 10 6 CD8 + T cells (B). PBMCs were incubated for 6 d with NY-ESO-1 157–165 peptide or with HIVpol 476–484 peptide and blocking anti–Tim-3 (aTim-3) and/or anti–PD-L1 (aPD-L1) mAbs or an isotype control antibody (IgG) before evaluating intracellular cytokine production of A2/NY-ESO-1 157–165 tet + CD8 + T cells in response to cognate peptide. (C) Fold change of the frequency of IFN-γ–, TNF-, and IL-2–producing A2/NY-ESO-1 157–165 tet + CD8 + T cells after 6-d IVS with cognate peptide and blocking anti–Tim-3 and/or anti–PD-L1 mAbs. The ratio of the frequency of cytokine-producing tet + CD8 + T cells from melanoma patients ( n = 9) in the presence of indicated antibody treatment and isotype control antibody is shown. The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: Blockade of the Tim-3–Tim-3L pathway alone or in combination with PD-1–PD-L1 blockade with prolonged antigen stimulation increases the frequency of cytokine-producing NY-ESO-1–specific CD8 + T cells. (A and B) Representative flow cytometry analysis from one melanoma patient showing percentages of IFN-γ-, TNF-, and IL-2–producing A2/NY-ESO-1 157–165 tet + CD8 + T cells among total CD8 + T cells (A) and pooled data from melanoma patients ( n = 9) showing the variation in the frequencies of IFN-γ–, TNF-, and IL-2–producing NY-ESO-1 tet + cells for 10 6 CD8 + T cells (B). PBMCs were incubated for 6 d with NY-ESO-1 157–165 peptide or with HIVpol 476–484 peptide and blocking anti–Tim-3 (aTim-3) and/or anti–PD-L1 (aPD-L1) mAbs or an isotype control antibody (IgG) before evaluating intracellular cytokine production of A2/NY-ESO-1 157–165 tet + CD8 + T cells in response to cognate peptide. (C) Fold change of the frequency of IFN-γ–, TNF-, and IL-2–producing A2/NY-ESO-1 157–165 tet + CD8 + T cells after 6-d IVS with cognate peptide and blocking anti–Tim-3 and/or anti–PD-L1 mAbs. The ratio of the frequency of cytokine-producing tet + CD8 + T cells from melanoma patients ( n = 9) in the presence of indicated antibody treatment and isotype control antibody is shown. The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Blocking Assay

Ex vivo blockade of the Tim-3–Tim-3L pathway enhances cytokine production by NY-ESO-1–specific CD8 + T cells. (A and B) Representative dot plots from one melanoma patient (A) and summary data for all melanoma patients ( n = 8; B) showing the percentages of A2/NY-ESO-1 157–165 tet + CD8 + T cells that produce IFN-γ and TNF among total NY-ESO-1–specific CD8 + T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). (C) Summary data for melanoma patients ( n = 6) showing the percentages of A2/CMV 495–503 tet + CD8 + T cells that produce IFN-γ and TNF among total CMV-specific CD8 + T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.
Figure Legend Snippet: Ex vivo blockade of the Tim-3–Tim-3L pathway enhances cytokine production by NY-ESO-1–specific CD8 + T cells. (A and B) Representative dot plots from one melanoma patient (A) and summary data for all melanoma patients ( n = 8; B) showing the percentages of A2/NY-ESO-1 157–165 tet + CD8 + T cells that produce IFN-γ and TNF among total NY-ESO-1–specific CD8 + T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). (C) Summary data for melanoma patients ( n = 6) showing the percentages of A2/CMV 495–503 tet + CD8 + T cells that produce IFN-γ and TNF among total CMV-specific CD8 + T cells after short ex vivo stimulation with cognate peptide in the presence of blocking anti–Tim-3 and/or anti–PD-L1 mAbs or an isotype control antibody (IgG). The p-values were calculated using the Wilcoxon signed rank test. Data shown are representative of two independent experiments performed in duplicate.

Techniques Used: Ex Vivo, Blocking Assay

Tim-3 is up-regulated and coexpressed with PD-1 on NY-ESO-1–specific CD8 + T cells. (A) Representative dot plots from melanoma patients (MP) showing ex vivo Tim-3 expression on A2/NY-ESO-1 157–165, A2/EBV BMLF-1 280–288, A2/CMV 495–503, and A2/Flu-M 58–66 tet + CD8 + T cells. As shown for one melanoma patient (MP1), CD8 + T cells stained with A2/HIVpol 476–484 tetramers or PE-labeled IgG control Ab were used to establish the threshold for identifying tet + cells and Tim-3 + cells, respectively. (B and C) Pooled data showing the percentage (%) and MFI of Tim-3 expression on NY-ESO-1–, CMV-, EBV-, and Flu-specific CD8 + T cells, as well as total effector (CD45RA + CCR7 − ) and effector/memory (CD45RO + CCR7 − ) CD8 + T cells from nine melanoma patients (B) and nine healthy donors (C). (D) Dot plots from one representative melanoma patient showing ex vivo Tim-3 and PD-1 expression on A2/NY-ESO-1 157–165, A2/EBV BMLF-1 280–288, A2/CMV 495–503 and A2/Flu-M 58–66 tet + CD8 + T cells. As control, A2/NY-ESO-1 157–165 tet + CD8 + T cells stained with PE-labeled and FITC-labeled IgG control antibodies are shown. (E) Pooled data showing the distribution of NY-ESO-1–, CMV-, EBV-, and Flu-specific CD8 + T cells, as well as total tet − CD8 + T cells according to Tim-3 and PD-1 expression. Horizontal bars depict the mean percentage or MFI of Tim-3 and/or PD-1 expression on tet + CD8 + T cells. The p- values were calculated using the Wilcoxon signed rank test. Data shown are representative of at least three independent experiments.
Figure Legend Snippet: Tim-3 is up-regulated and coexpressed with PD-1 on NY-ESO-1–specific CD8 + T cells. (A) Representative dot plots from melanoma patients (MP) showing ex vivo Tim-3 expression on A2/NY-ESO-1 157–165, A2/EBV BMLF-1 280–288, A2/CMV 495–503, and A2/Flu-M 58–66 tet + CD8 + T cells. As shown for one melanoma patient (MP1), CD8 + T cells stained with A2/HIVpol 476–484 tetramers or PE-labeled IgG control Ab were used to establish the threshold for identifying tet + cells and Tim-3 + cells, respectively. (B and C) Pooled data showing the percentage (%) and MFI of Tim-3 expression on NY-ESO-1–, CMV-, EBV-, and Flu-specific CD8 + T cells, as well as total effector (CD45RA + CCR7 − ) and effector/memory (CD45RO + CCR7 − ) CD8 + T cells from nine melanoma patients (B) and nine healthy donors (C). (D) Dot plots from one representative melanoma patient showing ex vivo Tim-3 and PD-1 expression on A2/NY-ESO-1 157–165, A2/EBV BMLF-1 280–288, A2/CMV 495–503 and A2/Flu-M 58–66 tet + CD8 + T cells. As control, A2/NY-ESO-1 157–165 tet + CD8 + T cells stained with PE-labeled and FITC-labeled IgG control antibodies are shown. (E) Pooled data showing the distribution of NY-ESO-1–, CMV-, EBV-, and Flu-specific CD8 + T cells, as well as total tet − CD8 + T cells according to Tim-3 and PD-1 expression. Horizontal bars depict the mean percentage or MFI of Tim-3 and/or PD-1 expression on tet + CD8 + T cells. The p- values were calculated using the Wilcoxon signed rank test. Data shown are representative of at least three independent experiments.

Techniques Used: Ex Vivo, Expressing, Staining, Labeling

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    Effects of PQQ treatment on Th1, Th2, Th17, and Treg cells in peripheral blood and lung tissues of asthmatic model mice. (a and b) The gating strategy of flow cytometry for blood and tissue. (c and d) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + IFN- γ + T cells in blood and lung tissues. (e and f) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + IL-4+ T cells in blood and lung tissues. (g and h) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + ROR γ t+ T cells in blood and lung tissues. (i and j) Representative scatter diagrams in flow cytometry and quantitative analysis of the percentages of CD4 + CD25 + FoxP3+ T cells in blood and lung tissues. The columns and error bars represent the means and SEMs ( n = 5 per group). ∗ P

    Journal: Mediators of Inflammation

    Article Title: Pyrroloquinoline Quinone Administration Alleviates Allergic Airway Inflammation in Mice by Regulating the JAK-STAT Signaling Pathway

    doi: 10.1155/2022/1267841

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    Techniques: Mouse Assay, Flow Cytometry