Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD3 , REA1151 , 50 , 130-120-267 , FITC (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Nature Communications

Article Title: Exposure to pesticides in utero impacts the fetal immune system and response to vaccination in infancy

doi: 10.1038/s41467-020-20475-8

Figure Lengend Snippet: a Cord blood non-naive (Supplementary Fig. ) CD4 T cell frequency in exposed (red, n = 98) and unexposed (blue, n = 68) infants. b Cord blood T reg frequency (CD25 high FoxP3 + CD127 lo ) in exposed (red, n = 98) and unexposed (blue, n = 68) infants. c Myeloid (Lin-2 – HLA-DR + CD11c + CD123 - ) and plasmacytoid (Lin-2 – HLA-DR + CD11c − CD123 + ) dendritic cell counts in whole cord blood, in exposed (red, n = 142) and unexposed (blue, n = 96) infants. d Cord blood plasma cytokine levels (unadjusted p values: TNF-RI ( p < 0.0001), IL-2 ( p < 0.0001), IL-4 ( p = 0.037), IL-5 ( p < 0.0001), IL-7 ( p < 0.0001), MDC ( p < 0.0001), TNF-RI ( p < 0.0001), IP-10 ( p = 0.0009), and MIP-1α ( p = 0.01), ran in duplicate) in exposed (red, n = 114) and unexposed (blue, n = 73) infants. Following Bonferroni adjustment for all cytokines and chemokines measured, all associations indicated above remained significant with the exception of IL-4 and MIP-1α. All comparisons were made by Wilcoxon rank-sum test. Bars indicate median with interquartile range. Two-sided p values were calculated for all test statistics.

Article Snippet: Dendritic cell staining was performed on 238 cord blood samples using FITC Lin-2 (clone MφP9/SJ25C1/SK7/L27/NCAM16.2, anti-CD3, CD14, CD19, CD20, CD56), PE CD123 (clone 9F5), PerCP HLA-DR (clone L243), and APC CD11c (clone S-HCL-3, BD Pharmingen).

Techniques:

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: The tetraspanin CD81 cooperates with TCR/CD3 to enhance HIV-1 transcriptional activity in Jurkat cells. Jurkat cells either transiently transfected with pLTRx-LUC (A and B), cotransfected with pLTRx-LUC and pCEP4-Tat (C), or infected with reporter HIV-1 pseudotyped with VSV-G (D) were either left untreated or incubated for 8 (A, B, and C) or 24 (D) h with the indicated cross-linked antibodies in various combinations (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml; 9.3 at 1 μg/ml). Cells were next lysed in order to assess HIV-1 LTR-encoded reporter gene activity. Results are shown as the increase over the activity of samples treated with GAM (used as negative controls) and are calculated from the mean ± SD for three different lysed cell samples in the same experimental setting. The mean luciferase counts of control cells were 2.2 (A), 10.5 (B), 80.5 (C), and 3.4 (D) RLU. Results are representative of three independent experiments. Asterisks indicate significant differences between cells stimulated with OKT3-5A6, OKT3-9.3, or OKT3-9.3-5A6 and cells treated with OKT3 alone (*, P < 0.05; **, P < 0.02).

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Activity Assay, Transfection, Infection, Incubation, Luciferase, Control

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: CD81 acts as a costimulatory molecule to enhance TCR/CD3-mediated induction of HIV-1 transcription and virus production in primary CD4+ T lymphocytes. (A) Flow cytometric analyses of CD81 surface expression on Jurkat cells and purified CD4+ T lymphocytes after staining with an isotype-matched control antibody (dotted lines) or the anti-CD81 antibody 5A6 (continuous lines). (B) Primary CD4+ T cells were inoculated with VSV-G-pseudotyped reporter viruses and were either left untreated or treated for 48 h with cross-linked 5A6 (2 μg/ml), OKT3 (0.25 μg/ml), and/or 9.3 (1 μg/ml). Cells were next lysed to assess HIV-1 LTR-encoded reporter gene activity. The mean luciferase activity in control cells was 10 RLU. (C) CD4+ T lymphocytes were infected with fully competent HIV-1 (strain NL4-3) and were either left untreated or treated with the listed cross-linked antibodies. Cell-free supernatants were harvested 96 h poststimulation, and p24 contents were measured by an enzymatic assay. Results are calculated from the mean ± SD of three different cell samples in the same experimental setting. Results are representative of three independent experiments. Asterisks indicate significant differences between cells stimulated with OKT3-5A6, OKT3-9.3, or OKT3-9.3-5A6 and cells treated with OKT3 only (*, P < 0.05; **, P < 0.01).

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Virus, Expressing, Purification, Staining, Control, Activity Assay, Luciferase, Infection, Enzymatic Assay

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: CD81-mediated costimulatory capacity enhances nuclear translocation of transcription factors that can bind to the HIV-1 enhancer region. Primary CD4+ T lymphocytes were incubated for 6 h either with goat anti-mouse IgG, used as a negative control (Ctrl), or with the indicated antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml) before nuclear extraction. Nuclear proteins were then incubated with a labeled HIV-1 enhancer probe (A) or the labeled negative internal control SP1 (B), and the complexes were resolved on a native 4% acrylamide gel. The specificities of the migrating complexes were verified by competition with a specific (HIV-1 enhancer [Cold probe]) or nonspecific (Oct-2A probe) probe. The intensities of the bands were quantified with an Alpha Imager 2000 digital imaging and analysis system. Results are representative of three independent experiments. Arrows on the left indicate the specific migrating complexes.

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Translocation Assay, Incubation, Negative Control, Extraction, Labeling, Control, Acrylamide Gel Assay, Imaging

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: CD81-directed costimulatory signal leads to augmented induction of NF-κB resulting in increased HIV-1 transcription in Jurkat and CD4+ T cells. Jurkat cells were transiently transfected with pNF-κB-LUC (A) or were transfected with pLTR-LUC or pmκB-LTR-LUC (B). In some instances, Jurkat cells (D) or primary CD4+ T lymphocytes (E) were used as targets for pseudotyped reporter HIV-1 particles. Jurkat and CD4+ T cells that had been inoculated with reporter HIV-1 particles pseudotyped with VSV-G envelope proteins were either left untreated or pretreated for 1 h with the proteasome inhibitor MG-132 at the indicated concentrations. Next, cells were either left untreated or treated for 8 (A, B, and D) or 24 (E) h with the listed cross-linked antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml; 9.3 at 1 μg/ml). Finally, cells were lysed to assess HIV-1 LTR-encoded reporter gene activity. Results are shown as the increase over the activity in samples treated with GAM (used as negative controls) and are calculated from the mean ± SD of three different lysed cell samples in the same experimental setting. The mean luciferase counts of control cells were 13 RLU in panel A, 19 (LTR-Luc) and 2.2 (mκBLTR-Luc) RLU in panel B, 61 (without MG-132) and 223 (1 μM MG-132) RLU in panel D, and 10 (without MG-132), 10.7 (0.1 μM MG-132), and 4.3 (1 μM MG-132) RLU in panel E. Results are representative of three independent experiments. Asterisks indicate significant differences between cells treated with MG-132 and untreated cells (*, P < 0.05). (C) Untreated or MG-132-treated Jurkat cells were stimulated with TNF-α (20 ng/ml) for the indicated times (in minutes) and then lysed in sample buffer. Samples were resolved by SDS-PAGE and immunoblotted with the indicated antibodies.

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Transfection, Activity Assay, Luciferase, Control, SDS Page

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: Nuclear translocation of NF-κB is augmented upon coligation of CD81 and the OKT3/CD3 complex in primary CD4+ T lymphocytes. (A and B) Primary CD4+ T lymphocytes were incubated for 6 h either with GAM, used as a negative control (Ctrl), or with the indicated antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml) before nuclear extraction. Nuclear proteins were then incubated with a labeled NF-κB-specific probe (A) or the labeled negative internal control SP1 (B), and the complexes were resolved on a native 4% acrylamide gel. The intensities of the bands were quantified with an Alpha Imager 2000 digital imaging and analysis system. (C) The labeled HIV-1 enhancer region was also used as a probe, and the resolved complexes were supershifted with anti-p50 or anti-p65 antibodies. The specificities of the migrating complexes were verified by competition with a specific (NF-κB specific [A] or HIV-1 enhancer [C]) or nonspecific (Oct-2A) probe. Results are representative of three independent experiments. Arrows indicate the antibody-mediated migrating and supershifted complexes.

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Translocation Assay, Incubation, Negative Control, Extraction, Labeling, Control, Acrylamide Gel Assay, Imaging

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: The cosignal provided by CD81 further enhances TCR/CD3-mediated induction of NFAT, an event that leads to increased HIV-1 transcription in Jurkat and primary CD4+ T cells. Jurkat cells were transiently transfected with pNFAT-LUC (A) or infected with VSV-G-pseudotyped reporter virus (B). In some experiments, primary CD4+ T lymphocytes were used as targets for pseudotyped reporter HIV-1 particles (C). Jurkat and CD4+ T cells inoculated with reporter HIV-1 particles pseudotyped with VSV-G envelope proteins were either left untreated or pretreated for 1 h with FK506 at the indicated concentrations. Next, cells were either left untreated or treated for 8 (A and B) or 24 (C) h with the indicated cross-linked antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml; 9.3 at 1 μg/ml). Finally, cells were lysed to assess HIV-1 LTR-encoded reporter gene activity. Results are shown as the increase over the activity in samples treated with goat-anti-mouse IgG (used as negative controls) and are calculated from the mean ± SD of three different lysed cell samples in the same experimental setting. The mean luciferase counts of control cells were 0.5 RLU in panel A, 7.5 (0 and 0.2 ng of FK506/ml) and 7.9 (2 ng of FK506/ml) RLU in panel B, and 10 (0 and 0.2 ng of FK506/ml) and 9 (2 ng of FK506/ml) RLU in panel C. Results are representative of three independent experiments. Asterisks indicate significant differences between cells treated with FK506 and untreated cells (*, P < 0.05; **, P < 0.005).

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Transfection, Infection, Virus, Activity Assay, Luciferase, Control

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: CD81 cooperates with TCR/CD3 to increase the translocation of NFAT to the nucleus, where it can bind to the HIV-1 enhancer region. Primary CD4+ T lymphocytes were incubated for 6 h either with GAM, used as a negative control (Ctrl), or with the indicated antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml) before nuclear extraction. Nuclear proteins were then incubated with a labeled NFAT-specific probe (A) or the labeled negative internal control SP1 (B), and the complexes were resolved on a native 4% acrylamide gel. The intensities of the bands were quantified with an Alpha Imager 2000 digital imaging and analysis system. Results are representative of three independent experiments.

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Translocation Assay, Incubation, Negative Control, Extraction, Labeling, Control, Acrylamide Gel Assay, Imaging

Journal:

Article Title: Tetraspanin CD81 Provides a Costimulatory Signal Resulting in Increased Human Immunodeficiency Virus Type 1 Gene Expression in Primary CD4 + T Lymphocytes through NF-?B, NFAT, and AP-1 Transduction Pathways

doi: 10.1128/JVI.79.7.4316-4328.2005

Figure Lengend Snippet: Coengagement of CD81 and TCR/CD3 leads to increased AP-1 DNA-binding activity and HIV-1 transcription in CD4+ T cells through ERK1/2- and JNK-dependent pathways. (A and B) Primary CD4+ T lymphocytes were incubated for 6 h either with GAM, used as a negative control (Ctrl), or with the indicated cross-linked antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml) before nuclear extraction. Nuclear proteins were then incubated with a labeled specific AP-1 probe (A) or the labeled negative internal control SP1 (B), and the complexes were resolved on a native 4% acrylamide gel. Arrows indicate the antibody-mediated migrating complexes. The intensities of the bands were quantified with an Alpha Imager 2000 digital imaging and analysis system. The specificities of the migrating complexes were verified by competition with a specific (AP-1 specific) or nonspecific (Oct-2A) probe. Results are representative of two independent experiments. (C and D) In some experiments, primary CD4+ T cells were infected with VSV-G-pseudotyped reporter virus and were either left untreated or pretreated for 1 h with the ERK inhibitor PD098059 (40 μM) (C) or the JNK inhibitor SP600125 (40 μM) (D). Next, cells were either left untreated or treated for 24 h with the listed cross-linked antibodies (5A6 at 2 μg/ml; OKT3 at 0.25 μg/ml; 9.3 at 1 μg/ml). Finally, cells were lysed to assess HIV-1 LTR-encoded reporter gene activity. Results are shown as the increase over the activity in samples treated with GAM (used as negative controls) and are calculated from the mean ± SD of three different lysed cell samples in the same experimental setting. The mean luciferase activity in control cells was 1.2 RLU for untreated cells, 1.3 RLU for PD098059-treated cells, and 0.6 RLU for SP600125-treated cells. Results are representative of three independent experiments. Asterisks indicate significant differences between cells treated with FK506 or SP600125 and untreated cells (*, P < 0.05; **, P < 0.02).

Article Snippet: The hybridoma cell line OKT3, which produces the anti-CD3ζ monoclonal antibody, was obtained from the American Type Culture Collection (ATCC) (Manassas, Va.).

Techniques: Binding Assay, Activity Assay, Incubation, Negative Control, Extraction, Labeling, Control, Acrylamide Gel Assay, Imaging, Infection, Virus, Luciferase