mouse anti chicken cd3 mab  (SouthernBiotech)

Journal: Veterinary research

Article Title: Revealing novel and conservative CD8 + T-cell epitopes with MHC B2 restriction on ALV-J.

doi: 10.1186/s13567-024-01426-3

Figure Lengend Snippet: Figure 1 Detection of ALV-J shedding, ALV-J viremia, ALV-J antibody and T lymphocyte percentage of B2 haplotype chickens after ALV-J infection. Six chickens were randomly selected for sampling every 7 days post-infection (dpi). A ALV-J shedding was monitored via detecting the p27 expression levels in cloacal swabs. S/P value below 0.2 indicated negative ALV-J shedding. B ALV-J viremia was monitored via virus isolation. An S/P value above 0.2 indicated positive ALV-J viremia. C The ALV-J antibody level in the serum was monitored using the commercial ALV-J antibody test kit. An S/P value above 0.6 was considered ALV-J antibody positive. Five days before infection (dbi) and each week after infection, PBMCs derived from five chickens of infected and control groups were isolated to detect the T lymphocyte percentage, including the percentage of the CD4+CD3+T cell (D), the CD8α+CD3+T cell (E), and the CD3+CD4+CD8α+T cell (F). Each sample collected 1 × 105 cells for flow cytometric analysis. The one-way test was used for statistical comparison among A, B and C. And the unpaired Student t test was used for statistical comparison among D, E and F. * P < 0.05, ** P < 0.01.

Article Snippet: PBMCs (1 × 106) were incubated with mouse anti-chicken CD3 mAb (SouthernBiotech, Birmingham, USA), mouse anti-chicken CD4 mAb (SouthernBiotech, Birmingham, USA) and mouse anti-chicken CD8α mAb (SouthernBiotech, Birmingham, USA), respectively, at 4 °C for 30 min in the dark.

Techniques: Infection, Sampling, Expressing, Virus, Isolation, Derivative Assay, Control, Comparison

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infection upregulates COX-2 expression and impairs T cell proliferation. (A) Graphical presentation for experimental setup for ex vivo proliferation assay: Splenocytes, harvested at 21 dpi from control, MDV infected (RB1B, 1,000 pfu/dose), and vaccinated birds (Rispens-CVI988, 1,000 pfu/dose), were stained with CFSE and stimulated with Con-A (5 µg/ml) for 3 days. (B) Representative dot plot and histograms showing the gating strategy used in acquisition and analysis of CFSE based proliferation data. The proliferation of 7AAD - (live cells) CD3 + T cells was analysed by flow cytometry. (C) Proliferation index showing proliferation in CD3+ T cells from control, MDV infected and vaccinated birds. (D) qRT-PCR data showing fold change in mRNA level for COX-2 gene in MDV infected and vaccinated birds which was calculated over the control birds. (E) Representative histograms and corresponding (F) proliferation index showing proliferation in CD3+ T cells from MDV infected birds stimulated with Con-A in presence of the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). Grey bars: No significant difference between mock and treatment and red bars: significant difference or restoration to non-significant level between mock and treatment group. Dot plot and histograms are representative of proliferation data from six individual infected birds. Each dot in proliferation index represents the average of three biological replicates from six individual chickens. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Expressing, Ex Vivo, Proliferation Assay, Control, Staining, Flow Cytometry, Quantitative RT-PCR, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infected CEFs and 265L tumour cells produce high PGE2 level resulting in downregulation of chIL2 and transferrin uptake in CD3+ T cells. Culture supernatants were harvested from control CEF (non-infected),CEF infected with virulent MDV (MDV supernatant) or vaccine strain of MDV (vaccine supernatant) at 72 hrs post-infection. Culture supernatant harvested from CD4+ T cells of naïve splenocytes and MDV-transformed CD4+ T cells (265L supernatant). (A) The levels of PGE2 were determined using an ELISA assay. (B) chIL2 expression level was determined in Con-A stimulated splenocytes treated with PGE2 (10 µg/ml), or the supernatants as described above using qRT-PCR, and fold change are shown. Inhibition of chIL-2 expression by (C) PGE2, (D) MDV supernatant, (E) 265L supernatant was rescued in the presence of the inhibitors of the COX-2/PGE2 pathway; TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). (F) Representative confocal images showing uptake of transferrin (red color) by CD3+ T cells in the Con-A stimulated splenocytes treated with control supernatant, PGE2, MDV supernatant, vaccine supernatant or 265L supernatant using confocal microscopy. (G) Graph shows mean fluorescent intensity (MFI) of transferrin uptake using confocal microscopy. (H) Bar graph shows the effects of a COX-2 inhibitor (SC-236) on transferrin uptake in CD3+ T cells treated with PGE2, MDV supernatant or 265L supernatant using confocal microscopy. Graphs are representative of three independent experiments, each performed with three biological replicates. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot in ELISA represents the average of three biological replicates from six individual culture groups. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Control, Transformation Assay, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Inhibition, Confocal Microscopy, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Expression of CD25 + on CD3 + T cells remained unaffected by PGE2 treatment. CD25 surface expression on CD3+ T cells were quantified in splenocytes stimulated with Con-A (5 µg/ml) in presence of cell culture media containing either recombinant PGE2 (5 µg/ml), the control supernatants, MDV supernatant, vaccine supernatant or the 265L supernatant. (A) Dot plot (B) bar graph showing percentage of CD3 + CD25 + T cells. Graph is representative of three independent experiments, each performed with three biological replicates per treatment. Each dot in ELISA represents the average of three biological replicates from six individual culture groups. Grey bars represent the experimental groups with no significant difference. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Expressing, Cell Culture, Recombinant, Control, Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: MDV infection drives PGE2/COX-2 dependent downregulation of T cell proliferation in vitro . Splenocytes were harvested from naïve birds and stained with CFSE and then they were stimulated with Con-A (5 µg/ml) in presence of PGE2, control supernatant, MDV supernatant, Vaccine supernatant, or 265L supernatant with or without the chemical the inhibitors of the PGE2-COX-2 pathway. After 3 days of culture, T cell proliferation was analysed by flow cytometry. (A) Representative histograms and corresponding (B) proliferation index showing dose-dependent effect of recombinant PGE2 (2.5, 5 and 10 µg/ml) on in vitro proliferation of CD3+ T cells. (C) Representative histograms and corresponding (D) proliferation index showing effect of control supernatant, MDV supernatant, Vaccine supernatant, or 256L supernatant on in vitro proliferation of CD3+ T cells. (E, G) Representative histograms and corresponding (F, H) proliferation index showing inhibitory effect of the chemical inhibitor: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL). Dot plot and histograms are representative of proliferation data from six individual birds. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot in proliferation index represents the average of three biological replicates from six individual chickens. Error bar represents the mean ± standard deviation. Statistical significance was estimated as p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, In Vitro, Staining, Control, Flow Cytometry, Recombinant, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Treatment of splenocytes with PGE2/MDV sup/265L sup induced PGE2-COX-2 pathway drives dysfunction of CD3 + T cells. (A) Splenocytes were cultured for 3 days in media containing recombinant PGE2 (5 µg/ml), control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The isolated CD3 + T cells were CFSE stained and co-cultured with freshly isolated CD3 negative cells. The co-culture was stimulated with Con-A (5 µg/ml) in presence or absence of the chemical inhibitors of the PGE2-COX-2 pathway. After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B) Representative histograms and corresponding (C) proliferation index. (D) Representative histograms and corresponding (E–G) proliferation index showing in vitro proliferation in CD3 + T cells from splenocytes cultured with PGE2, MDV Supernatant and 265L supernatant in presence of the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL), respectively. Data is representation of three independent experiments, each performed with three biological replicates per treatment. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Statistical significance was estimated by p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Cell Culture, Recombinant, Control, Isolation, Staining, Co-Culture Assay, Flow Cytometry, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: PGE2-COX-2 drives dysfunction of CD3 + T cells in MDV infected chickens. (A) CD3 + T cells were isolated from splenocytes harvested at 21 dpi from control (n=5), MDV infected (n=5) and vaccinated birds (n=5) and stained with CFSE before co-culturing with CD3 negative T cells isolated from splenocytes of naïve birds. The co-culture was stimulated with Con-A (5 µg/ml) in presence or absence of the chemical inhibitors of PGE2-COX-2 pathway. After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B, D) Representative histograms showing proliferation in CD3+ T cells from the control, vaccinated birds, and MDV infected birds with or without the chemical inhibitors: TG4-155 (4 μM), ER-819762 (8 μM) and SC-236 (5 μg/mL) and corresponding (C) proliferation index from the experimental groups of birds. (E) Proliferation index for CD3 + T cells from the MDV infected birds in presence of the chemical inhibitors of the PGE2-COX-2 pathway. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Statistical significance was estimated by p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Isolation, Control, Staining, Co-Culture Assay, Flow Cytometry, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: PGE2/MDV supernatant directly affected CD3+ T cells and impaired CD3 + T cells is independent of CD3 negative T cells and BMDCs. (A) CD3 negative cells were isolated from splenocytes cultured for 3 days in media containing recombinant PGE2 (5 µg/ml), the control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The isolated CD3 negative cells were co-cultured with CFSE stained CD3 + T cells isolated from naive birds in presence of Con-A (5 µg/ml). After 3 days of co-culture, T cell proliferation was analysed by flow cytometry. (B, C) Representative histograms and corresponding (C) proliferation index showing in vitro proliferation of CD3+ T cells from the experimental groups. (D) BMDCs were cultured for 3 days with media containing recombinant PGE2 (5 µg/ml), the control supernatant, MDV supernatant, vaccine supernatant or 265L supernatant. The treated BMDCs were harvested, washed, and cultured with CFSE stained CD3 + T cells isolated from naïve birds in fresh medium containing Con-A (5 µg/ml) for 3 days. (E) Representative histograms and corresponding (F) proliferation index for CD3 + T cells from the different experimental groups. Data is representation of three independent experiments, each performed with three biological replicates per treatment. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Statistical significance was estimated by p value calculated by ANOVA test. NS, not significant.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Isolation, Cell Culture, Recombinant, Control, Staining, Co-Culture Assay, Flow Cytometry, In Vitro, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Marek’s Disease Virus Modulates T Cell Proliferation via Activation of Cyclooxygenase 2-Dependent Prostaglandin E2

doi: 10.3389/fimmu.2021.801781

Figure Lengend Snippet: Administration of meloxicam rescues T cell proliferation in MDV infected chickens. (A) Schematic diagram showing the experimental groups; non-infected mock controls, MDV infected birds (RB1B, 1,000 pfu/dose), and MDV infected birds that received daily oral administration of meloxicam. (B) COX-2 expression levels in splenocytes of the experimental groups were analysed using RT-PCR assay. CFSE-labelled splenocytes were stimulated with Con-A and cultured for 3 days in vitro to assess T cell proliferation using flow cytometry. (C) Representative histograms and corresponding (D) proliferation index showing ex vivo proliferation of CD3+ T cells in mock control and MDV infected with and without meloxicam treated birds. Grey bars are used in the experimental groups in which no significant difference are found, while red bars are used to represent significant differences. Each dot represents the data from individual chicken and the graph represents the mean ± standard deviation of five independent replicates from individual chickens. Statistical significance was estimated by p value calculated by ANOVA test.

Article Snippet: Briefly, chicken splenocytes (1 × 10 7 ) were incubated (30 min at 4°C) with PE-conjugated mouse anti-chicken CD3 mAbs or PE-conjugated mouse anti-chicken CD4 mAb (10 μl/10 7 cells) (Cambridge Bioscience, Cambridge, UK).

Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Cell Culture, In Vitro, Flow Cytometry, Ex Vivo, Control, Standard Deviation

Journal: Veterinary Research

Article Title: High-frequency and activation of CD4 + CD25 + T cells maintain persistent immunotolerance induced by congenital ALV-J infection

doi: 10.1186/s13567-021-00989-9

Figure Lengend Snippet: Congenital ALV-J infection causes stunting of immune organs and immunotolerance. A Detection of ALV-J antigen by ELISA in the cloacal swabs of chickens. B Detection of ALV-J antigen in peripheral blood by ELISA. C Anti-ALV-J neutralizing antibody titers in blood by ELISA. D Gross lesions of immune organs. E Dynamic changes in the immune index. F Histopathological detection of immune organs. G CLSM analysis of CD3 + T cells in the spleen. H FACS analysis of CD3 + T cells in the thymus and spleen. ** p < 0.01, *** p < 0.001; the unpaired t -test was performed, and the data were expressed as mean ± SEM.

Article Snippet: Then, these sections were incubated with PE-conjugated mouse anti-chicken CD4 monoclonal antibody (mAb; Southern Biotech, 1:500), FITC-conjugated mouse anti-chicken CD25 mAb (Bio-Rad, 1:500), or FITC-conjugated mouse anti-chicken CD3 mAb (Southern Biotech, 1:500) for 30 min at 37 °C.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Veterinary Research

Article Title: High-frequency and activation of CD4 + CD25 + T cells maintain persistent immunotolerance induced by congenital ALV-J infection

doi: 10.1186/s13567-021-00989-9

Figure Lengend Snippet: Congenital ALV-J infection causes stunting of immune organs and immunotolerance. A Detection of ALV-J antigen by ELISA in the cloacal swabs of chickens. B Detection of ALV-J antigen in peripheral blood by ELISA. C Anti-ALV-J neutralizing antibody titers in blood by ELISA. D Gross lesions of immune organs. E Dynamic changes in the immune index. F Histopathological detection of immune organs. G CLSM analysis of CD3 + T cells in the spleen. H FACS analysis of CD3 + T cells in the thymus and spleen. ** p < 0.01, *** p < 0.001; the unpaired t -test was performed, and the data were expressed as mean ± SEM.

Article Snippet: Meanwhile, these lymphocytes were stained with AF700-conjugated mouse anti chicken CD3 mAb, FITC-conjugated mouse anti chicken CD4 mAb, PE-conjugated mouse anti chicken CD8 mAb (Southern Biotech, Birmingham, AL, USA), or FITC-conjugated mouse anti chicken CD25 mAb (Bio-Rad, Hercules, CA, USA) for 30 min at 4 °C.

Techniques: Infection, Enzyme-linked Immunosorbent Assay