pbs anti cd4  (Novus Biologicals)

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: High CD25 expression correlates with robust FOXP3 expression in CD4+ positive canine T lymphocytes A ) Representative flow cytometric plot illustrating a low percentage (26.1%) of FOXP3+ cells when gating includes all CD25+CD4+ cells. B ) Representative flow cytometric plot illustrating a positive correlation between FOXP3 and CD25 expression in CD4+ canine T lymphocytes. In this example, FOXP3 expression was identified in 70.7% of cells with the highest CD25 expression (1.83%; red box). C ) Scatter dot plot demonstrating the positive correlation between FOXP3 and CD25 expression. D ) FOXP3 geometric mean fluorescence intensity and E ) mRNA expression was significantly increased in CD4+ CD25high T lymphocytes compared to CD4+ CD25low and CD8+ T lymphocytes. F ) Positive immunoreactivity for FOXP3 was observed co-localizing with nuclei of CD4+CD25high T lymphocytes. Three biological replicates were tested ( n =3 dogs) with three technical replicates per sample. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test and unpaired two-tailed student’s t-test, where bars represent the group mean with the standard error of the mean (SEM). * p <0.05

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Fluorescence, Two Tailed Test

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: Canine Tregs has the highest expression of CCR4 amongst T-cells. A ) Representative flow cytometric plot illustrating the percentage of CCR4+ cells within CD4+CD25+ and CD8+ canine T lymphocytes. In this example, CCR4 expression was identified in 28.5% of CD4+ CD25high cells and 8.6% of CD4+ CD25low cells. The expression of CCR4 in CD8+ T lymphocytes was minimal, 1.71%. B ) The percentage of CCR4+ cells identified via flow cytometry and C ) mRNA expression was significantly increased in CD4+CD25high T lymphocytes (2.43-fold increase) compared to CD4+CD25low and CD8+ T lymphocytes. Three biological replicates were tested ( n =3 dogs) with three technical replicates per sample. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test and unpaired two-tailed student’s t-test, where bars represent the group mean with standard error of the mean (SEM). * p <0.05

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Flow Cytometry, Two Tailed Test

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: The CCL2-CCR4 axis induces chemotaxis in canine Tregs. A ) Acutely isolated canine Tregs demonstrated robust chemotaxis toward human recombinant CCL2 (rhCCL2; 0.5 ug/ml) relative to media alone ( p =0.0007). This effect was normalized by adding anti-CCL2 antibody (5.0 ug/ml; p =0.15). B ) Chemotaxis of CD4+ helper T-cells was not affected by rhCCL2 ( p =0.2) or anti-CCL2 antibody ( p =0.4). C ) Chemotaxis of acutely isolated canine Tregs (CD4+CD25high) increased toward GSC0514 derived supernatant ( p <0.0001) and E ) J3T Bg derived supernatant ( p <0.0001). This effect was mitigated with the addition of anti CCL2 antibody (GSC0514, p =0.006; J3T Bg, p =0.0001) and abolished following Treg pre-treatment with CCR4 inhibitor (GSC0514 p <0.0001; J3T Bg p <0.0001) and a combination of anti-CCL2 and CCR4 inhibition (GSC0514 p <0.0001; J3T Bg p <0.0001). Migration of CD4+ helper T-cells was not affected by D ) GSC0514 derived supernatant ( p =0.06) nor perturbation of CCL2 ( p =0.8), CCR4 ( p =0.8) vs. combination of the two signaling ( p =0.09). Chemotaxis of CD4+ helper T-cells towards J3T Bg supernatant was induced, F ) ( p =0.02), and this effect was mitigated by anti-CCL2 antibody ( p =0.016); CCR4 inhibitor ( p =0.03) and combination of both ( p =0.001). Two independent experiments with technical duplicates per each condition were performed. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test; bars represent the group mean with the standard error of the mean (SEM). **** p <0.0001

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Chemotaxis Assay, Isolation, Recombinant, Derivative Assay, Inhibition, Migration

Journal: Journal of Neuro-Oncology

Article Title: The CCL2-CCR4 axis promotes Regulatory T cell trafficking to canine glioma tissues

doi: 10.1007/s11060-024-04766-4

Figure Lengend Snippet: Canine glioma CCL2 mRNA expression increases when exposed to Tregs but not conventional T-cells. mRNA levels of CCL2 were increased in A ) GSC0514, 0.95 ± 0.17- fold increased, p <0.001; B ) J3T Bg, 1.02 ± 0.33- fold increased, p <0.05; C ) GSC0111, 0.55 ± 0.11-fold increase, p <0.1; D ) G06A, 0.56 ± 0.19-fold increase, p <0.05, co-cultured with CD4+CD25high T lymphocytes relative to GSC0514, J3T Bg, GSC0111 and G06A cell lines without exposure to CD4+CD25high. mRNA levels of CCL2 were not significantly different when glioma cells were co-cultured with conventional T-cells. A ) 0.08 ± 0.05- fold decrease in GSC0514, p =0.8 B ) 0.30 ± 0.15 - fold increase in J3T-Bg, p =0.6 C ) 0.47 ± 0.29- fold decrease in GSC1110, p =0.2 and D ) 0.02 ± 0.1- fold decrease in G06A cell line, p =0.9. All reactions were run as 20μl triplicates per each condition. Comparisons are based on one-way ANOVA with Tukey’s multiple comparisons test; bars represent the group mean with the standard error of the mean (SEM). **** p <0.0001

Article Snippet: Freshly isolated PBMCs were stained with mixtures of Pacific Blue (PB) conjugated anti-dog CD4 (clone YKIX302.9; Thermo Fischer Scientific) and Phycoerythrin (PE) conjugated anti-dog CD25 (clone P4A10; Thermo Fischer Scientific).

Techniques: Expressing, Cell Culture

Journal: Frontiers in Immunology

Article Title: Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion

doi: 10.3389/fimmu.2023.1208814

Figure Lengend Snippet: Structure of the joint models. Graphical description of the two joint models. Joint model I (A) starts at time of alloSCT, joint model II (B) at time of the early low-dose DLI. Each model consists of a longitudinal and a time-to-event submodel and was run in turn for each T-cell subset, considering either the CD3+, CD4+ or CD8+ T-cell counts, and the NK cell counts. These are the outcome of the longitudinal submodel and a time-dependent covariate in the time-to-event submodel. All other variables in each submodel are baseline covariates. Per endpoint of the time-to-event submodels, the clinical events that occurred during the relevant time period (first 6 months after alloSCT or first 3 months after the early low-dose DLI) are described. The NK cells were only analyzed in model I. See the Statistical Supplement for a detailed description of the model structures.

Article Snippet: Samples were measured either on a FACSCalibur using anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD45-PerCP or with anti-CD3-FITC, anti-CD16-PE, anti-CD19-APC, anti-CD45-PerCP, and anti-CD56-PE, or on a FACSCanto using anti-CD3-APC, anti-CD4-PB, anti-CD8-FITC, anti-CD16-PE, anti-CD19-PE Cy7, anti-CD45-PerCP, and anti-CD56-PE (all from BD).

Techniques:

Journal: Frontiers in Immunology

Article Title: Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion

doi: 10.3389/fimmu.2023.1208814

Figure Lengend Snippet: Model-based T-cell count trajectories after alloSCT. Predicted average trajectories of the total, CD4+ and CD8+ T-cell counts during the first 6 months after alloSCT, based on the longitudinal submodel of model I. For all predicted trajectories, the patient/donor CMV status was set to -/-. 95% confidence intervals are shown in grey. The right column zooms in on a specific part of the total trajectory.

Article Snippet: Samples were measured either on a FACSCalibur using anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD45-PerCP or with anti-CD3-FITC, anti-CD16-PE, anti-CD19-APC, anti-CD45-PerCP, and anti-CD56-PE, or on a FACSCanto using anti-CD3-APC, anti-CD4-PB, anti-CD8-FITC, anti-CD16-PE, anti-CD19-PE Cy7, anti-CD45-PerCP, and anti-CD56-PE (all from BD).

Techniques: Cell Counting

Journal: Frontiers in Immunology

Article Title: Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion

doi: 10.3389/fimmu.2023.1208814

Figure Lengend Snippet: Model-based T-cell count trajectories after early low-dose DLI. Predicted average trajectories of the total, CD4+ and CD8+ T-cell counts during the first 3 months after early low-dose DLI. These are based on the longitudinal submodel of model II. 95% confidence intervals are shown in grey. The distance between the two lines in each panel (and further visualized by the adjacent arrows) corresponds to the CMV patient/donor effect on the trajectories. Namely, higher cell counts are predicted for patient/donor pairs where at least one is CMV seropositive, relative to a pair where both are CMV seronegative.

Article Snippet: Samples were measured either on a FACSCalibur using anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD45-PerCP or with anti-CD3-FITC, anti-CD16-PE, anti-CD19-APC, anti-CD45-PerCP, and anti-CD56-PE, or on a FACSCanto using anti-CD3-APC, anti-CD4-PB, anti-CD8-FITC, anti-CD16-PE, anti-CD19-PE Cy7, anti-CD45-PerCP, and anti-CD56-PE (all from BD).

Techniques: Cell Counting

Journal: Frontiers in Immunology

Article Title: Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion

doi: 10.3389/fimmu.2023.1208814

Figure Lengend Snippet: Forest plot for ITT analysis. Hazard ratios with associated 95% confidence intervals for donor type, disease risk and current value of the log of total, CD4+ or CD8+ T-cell counts on the events of interest. These are based on the time-to-event submodel of model I (see Figure 1A ).

Article Snippet: Samples were measured either on a FACSCalibur using anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD45-PerCP or with anti-CD3-FITC, anti-CD16-PE, anti-CD19-APC, anti-CD45-PerCP, and anti-CD56-PE, or on a FACSCanto using anti-CD3-APC, anti-CD4-PB, anti-CD8-FITC, anti-CD16-PE, anti-CD19-PE Cy7, anti-CD45-PerCP, and anti-CD56-PE (all from BD).

Techniques:

Journal: Frontiers in Immunology

Article Title: Joint models quantify associations between immune cell kinetics and allo-immunological events after allogeneic stem cell transplantation and subsequent donor lymphocyte infusion

doi: 10.3389/fimmu.2023.1208814

Figure Lengend Snippet: Forest plot for postDLI models. Hazard ratios with associated 95% confidence intervals for donor type and current value of the log of total, CD4+ or CD8+ T-cell counts on the events of interest. These are based on the time-to-event submodel of model II (see Figure 1B ).

Article Snippet: Samples were measured either on a FACSCalibur using anti-CD3-APC, anti-CD4-FITC, anti-CD8-PE, and anti-CD45-PerCP or with anti-CD3-FITC, anti-CD16-PE, anti-CD19-APC, anti-CD45-PerCP, and anti-CD56-PE, or on a FACSCanto using anti-CD3-APC, anti-CD4-PB, anti-CD8-FITC, anti-CD16-PE, anti-CD19-PE Cy7, anti-CD45-PerCP, and anti-CD56-PE (all from BD).

Techniques: