Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 triggering enhances HIV-1 replication in CD3/CD28-costimulated CD4+ T cells. Purified primary human CD4+ T cells were subjected to CD3/CD28 costimulation either in the absence or presence of the TLR2 agonist Pam3CSK4. (A) Cells were incubated with NL4.3 Balenv, and virus production was estimated at the indicated time points by measuring the p24 content in cell-free supernatants. (B) Cells were inoculated with NL4.3 Bal-IRES-HSA reporter virus, and the percentages of cells productively infected with HIV-1 (i.e., HSA+) were evaluated by flow cytometry. Data shown in panel A represent the means ± standard deviations (SD) of duplicates for a representative donor out of four. Each symbol shown in panel B represents a different donor, with the horizontal line depicting the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (**, P ≤ 0.01).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Purification, Incubation, Infection, Flow Cytometry

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 ligation does not modulate proliferation and activation profiles in CD3/CD28-costimulated CD4+ T cells. Purified primary human CD4+ T cells were treated as indicated in the legend to Fig. 1. (A) A CFSE-based dilution assay was performed by flow cytometry to evaluate cell proliferation (as expressed by a division index) following 72 h and 6 days of stimulation. Representative proliferation profiles are depicted in the panels on the left, whereas division indices are shown in the panels on the right. (B) Surface expression of activation markers CD25, CD69, and CD154 was evaluated by flow cytometry following 72 h of stimulation (%*MFI, percentage of positive cells × mean fluorescence intensity). The data shown were obtained from CD4+ T cell preparations isolated from the peripheral blood of four (A) or seven (B) healthy participants. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Ligation, Activation Assay, Purification, Dilution Assay, Flow Cytometry, Expressing, Fluorescence, Isolation

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 engagement increases susceptibility of the CD3/CD28-costimulated CCR6+ CD4+ T cell subset to productive HIV-1 infection. Purified primary human CD4+ T cells were treated as indicated in the legend to Fig. 1. (A) Surface expression of the CCR6 subset marker was analyzed by flow cytometry in the total CD4+ T cell population. (B) Cells were infected with NL4.3 Bal-IRES-HSA reporter virus, and percentages of HSA+ cells were estimated by flow cytometry in both CCR6− CD4+ and CCR6+ CD4+ T cell subsets. The data shown were obtained from CD4+ T cell preparations isolated from the peripheral blood of five healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (**, P ≤ 0.01).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Infection, Purification, Expressing, Marker, Flow Cytometry, Isolation

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 engagement does not affect CCR5 expression in CD3/CD28-costimulated CCR6+ CD4+ T cells. Purified primary human CD4+ T cells were treated as indicated in the legend to Fig. 1. (A) Surface expression of CCR5 in the total CD4+ T cell population following activation was evaluated by flow cytometry. (B) Expression of CCR5 was also assessed in both CCR6− CD4+ and CCR6+ CD4+ T cell subpopulations. The data shown were obtained from CD4+ T cell preparations isolated from the peripheral blood of five healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (**, P ≤ 0.01).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Expressing, Purification, Activation Assay, Flow Cytometry, Isolation

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 triggering augments virus entry in CD3/CD28-costimulated CCR6+ CD4+ T cells. Purified primary human CD4+ T cells were treated as indicated in the legend to Fig. 1, and virus entry was estimated either by quantifying intracellular p24 contents by ELISA in the total cell population (A) or monitoring the percentage of p24-expressing cells by flow cytometry in both CCR6− CD4+ and CCR6+ CD4+ T cell subsets (B). Results depicted in panel A were obtained from cell preparations of seven healthy donors and show the percentage of increase of cell-associated p24 over the control ± standard errors of the means (SEM) (i.e., cells subjected to CD3/CD28 costimulation only) for each donor. Results depicted in panel B were obtained from cell preparations of 11 healthy donors and show the percentage of p24-positive cells ± SEM in CCR6− and CCR6+ CD4+ T cell populations stimulated with Pam3CSK4 or left unstimulated. Statistical analyses were made using ratio paired t test between selected pairs of data. Asterisks denote statistically significant data (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Purification, Enzyme-linked Immunosorbent Assay, Expressing, Flow Cytometry

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 agonist does not impact expression of its cognate receptor in CD3/CD28-costimulated CCR6+ CD4+ T cells. Purified primary human CD4+ T cells were treated as indicated in the legend to Fig. 1. (A) Surface expression of TLR2 was then analyzed by flow cytometry in the total CD4+ T cell population. (B) Expression of TLR2 was also monitored in both CCR6− CD4+ and CCR6+ CD4+ T cell subsets. The data shown were obtained from CD4+ T cell preparations isolated from the peripheral blood of seven healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Expressing, Purification, Flow Cytometry, Isolation

Journal: Journal of Virology

Article Title: Toll-Like Receptor 2 Ligation Enhances HIV-1 Replication in Activated CCR6 + CD4 + T Cells by Increasing Virus Entry and Establishing a More Permissive Environment to Infection

doi: 10.1128/JVI.01402-16

Figure Lengend Snippet: TLR2 agonist augments intracellular levels of phosphorylated p65 in CD3/CD28-costimulated CCR6+ CD4+ T cells. Purified primary human CD4+ T cells were subjected to a CD3/CD28 costimulation in the absence or presence of Pam3CSK4. Intracellular expression of phosphorylated p65 was then analyzed by flow cytometry in both CCR6− CD4+ and CCR6+ CD4+ T cell subpopulations. The data shown were obtained from CD4+ T cell preparations isolated from the peripheral blood of five healthy donors. Each symbol represents a different donor, and the horizontal line depicts the means for all donors tested. Statistical analyses were made using ratio paired t test and one-way ANOVA, followed by a Dunnett's multiple-comparison test. Asterisks denote statistically significant data (*, P ≤ 0.05; **, P ≤ 0.01).

Article Snippet: Allophycocyanin (APC)-Cy7-conjugated anti-human CD196 (CCR6) was obtained from BioLegend (San Diego, CA), whereas APC-conjugated anti-mouse CD24 (HSA), APC-conjugated anti-human CD282 (TLR2), and eFluor660-conjugated anti-human phospho-NF-κB p65 were purchased from eBioscience (San Diego, CA).

Techniques: Purification, Expressing, Flow Cytometry, Isolation