Article Figures & Data
Figures
- Fig. 1.
Healthy and HIV+ donors have comparable T-cell composition. (A) T cells were identified as CD3+ lymphocytes, and then classified as helper or cytotoxic T lymphocytes based upon the surface expression of CD4 and CD8, respectively. Memory T cells were identified as CD45RO+ and nonmemory T cells were identified as CD45RO− for both CD4+ and CD8+ T cells. (B) HIV+ donors possessed CD4+ and CD8+ T-cell numbers that were comparable to those of the healthy donors, and comparable memory (CD45RO+)/nonmemory (CD45RO−) cell composition. FSC-A, forward scatter area; RO−, CD45RO−; RO+, CD45RO+; SSC-A, side scatter area.
- Fig. 2.
T cells from healthy and HIV+ donors exhibit different profiles of IFNΑR2 expression and IFNα-induced STAT1 phosphorylation, which is suppressed by THC. PBMCs from healthy and HIV+ donors were isolated by Ficoll Paque density gradient centrifugation and used for the determination of either IFNΑR2 surface expression or IFNα-induced STAT1 phosphorylation (pSTAT1). (A) IFNAR2 expression was quantified by flow cytometry using the mean fluorescence intensity (MFI). For pSTAT1, PBMCs were treated with either vehicle (0.03% EtOH) or THC (1, 5, or 10 µM) in 0.03% EtOH for 30 minutes before stimulation with 100 U/ml IFNα for 30 minutes. (B) Representative experiment of IFNα-mediated pSTAT1 induction and THC (10 µM)-mediated suppression in T cells from a healthy donor. The effects of THC on IFNα-pSTAT1 induction in: total (C), memory (D) and nonmemory (E) CD4+ T cells; and total (F), memory (G), and nonmemory (H) CD8+ T cells. For IFNΑR2 expression, asterisks indicate statistically significant differences (*P < 0.05) of MFI in HIV compared with type-matched T cells from healthy donors. For pSTAT1, asterisks indicate statistically significant differences of the treatment with the HIV status–matched VC (0 THC) (*P < 0.05; **P < 0.01; ***P < 0.001). Daggers indicate statistically significant differences of treatment-matched groups between T cells from healthy and HIV+ donors (†P < 0.05) (two-way analysis of variance with Bonferroni multiple-comparisons post-test). SSC-A, side scatter area.
- Fig. 3.
THC suppresses IFNα-induced expression of IL-7Rα mRNA in T cells from healthy donors but differentially affects IFNα-induced surface expression of IL-7Rα in T cells from healthy vs. HIV+ T donors. (A) To determine the effects of IFNα and THC on IL-7Rα mRNA expression, T cells were purified from healthy donors, and treated with either vehicle (0.03% EtOH) or various concentrations of THC (1, 5, or 10 µM) for 30 minutes. After treatment, cells were stimulated with IFNα (100 U/ml), incubated for 48 hours, and harvested for quantification of IL-7Rα mRNA levels by real-time quantitative PCR. For the determination of IL-7Rα surface expression, PBMCs from healthy and HIV+ donors were isolated through Ficoll Paque density gradient centrifugation and either immediately stained for CD3, CD4, CD8, CD45RO, and IL-7Rα (D0) or treated with THC and IFNα, as described above, and measured for IL-7Rα expression after 48 hours. (B) Example of IFNα-mediated IL-7Rα expression and THC (10 µM)-mediated suppression in a healthy donor. The effects of THC on the expression level [mean fluorescence intensity (MFI)] of IL-7Rα in T cells from healthy and HIV+ donors in total (C), memory (D), and nonmemory (E) CD4+ cells; and total (F), memory (G), and nonmemory (H) CD8+ cells. Asterisks indicate statistically significant differences of the treatment with the HIV status–matched VC (0 THC) (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Daggers indicate statistically significant differences of treatment-matched groups between Healthy and HIV+ T cells (†P ≤ 0.05; ††P ≤ 0.01) (two-way analysis of variance with Bonferroni multiple-comparisons post-test). D0, day 0; SSC-A, side scatter area.
- Fig. 4.
THC suppresses IFNα-mediated augmentation of IL-7–induced STAT5 phosphorylation. PBMCs from healthy and HIV+ donors were isolated through Ficoll Paque density gradient centrifugation. Cells were either: 1) immediately used for the detection of IL-7–induced pSTAT5 (D0) by treating with IL-7 (10 ng/ml) for 15 minutes then rapidly fixed; or 2) treated with either vehicle (0.03% EtOH) or various concentrations of THC (1, 5, or 10 µM) for 30 minutes, stimulated with IFNα (100 U/ml), incubated for 48, and then used for the detection of IL-7–induced pSTAT5, as described above. (A) Representative experiment of IL-7Rα–induced pSTAT5- and THC (10 µM)-mediated suppression in a healthy donor. (B) Levels [mean fluorescence intensity (MFI)] of pSTAT5 in T cells was determined by flow cytometry on day 0. The effects of THC on the IL-7–induced pSTAT5 level after IFNα stimulation of T cells from healthy and HIV+ donors in: total (C), memory (D), and nonmemory (E) CD4+ cells; and total (F), memory (G), and nonmemory (H) CD8+ cells. Asterisks indicate statistically significant differences between treatments with the HIV status–matched VC (0 THC) (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Daggers indicate statistically significant differences of treatment-matched groups between healthy and HIV+ T cells (†P ≤ 0.05) (two-way analysis of variance with Bonferroni multiple-comparisons post-test). D0, day 0; SSC-A, side scatter area.
- Fig. 5.
IL-7 augmented CD3/CD28/IFNα-induced T-cell proliferation is suppressed by THC. PBMCs from healthy and HIV+ donors were isolated through Ficoll Paque density gradient centrifugation. Cells were stained with violet CellTrace dye and then treated with either vehicle (0.03% EtOH) or various concentrations of THC (1, 5, or 10 µM) for 30 minutes. After treatment, cells were stimulated with IFNα (100 U/ml) and anti-CD3 and anti-CD28 antibodies (2.5 μg/ml each) for 48 hours, treated with IL-7 (10 ng/ml) or vehicle (endotoxin-free H2O), and incubated for another 48 hours before harvesting. T-cell proliferation is represented as the DI, as determined by the FlowJo proliferation tool. (A) Representative experiment of IL-7–mediated augmentation of T-cell proliferation in CD3/CD28/IFNα-stimulated T cells and THC (10 µM)-mediated suppression in a healthy donor. The effects of treatment with IFNα and IL-7 on anti-CD3/CD28-mediated T-cell proliferation in CD4+ (B) and CD8+ (C) T cells from healthy and HIV+ donors. The effect of IL-7 stimulation on total CD3+ T-cell composition and between memory (CD45RO+) and nonmemory (CD45RO−) cells in CD4+ (D) and CD8+ (E) T cells. The effects of THC on the IL-7–induced augmentation of CD3/CD28/IFNα-induced proliferation of T cells from healthy and HIV+ donors in total (F), memory (G), and nonmemory (H) CD4+ cells; and total (I), memory (J), and nonmemory (K) CD8+ cells. Pound signs indicate statistically significant differences in the proliferation of IL-7 treated T cells compared to T cells without IL-7 treatment (#P ≤ 0.05). Asterisks indicate statistically significant differences of the treatment with the HIV status–matched VC (0 THC or D0) (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001). Daggers indicate statistically significant differences of treatment-matched groups between healthy and HIV+ T cells (†P ≤ 0.05; ††P ≤ 0.01) (two-way analysis of variance with Bonferroni multiple-comparisons post-test). D0, day 0; SSC-A, side scatter area.
Additional Files
Data Supplement
- Supplemental Figure -
1. Stimulation of T cells with IFN alpha reduces IFNAR2 expression of CD4+ cells and enhances expression on CD8+ T cells...
- Supplemental Figure -
In this issue
THC Suppresses T-Cell Activation by IFNα and IL-7
