Programmed death-1 affects suppressor of cytokine signaling-1 expression in T cells during hepatitis C infection

Abstract

Chronic hepatitis C virus (HCV) infection is associated with T-cell exhaustion that is mediated through upregulation of the PD-1 negative regulatory pathway. PD-1 expression is induced by HCV core protein, which also induces upregulation of SOCS-1, a key modulator that controls the Jak/STAT pathway regulating cytokine expression. To determine whether these two negative regulatory pathways are linked during T-cell signaling, SOCS-1 expression was examined by blocking the PD-1 pathway in T cells stimulated with anti-CD3/CD28 in the presence of HCV core protein. T cells isolated from healthy subjects or HCV-infected individuals were treated with anti-PD-1 or anti-PDL-1 antibodies in the presence or absence of HCV core protein, and SOCS-1 gene expression was detected by RT-PCR or immunoblotting, while T-cell functions were assayed by flow cytometric analyses. Both PD-1 and SOCS-1 gene expression were upregulated in healthy T cells exposed to HCV core protein, and blocking the PD-1 pathway downregulated SOCS-1 gene expression in these cells. Additionally, T cells isolated from chronically HCV-infected subjects exhibited increased PD-1 and SOCS-1 expression compared to healthy subjects, and SOCS-1 expression in T cells isolated from HCV-infected subjects was also inhibited by blocking PD-1 signaling; this in turn enhanced the phosphorylation of STAT-1, and improved the impaired T-cell proliferation observed in the setting of HCV infection. These data demonstrate that PD-1 and SOCS-1 are linked in dysregulating T-cell signaling during HCV infection, and their cross-talk may coordinately inhibit T-cell signaling pathways that lead to T-cell exhaustion during chronic viral infection.

FIG. 1.

Blocking the PD-1 pathway inhibits SOCS-1 mRNA expression in healthy T cells exposed to HCV core protein. (A) PD-1 upregulation in healthy T cells exposed to HCV core protein. CD4⁺ T lymphocytes isolated from healthy donors using antibody-conjugated magnetic beads, as described in the text, were stimulated with (top) or without (middle) anti-CD3/CD28 in the presence or absence of HCV core protein for 24 h. Total RNA was isolated from the treated cells, and PD-1 gene expression was detected by RT-PCR. β-Actin served as a control. The bar graph at the bottom shows the relative PD-1 expression in untreated and HCV core-treated T cells in the absence of TCR stimulation. (B) SOCS-1 upregulation in healthy T cells exposed to HCV core protein. CD4⁺ T lymphocytes isolated from healthy donors as above were stimulated with (top) or without (middle) anti-CD3/CD28 in the presence or absence of HCV core protein for 24 h. SOCS-1 gene expression was detected by RT-PCR, and β-actin served as a control. The bar graph at the bottom shows relative SOCS-1 expression in untreated and HCV core-treated T cells in the absence of TCR stimulation. (C) PD-1 blockade inhibits HCV core-induced SOCS-1 expression in PBMCs. PBMCs isolated from healthy donors were treated with anti-PD-1, anti-PDL-1, or control antibody for 6 h, followed by stimulation with HCV core protein and anti-CD3/CD28 for 24 h. SOCS-1 gene expression was detected by RT-PCR, and β-actin served as a control. (D) PD-1 blockade inhibits HCV core-induced SOCS-1 expression in purified lymphocytes. CD4⁺ T lymphocytes isolated from healthy donors as above were treated with anti-PD-1, anti-PDL-1, or control antibody for 6 h, followed by stimulation with HCV core protein and anti-CD3/CD28 (top) or anti-CD3/CD28 alone (middle) for 24 h. SOCS-1 gene expression was detected by RT-PCR, and β-actin served as a control. A graphic representation of the CD3/CD28-stimulated SOCS-1 expression is shown at bottom. (E) PD-1 blockade regulates SOCS-1 gene expression as detected by real-time RT-PCR. Healthy PBMCs were treated with or without anti-PD-1 or anti-PDL-1 antibody for 6 h, followed by stimulation with anti-CD3/CD28 in the presence or absence of HCV core protein for 24 h. SOCS-1 gene expression was detected by quantitative real-time RT-PCR, and β2M gene served as an internal control. The average cycle threshold of three samples measured for SOCS-1 after normalizing to β2M gene is shown with error bars.

FIG. 2.

Blocking the PD-1 pathway inhibits SOCS-1 protein expression in healthy T cells exposed to HCV core protein. (A) SOCS-1 upregulation in healthy T cells exposed to HCV core protein. CD4⁺ T cells isolated from healthy donors using antibody-conjugated magnetic beads were stimulated with anti-CD3/CD28 in the presence or absence of HCV core protein for 24 h. SOCS-1 protein expression was detected by Western blot, and β-actin served as a control. (B) PD-1 blocking inhibits HCV core-induced SOCS-1 expression in activated T cells. T cells isolated from healthy donors as above were treated with anti-PD-1, anti-PDL-1, or control antibody for 6 h, followed by stimulation with anti-CD3/CD28 and HCV core protein for 24 h. SOCS-1 protein expression was detected by Western blot, and β-actin served as a control.

FIG. 3.

Blocking the PD-1 pathway inhibits SOCS-1 expression in T lymphocytes from chronically HCV-infected patients. (A) PD-1 and SOCS-1 mRNA are upregulated in T cells from chronically HCV-infected patients. T cells were isolated from HCV-infected patients as well as healthy subjects using antibody-conjugated magnetic beads as described in the text, and stimulated with anti-CD3/CD28 for 24 h. PD-1 and SOCS-1 were detected by RT-PCR, and human β2M served as a loading control. (B) PD-1 and SOCS-1 protein expression is upregulated in T cells from chronically HCV-infected patients. T lymphocytes were isolated from HCV-infected patients as well as healthy subjects as above, and stimulated with anti-CD3/CD28 for 24 h. PD-1 and SOCS-1 were detected by Western blot, and β-actin served as a loading control. (C) PD-1 blockade inhibits SOCS-1 gene expression in T cells from HCV-infected subjects. T lymphocytes isolated from HCV patients were treated with anti-PDL-1 or control antibody overnight, followed by stimulation with anti-CD3/CD28 for 5 d. SOCS-1 gene expression was detected by RT-PCR, and β-actin served as a control. (D) PD-1 blockade inhibits SOCS-1 protein expression and increases STAT-1 phosphorylation in T cells from HCV-infected patients. T lymphocytes isolated from HCV patients were treated with anti-PDL-1 or control antibody overnight, followed by stimulation with anti-CD3/CD28 for 5 d. SOCS-1 protein expression and STAT-1 phosphorylation were detected by Western blot. β-Actin served as a control. Data were reproducible in independent experiments using T cells isolated from at least three different subjects.

FIG. 4.

Blocking the PD-1 pathway can improve the proliferative capability of T cells from chronically HCV-infected individuals. (A) Improved T-cell proliferation by blocking the PD-1 pathway in T lymphocytes stimulated by anti-CD3/CD28. PBMCs isolated from HCV patients were CFSE-labeled, followed by anti-PDL-1 or control antibody treatment overnight, and anti-CD3/CD28 stimulation for 5 d. After double staining, the T lymphocytes were gated and CFSE dilution, which represents T-cell proliferation, as well as the percentage of cells left shifted on each gating, are shown in the histograms. (B) Improved T-cell proliferation by blocking the PD-1 pathway in T lymphocytes stimulated by HCV peptides. PBMCs isolated from HCV-infected subjects were CFSE-labeled, followed by anti-PDL-1 or control antibody treatment overnight, and HCV NS3 peptide stimulation for 5 d. T-cell proliferation was analyzed as described above. (C) Improved T-cell proliferation by blocking the PD-1 pathway in T lymphocytes stimulated by healthy T cells. PBMCs isolated from three individual HCV-infected subjects were CFSE-labeled, and treated with anti-PDL-1 or control antibody overnight, followed by stimulation with healthy lymphocytes for 5 d. T-cell proliferation was analyzed as above.

FIG. 5.

Proposed model for immunodysregulation via PD-1/SOCS-1 during chronic HCV infection. This model is based on multiple studies involving these molecules (7,16–21).