Hepatitis C virus (HCV)-specific CD8+ cells produce transforming growth factor beta that can suppress HCV-specific T-cell responses

Abstract

Hepatitis C virus (HCV)-specific T-cell responses are rarely detected in peripheral blood, especially in the presence of human immunodeficiency virus (HIV) coinfection. Based on recent evidence that T-regulatory cells may be increased in chronic HCV, we hypothesized that functional blockade of regulatory cells could raise HCV-specific responses and might be differentially regulated in the setting of HIV coinfection. Three groups of subjects were studied: HCV monoinfected, HCV-HIV coinfected, and healthy controls. Frequencies of peripheral T cells specific for peptides derived from HCV core, HIV type 1 p24, and recall antigens were analyzed by gamma interferon (IFN-gamma) enzyme-linked immuno-spot assay. HCV-specific T-cell responses were very weak in groups with HCV and HCV-HIV infections. Addition of blocking antibodies against transforming growth factor beta1 (TGF-beta1), -2, and -3 and interleukin-10 specifically increased the HCV-specific T-cell responses in both infected groups; however, this increase was attenuated in the group with HCV-HIV coinfection compared to HCV infection alone. No increase in recall antigen- or HIV-specific responses was observed. Flow cytometric sorter analysis demonstrated that regulatory-associated cytokines were produced by HCV-specific CD3(+)CD8(+)CD25(-) cells. Enhancement of the IFN-gamma effect was observed for both CD4 and CD8 T cells and was mediated primarily by TGF-beta1, -2, and -3 neutralization. In conclusion, blockade of TGF-beta secretion could enhance peripheral HCV-specific T-cell responses even in the presence of HIV coinfection.

FIG. 1.

IFN-γ ELISPOT assay against HCV core peptides in the presence of isotype controls and the blocking Abs anti-TGF-β and anti-IL-10. Each line corresponds to a subject. The results are shown as SFCs/10⁶ PBMC over background (unstimulated cells). After the addition of blocking Abs, significant enhancement of median HCV-specific T-cell responses was observed in both groups of subjects with HCV monoinfection (n = 11) (P = 0.003) and with HCV-HIV coinfection (n = 12) (P = 02). Similarly, the number of responders to HCV, defined as greater than 50 SFCs/10⁶ cells, was increased from two to nine HCV responders in the HCV-monoinfected group and from one to four responders in the HCV-HIV-coinfected group. No controls had positive responses to HCV. HCV-specific T-cell responses were not different between the two groups, HCV monoinfected and HCV-HIV coinfected, when isotype controls were added, while after the addition of blocking Abs, these responses became significantly higher in the HCV-monoinfected group (P = 0.05).

FIG. 2.

IFN-γ ELISPOT assays against CEF (top) and HIV p24 (bottom) peptides in the presence of isotype controls and anti-TGF-β and anti-IL-10 Abs. Each line corresponds to a subject. The results are shown as SFCs/10⁶ PBMC over background (unstimulated cells). No enhancement of median T-cell responses toward non-HCV antigens was observed after the addition of blocking Abs. Neither controls nor subjects with HCV monoinfection had positive responses to HIV peptides.

FIG. 3.

Frequencies of CD4 and CD8 T cells producing IFN-γ, by intracellular staining, in response to medium alone or to the pool of HCV core peptides before and after the addition of the blocking Abs anti-TGF-β and anti-IL-10 or the appropriate isotype controls in one representative subject, 002P (HCV monoinfected). After 6 h of appropriate stimulation (in the presence of Golgi Plug added 1 h after the start of stimulation), PBMC were stained with anti-CD3-PE-Cy5, anti-CD4-ECD, and anti-CD8-PE-Cy7 and then fixed/permeabilized for intracellular staining with anti-IFN-γ-PE. The dot plots are gated on CD3⁺ cells. The corresponding isotype control staining was <0.04 (not shown).

FIG. 4.

Frequencies of T cells producing TGF-β or IL-10, in response to medium alone or to HCV core peptides, by intracellular staining in one representative subject, 0090 (HCV monoinfected). After 6 h of appropriate stimulation (in the presence of Golgi Plug added 1 h after the start of stimulation), PBMC were stained with anti-CD3-PE-Cy5, anti-CD4-ECD, anti-CD8-PE-Cy7, and anti-CD25-FITC and then fixed/permeabilized for intracellular staining with anti-TGF-β-PE, anti-IL-10-PE, or isotype-PE. The dot plots are gated on (A) CD3⁺CD4⁺ and (B) CD3⁺CD8⁺ cells. Neither CD3⁺CD8⁺CD25⁺ nor CD3⁺CD4⁺ cells produced regulatory cytokines.

FIG. 5.

IFN-γ ELISPOT assay against HCV core peptides in the presence of medium alone or either both or one of each blocking Ab (anti-TGF-β and/or anti-IL-10) or their corresponding isotype controls. The bars represent the results as SFCs/10⁶ PBMC over background (unstimulated cells), shown for five HCV-monoinfected and two HCV-HIV-coinfected subjects for each stimulation condition. A responder was defined as greater than 50 SFCs/10⁶ PBMC over background, as shown by the horizontal dotted lines. The blocking effect was mediated by TGF-β and was IL-10 independent in six out of seven subjects.

FIG. 6.

IL-10 ELISPOT assay against the three HCV core pools, P1, P2, and P3, and CEF in the presence of medium, anti-TGF-β blocking MAb, or the corresponding isotype control IgG1. The bars represent the results as SFCs/10⁶ PBMC over background (unstimulated cells), shown for two subjects, 0055 (HCV monoinfected) and 0186 (HIV-HCV coinfected). Addition of the blocking Ab anti-TGF-β enhanced the IL-10 response to HCV pools, but not to CEF.

FIG. 7.

Frequencies of T cells producing TGF-β in response to medium alone or HCV core peptides by intracellular staining in two subjects, 0186 (HCV-HIV coinfected) and 9136 (HCV monoinfected). After 6 h of appropriate stimulation (in the presence of Golgi Plug added 1 h after the start of stimulation), PBMC were stained with anti-CD3-PE-Cy5, anti-CD4-ECD, and anti-CD8-PE-Cy7 and then fixed/permeabilized for intracellular staining with anti-Foxp3-FITC and anti-TGF-β-PE or their isotype controls. The dot plots are gated on (A) CD3⁺CD4⁺ and (B) CD3⁺CD8⁺ cells. Of note, the staining rates for all the isotype controls were low (<0.02%) (not shown). Only CD4⁺ cells expressed Foxp3, but they did not produce TGF-β in response to HCV (A). CD3⁺CD8⁺ cells producing TGF-β in response to HCV were Foxp3 negative (B).

FIG. 8.

Frequencies of T cells producing TGF-β in response to medium alone, to HCV core peptides, or to CEF peptides by intracellular staining in three subjects: 0186 and 0992 with HCV/HIV coinfection and 9136 with HCV monoinfection. After 6 h of appropriate stimulation (in the presence of Golgi Plug added 1 h after the start of stimulation), PBMC were stained with anti-CD3-PE-Cy5, anti-CD4-ECD, and anti-CD8-PE-Cy7 and then fixed/permeabilized for intracellular staining with anti-TGF-β-PE or isotype-PE. The dot plots are gated on CD3⁺CD8⁺ cells. CD3⁺CD4⁺ cells did not produce the regulatory cytokine, and the isotype controls were low (<0.02). TGF-β production was observed in response to HCV, but not to CEF.

FIG. 9.

Overall frequencies of CD3⁺CD8⁺CD25⁻ cells producing TGF-β by intracellular staining in 10 studied subjects. Each line corresponds to a subject. TGF-β production by CD8 cells was significantly higher in response to HCV stimulation than in medium alone (P = 0.01) (A). There was no significant (P = 0.12) difference between TGF-β-PE and Iso-PE staining in medium alone, while after HCV stimulation, TGF-β-PE staining was significantly (P = 0.01) higher than Iso-PE (B).

FIG. 10.

Effects of CD8 depletion on IFN-γ responses. (A) Frequencies of CD4-producing IFN-γ, by intracellular staining, in response to medium or to the pool of HCV core peptides before and after CD8 depletion in one coinfected subject, 0186. After 6 h of appropriate stimulation (in the presence of Golgi Plug added 1 h after the start of stimulation), PBMC or CD8⁻ cells were stained with anti-CD3-PE-Cy5 and anti-CD4-ECD and then fixed/permeabilized for intracellular staining with anti-IFN-γ-PE and isotype-PE. The dot plots are gated on CD3⁺CD4⁺ cells. (B) Frequencies of CD4-producing IFN-γ, by intracellular staining, in response to the pool of HCV core peptides before and after CD8 depletion in three subjects. In subject 9136, the same experiment was performed once antigen-presenting cells were removed and then added back at equal numbers to the PBMC and CD8-depleted cells. The bars represent the results as frequencies of CD4⁺IFN-γ⁺ cells gated on CD3⁺CD4⁺ cells after background subtraction (unstimulated cells, or medium). The CD8 depletion increased intracellular IFN-γ production in CD4 cells in response to HCV.