Background:  We previously showed that blocking regulatory T cell (Treg) -associated immunoregulatory cytokines, transforming growth factor-beta (TGF-β), and interleukin-10 (IL-10) significantly enhance the detectability of very weak HCV-specific T-cell responses of peripheral blood mononuclear cells (PBMC) in chronic HCV infection, even with co-infection by HIV. Most studies of suppression of such responses have focused on the CD4⁺CD25⁺, IL-10⁺ Treg population. We hypothesized that other cell types and cytokines may also play a role in suppression of HCV-specific responses in chronic infection and might be differentially regulated in the setting of HIV co-infection.

Methods:  We studied 9 subjects in whom TGF-β and IL-10 blockade raised the HCV-specific interferon-gamma (IFN-γ) ELISpot response: 5 HCV mono-infected and 4 HCV/HIV-co-infected. Frequencies of peripheral T cells specific for peptides derived from HCV core, and viral recall antigens (“CEF” derived from cytomegalovirus [CMV], Epstein-Barr virus [EBV], and influenza) were analyzed by IFN-γ and IL-10 ELISpot in the presence of blocking antibodies against TGF-β-1,2,3 or IL-10. The phenotype of T cells producing IFN-γ, TGF-β, and IL-10 in response to HCV-core peptides was analyzed using 5-color FACS analysis. Assays were performed before and after CD8 or CD25 depletion.

Results:  As expected, addition of blocking antibodies specifically increased the HCV-specific IFN-γ ELISpot T-cell responses in all subjects with no increase in recall antigen-specific responses. IL-10 ELISpot responses were also increased in response to HCV. IFN-γ production by both CD4 and CD8 T cells was mediated primarily by TGF-β neutralization. TGF-β was produced by HCV-specific CD3⁺CD8⁺CD25^– cells in 8 of 9 studied subjects. CD25 depletion did not raise the IFN-γ response. CD8 depletion also did not raise the IFN-γ ELISpot response and even decreased the CEF-specific IFN-γ response. However, by flow cytometry it increased the frequency of CD4⁺IFN-γ⁺ T cells in response to HCV.    

Conclusions:  Blockade of TGF-β produced by CD8⁺CD25^– could enhance detection of peripheral HCV-specific T-cell responses, even in presence of HIV co-infection.