Expression of programmed cell death protein 1 and T-cell immunoglobulin- and mucin-domain-containing molecule-3 on peripheral blood CD4+CD8+ double positive T cells in patients with chronic hepatitis C virus infection and in subjects who spontaneously cleared the virus

Abstract

Chronic hepatitis C virus (HCV) infection is characterized by increased proportion of CD4+CD8+ double positive (DP) T cells, but their role in this infection is unclear. In chronic hepatitis C, immune responses to HCV become functionally exhausted, which manifests itself by increased expression of programmed cell death protein 1 (PD-1) and T-cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) on T cells. The aim of our study was to determine PD-1 and Tim-3 phenotype of DP T cells in subjects with naturally resolved and chronic HCV infection. Peripheral blood mononuclear cells from 16 patients with chronic infection and 14 subjects who cleared HCV in the past were stained with anti-CD3, anti-CD4, anti-CD8, anti-PD-1 and anti-Tim-3 antibodies and, in 12 HLA-A*02-positive subjects, MHC class I pentamer with HCV NS3₁₄₀₆ epitope. In chronic and past HCV infection, proportions of total DP T cells and PD-1+ DP T cells were similar but significantly higher than in healthy controls. DP T cells were more likely to be PD-1+ than either CD4+ or CD8+ single positive (SP) T cells. HCV-specific cells were present in higher proportions among DP T cells than among CD8+ SP T cells in both patient groups. Furthermore, while the majority of HCV-specific DP T cells were PD-1+, the proportion of HCV-specific CD8+ T cells which were PD-1+ was 4.9 and 1.9 times lower (chronic and past infection, respectively). PD-1 and Tim-3 were predominantly expressed on CD4^high CD8^low and CD4^low CD8^high cells, respectively, and co-expression of both markers was uncommon.

Keywords: DP T cells; PD-1; Tim-3; hepatitis C virus.

Figure 1

Gating strategy of flow cytometric phenotyping of double positive (DP) (CD4+ CD8+) T cells. A, Dot plot (SSC‐A vs FSC‐A) representing population of acquired PBMC with gated lymphocytes. B, Dot plot (SSC‐A vs Per‐CP) showing CD3+ lymphocytes gated on lymphocytes. C, Dot plot (FITC vs AmCyan) showing population of CD8+ and CD4+ cells gated on CD3+ cells. D, Dot plot (PE vs APC) representing expression of PD‐1 and Tim‐3 on gated DP T cells

Figure 2

Double positive (DP) (CD4+ CD8+) T cells populations gated on CD3+ cells showing predominant CD4^highCD8^low population in patients #27 (A) and #26 (B). Both patients had chronic HCV infection

Figure 3

Percentage of double positive (DP) T (CD4+ CD8+) cells expressing PD‐1 (A), Tim‐3 (B) and both PD‐1 and Tim‐3 (C) in peripheral blood of 16 patients with chronic HCV infection, 14 subjects who spontaneously cleared HCV and in 11 healthy controls. Horizontal lines represent median values

Figure 4

Percentage of CD4^highCD8^low and CD4^lowCD8^high double positive (DP) (CD4+ CD8+) T cells expressing PD‐1 (A), Tim‐3 (B) and both PD‐1 and Tim‐3 (C) in 16 patients with chronic HCV infection, 14 subjects who spontaneously cleared HCV and in 11 healthy controls. Horizontal lines represent median values

Figure 5

Population of double positive (DP) (CD4+ CD8+) T cells specific for HLA‐A*02‐restricted HCV NS3₁₄₀₆ KLSGLGLNAV epitope (A) together with PD‐1 expression (B) in patient #29 who cleared HCV infection in the past