Hepatitis C virus-specific CD4+ T cell phenotype and function in different infection outcomes

Abstract

CD4+ T cell failure is a hallmark of chronic hepatitis C virus (HCV) infection. However, the mechanisms underlying the impairment and loss of virus-specific CD4+ T cells in persisting HCV infection remain unclear. Here we examined HCV-specific CD4+ T cells longitudinally during acute infection with different infection outcomes. We found that HCV-specific CD4+ T cells are characterized by expression of a narrower range of T cell inhibitory receptors compared with CD8+ T cells, with initially high expression levels of PD-1 and CTLA-4 that were associated with negative regulation of proliferation in all patients, irrespective of outcome. In addition, HCV-specific CD4+ T cells were phenotypically similar during early resolving and persistent infection and secreted similar levels of cytokines. However, upon viral control, CD4+ T cells quickly downregulated inhibitory receptors and differentiated into long-lived memory cells. In contrast, persisting viremia continued to drive T cell activation and PD-1 and CTLA-4 expression, and blocked T cell differentiation, until the cells quickly disappeared from the circulation. Our data support an important and physiological role for inhibitory receptor-mediated regulation of CD4+ T cells in early HCV infection, irrespective of outcome, with persistent HCV viremia leading to sustained upregulation of PD-1 and CTLA-4.

Keywords: Adaptive immunity; Hepatitis; Immunology; Infectious disease; T cells.

Figure 1. Frequencies of HCV-specific CD4⁺ T cells over time and inhibitory receptor expression on HCV-specific CD4⁺ versus CD8⁺ T cells.

(A) HCV-specific CD4⁺ T cell frequencies from longitudinally sampled PBMCs of 33 patient progressors (top) and 22 resolvers (bottom). At least one HCV-specific CD4 response was detected in 27 of 33 (81%) progressors and 22 of 22 (100%) resolvers during the earliest phase of infection. However, HCV-specific CD4⁺ T cells quickly disappeared in blood of progressors, whereas a small population of these cells remained detectable directly ex vivo in resolvers years after resolution of infection. (B) Representative flow contour plots demonstrating expression of inhibitory receptors on HCV-specific CD4⁺ (top) or CD8⁺ T cells (bottom). (C) Cumulative data of the percentages of HCV-specific CD4⁺ and CD8⁺ T cells expressing each indicated inhibitory receptor. CD4⁺ T cell inhibitory receptor phenotypes were analyzed from a total of 15 progressors and 8 resolvers, while HCV-specific CD8⁺ T cells were examined from a total of 11 progressors and 8 resolvers. Each patient was analyzed at 2 to 3 time points.

Figure 2. HCV-specific CD4⁺ T cell phenotype during the initial 16 weeks of infection.

(A) Ex vivo phenotypic analysis of HCV-specific CD4⁺ T cells (dot plot) or total CD4⁺ T cells (contour plot) from the first available time point of resolvers and progressors. (B) Percentages of HCV-specific CD4⁺ T cells expressing each indicated immune markers are plotted for 10 progressors and 11 resolvers. No significant difference was found between the 2 groups of patients for any given marker. (C) PD-1 and CTLA-4 expression levels were also analyzed by median MFI. Statistical analysis was performed using the Kruskal-Wallis test.

Figure 3. HCV-specific CD4⁺ T cells diverge in phenotypes once viral loads diverge.

Immune phenotypes of HCV-specific CD4 cells were longitudinally examined in 15 progressors and 20 resolvers. Expression levels of PD-1 (A), CTLA-4 (B), CD38 (C), CD95 (D), and CD127 (E) in percentages or MFI in HCV-specific CD4⁺ cells from different infection outcomes are plotted against weeks after infection. Differences in slopes or elevation between progressors and resolvers (after 16 weeks) were calculated based on linear regression analysis. *P < 0.05; ****P < 0.001.

Figure 4. Functional analysis of HCV-specific CD4⁺ T cells.

(A) Representative flow cytometry plots for Th1 cytokine secretion by HCV-specific CD4⁺ T cells after stimulation with HCV proteins. Antigen-specific CD4 responses were identified as CD40L⁺CD4⁺ T cells. (B) Higher frequency of CD40L⁺ expressing CD4⁺ T cells (of total CD4⁺ cells) in response to HCV protein stimulation in 13 patients with resolving versus 9 with progressing infections (P = 0.016, 2-tailed Mann-Whitney U test). (C) (Poly)functional profiles of the CD40L-expressing cells. Progressors, red; resolvers, blue. (D) The mean number of CD40L⁺ CD4 populations secreting 0, 1, 2, or 3 cytokines. Representative flow cytometry plot demonstrating detection of tetramer-positive CD4⁺ T cells directly ex vivo and proliferation capacity of these cells upon cognate peptide stimulation in the absence or presence of rIL-2. After 14 days of culture, proliferating antigen-specific CD4⁺ T cells were identified as CD3⁺CD4⁺ Class II tetramer⁺ CellTrace Far Red by flow cytometry. Expression levels of PD-1 (E) and CTLA-4 (F) in samples that proliferated versus those that did not in the absence of rIL-2 (***P < 0.001, 2-tailed Mann-Whitney U test).