Clearance of chronic HCV infection leads to an expansion of a follicular T helper cell memory phenotype in HCV-specific CD4 T cells

80% of Hepatitis C virus (HCV) infected patients establish chronicity, which causes progressive liver disease. CD4 T cells are well known key regulators of the antiviral immune response. It has been previously shown that the follicular T helper (Tfh) cell subset increases over time due to ongoing antigen stimulation during persistent viral infection in mice and that this was required for pathogen control. During human acute HCV infection HCV-specific Tfh cell frequencies decrease over time. However, the fate of HCV-specific CD4 T cells during chronic HCV (cHCV) infection in humans is largely unknown. Additionally, the regulation of human HCV-specific CD4 T cells has not been studied in the context of antigen removal. The approval of direct acting antivirals (DAA), that can successfully eliminate cHCV infection, provides therefore a unique opportunity to study the effect of antigen removal on the phenotypical and functional characteristics of HCV-specific T cell subsets.

In this study, we aimed to determine the effect of antigen removal by DAA therapy on the HCV-specific CD4 T cell population.

HCV-specific CD4 T cells of 23 chronically infected patients were analyzed at 4 time points of DAA therapy (chronic (week 0, W0), week 2 of therapy (W2), end of therapy (EOT), 24 weeks after end of therapy (follow up, FU) using MHC class II tetramer technology. Surface markers of CD4 T cell subsets, relevant costimulatory and coinhibitory surface receptors as well as cytokine secretion (in selected patients) were determined by flow cytometry. The transcriptome of HCV-specific CD4 T cells from 3 patients at W0, W2 and FU was analysed by RNA sequencing. Since Tfh cells are professional B helper cells, we further analyzed the B cell response of 10 patients by flow cytometry. Moreover, plasma samples of 37 patients were analyzed for neutralizing antibody titers. Plasma samples of 33 patients were analyzed for CXCL13, a biomarker for germinal center activity.

The frequency of HCV-specific CD4 T cells increased significantly to W2. At W0 most of the HCV-specific CD4 T cells presented a Th1 phenotype (CXCR3+CCR6-), while under DAA therapy HCV-specific Tfh cells (CXCR5+PD-1+) appeared to expand. This effect was absent in bulk CD4 T cells, suggesting an antigen-specific effect. Correspondingly, transcriptome analysis revealed that after therapy the HCV-specific CD4 T cells more closely resemble Tfh rather than Th1 cells from the same patient. Changes in the expression of different surface markers on HCV-specific CD4 T cells suggest that during DAA therapy the activation status declines, while the cells acquire a central memory phenotype (CCR7+CD45RA-). Moreover, CXCL13 was elevated in cHCV infection and decreases quickly to W2. In addition, neutralizing antibody titers gradually decline after DAA treatment. Surprisingly, no changes could be detected in the B cell compartment.

Collectively, our results show a decrease of the immunological activity against HCV and suggest the formation of Tfh cell memory within HCV-specific CD4 T cells due to the antigen removal.