Memory CD8+ T cells are required for protection from persistent hepatitis C virus infection

Abstract

Few hepatitis C virus (HCV) infections resolve spontaneously but those that do appear to afford protective immunity. Second infections are usually shorter in duration and are less likely to persist but mechanisms of virus control in immune individuals have not been identified. In this study we investigated whether memory helper and/or cytotoxic T lymphocytes provide protection in chimpanzees serially reinfected with the virus. Clearance of the first infection took 3-4 mo and coincided with the delayed onset of CD4+ and CD8+ T cell responses. High frequencies of memory T cells targeting multiple HCV proteins were stable over 7 yr of follow-up. Animals were infected for a second time to assess the protective role of memory T cells. In contrast to the prolonged course of the first infection, viremia was terminated within 14 d. Control of this second infection was kinetically linked to rapid acquisition of virus-specific cytolytic activity by liver resident CD8+ T cells and expansion of memory CD4+ and CD8+ T cells in blood. The importance of memory CD8+ T cells in control of HCV infection was confirmed by antibody-mediated depletion of this lymphocyte subset before a third infection. Virus replication was prolonged despite the presence of memory CD4+ T helper cells primed by the two prior infections and was not terminated until HCV-specific CD8+ T cells recovered in the liver. These experiments demonstrate an essential role for memory CD8+ T cells in long-term protection from chronic hepatitis C.

Figure 1.

Course of acute resolving HCV-1 infection in chimpanzees CB0556 and CB0572. Virus replication and HCV-specific immune responses after primary infection of CB0556 (A) and CB0572 (B). HCV RNA genome equivalents (GE) per milliliter of plasma (GE/ml) are shown. Spot forming cells (SFCs) (×10⁻³) represent the sum of responses to pools of overlapping peptides. Day 2541 (7 yr) is representative of four independent time points. (C) Detection of E2₄₄₅-specific CD8⁺ T cells using IFN-γ ELISPOT and a Patr B2301 tetramer during primary infection of CB0572.

Figure 2.

Virus replication and HCV-specific immune responses after rechallenge with HCV-1 at 7 yr after primary infection IFN-γ SFC after a second infection of CB0556 (A) and CB0572 (B) represent the sum of responses to pools of overlapping peptides or the individual CD8⁺ T cell epitopes P7₇₅₆ (A) and E2₄₄₅ (B). Prechallenge ELISPOT represents the mean SFC/10⁶ PBMC at four independent time points. Frequency of E2₄₄₅ specific T cells measured by tetramer staining is represented as percent of CD8⁺ T cells. (C) Detection of E2₄₄₅-specific T cells using a Patr B2301 tetramer. PBMCs from CB0556 (Patr B2301−) (left panel) and from CB0572 (Patr B2301+) at ∼7 yr after the first infection (middle panel) and at day 14 after the second infection (right panel) were stained with E2₄₄₅/Patr B2301 tetramer. A total of 100,000 events were acquired in the CD8⁺ lymphocyte gate and the frequency is presented as percent of CD8⁺ tetramer⁺ T cells. (D) Peptide activation of E2₄₄₅-specific T cells from blood of CB0572 collected on day 14 after infection.

Figure 3.

Intrahepatic HCV-specific responses upon rechallenge with HCV-1. (A) Detection of E2₄₄₅ specific T cells using a Patr B2301 tetramer on in vitro expanded CD8⁺ T cells (IHL). CD8⁺ lymphocytes isolated from the liver of CB0572 at the indicated time points were expanded with anti-CD3 antibodies for 2–3 wk and stained with the Patr B2301/E2₄₄₅ tetramer. Frequency is presented as percent of CD8⁺ tetramer⁺ T cells. Acquisition of HCV-specific CTL activity by intrahepatic CD8⁺ T cells (IHL) isolated from CB0572 (B) and CB0556 (C). Intrahepatic CD8⁺ T cells expanded with anti-CD3 antibodies were tested for cytolytic activity against autologous B lymphoblastoid cell lines infected with recombinant vaccinia viruses expressing all regions of the HCV polyprotein. Killing was detected only against targets that expressed E2-P7-NS2-NS3 (vvE2-NS3; aa 364–1618) and NS5B (vvNS5B; aa 2396–3011) and targets pulsed with the E2₄₄₅ peptide. Cytolytic activity was scored as positive (*) when it exceeded baseline (preinfection) values by 10% at two effector to target (E:T) cell ratios. Data shown are for a 25:1 E:T ratio.

Figure 4.

Tissue distribution and phenotype of Patr B2301/E2₄₄₅ tetramer-reactive CD8⁺ T cells. Lymphocytes were isolated directly from blood and liver of CB0572 at the indicated time points and stained (without anti-CD3 antibody expansion) with anti-CD8 antibodies and Patr B2301/E2₄₄₅ tetramer (A). Cells were also stained with anti-CD69 antibodies (B). 10,000–15,000 events were acquired in the CD8⁺ T lymphocytes gate and frequency is represented as percent of CD8⁺ tetramer⁺ cells (A) or percent of tetramer⁺ CD69⁺ cells (B).

Figure 5.

Virus replication and intrahepatic immune responses following antibody-mediated depletion of CD8⁺ T cells. (A) CD4⁺ and CD8⁺ T cell subsets were monitored in the blood of CB0556 (top panel) and CB0572 (bottom panel) after three doses of cMT-807 (vertical dashed lines). Anti-CD3 expanded CD8⁺ T cells from the liver of CB0556 (B) and CB0572 (C) were tested for IFN-γ production in an ELISPOT assay using HCV-1 peptide pools and individual epitopes. * Indicates inability to recover and expand CD8⁺ T cells from liver tissue until days 42 and 28 after infection from CB0556 (B) and CB0572 (C), respectively. (NS) indicates that no liver sample was obtained from either chimpanzee on day 35 after infection.

Figure 6.

Comparison of virus replication patterns after three consecutive infections of CB0556 and CB0572 with HCV-1.