Kinetics of CD4+ and CD8+ memory T-cell responses during hepatitis C virus rechallenge of previously recovered chimpanzees

Abstract

The immunological correlates of hepatitis C virus (HCV)-specific immunity are not well understood. Antibodies to HCV structural proteins do not appear to play a key role in clearance of the virus and do not persist after recovery. Here, we studied the kinetics of the cellular immune responses of three HCV-recovered chimpanzees during rechallenge with increasing doses of homologous HCV. Although HCV envelope antibodies remained undetectable throughout the rechallenge, all animals mounted rapid HCV-specific T-cell responses. The pattern of the cellular immune response in blood and liver correlated with the virological outcome. The animal that most rapidly cleared circulating HCV as determined by nested reverse transcription-PCR (RT-PCR) displayed the most vigorous and sustained response of gamma interferon (IFN-gamma)-producing and proliferating CD4(+) T cells in the blood. Vigorous CD4(+) T-cell proliferation during viremia was followed by an increased frequency and a phenotypic and functional change of the tetramer(+) CD8(+) T-cell population. The second animal cleared HCV initially with strong peripheral and intrahepatic CD4(+) T-cell responses but experienced low-level HCV recrudescence 12 weeks later, when HCV-specific T cells became undetectable. The third animal maintained minute amounts of circulating HCV, detectable only by nested RT-PCR, in the face of a weak IFN-gamma(+) T-cell response. Collectively, the results suggest protective rather than sterilizing immunity after recovery from hepatitis C. The rate of HCV clearance following reexposure depends on the cellular immune response, the quality and quantity of which may vary among chimpanzees that recovered from HCV infection.

FIG. 1.

HCV-specific CD4⁺ and CD8⁺ T-cell responses of Ch4X0186 during HCV rechallenge. (A) Arrows, time points of intravenous inoculation. All sera tested negative by real-time PCR (detection limit, 400 HCV RNA copies/ml). Presence (+) or absence (−) of virus below the detection limit of real-time PCR was assessed by nested RT-PCR. HCV antibody RIBA scores: indeterminate (), a single HCV band with an intensity equal to or greater than that of the IgG control band was detected; positive (▨), at least two HCV bands with intensities equal to or greater than that of the IgG control band were detected. Only antibodies against HCV NS3 and NS4B were detected; all other antigens, including the E1/E2 glycoproteins, tested negative. (B) Frequency of HCV-specific T cells that produce IFN-γ in response to HCV proteins (ELISPOT analysis). Responses are indicated as specific spot-forming cells (SFC) per 1.2 × 10⁵ peripheral blood lymphocytes (PBL) (number of SFC in the presence of antigen − number of SFC in the absence of antigen). (C) Proliferative T-cell response to HCV proteins. The stimulation index represents proliferation in the presence of antigen divided by proliferation in the absence of antigen. Dotted line, cutoff of positivity; n.d., not done. (D) Frequency of HCV-specific CD8⁺ T cells that produce IFN-γ in response to eight HCV peptide epitopes (ELISPOT analysis). HCV peptides were selected according to the animal's Patr haplotype (38). ∗, time point (week 23) with the highest number of tetramer⁺ T cells (see Fig. 2C).

FIG. 2.

Frequency and phenotype of tetramer⁺ T cells in the blood of Ch4X0186 during rechallenge. (A) Specificity of Patr tetramers was confirmed by staining peptide-specific and nonspecific in vitro-expanded T-cell lines. (B) Direct ex vivo staining of PBMC from chimpanzee Ch4X0186. Staining was performed with a tetramer mixture due to the limited number of available PBMC. Tetramer 14 was part of this mixture because T cells of this specificity produced IFN-γ during rechallenge (Fig. 1D). A tetramer with peptide 1, the other peptide recognized by IFN-γ-producing T cells (Fig. 1D), did not fold and therefore could not be used. The frequency of tetramer⁺ T cells increased at week 23, coinciding with detectable HCV RNA in the serum (Fig. 1A). n.t., not tested. (C) PBMC were stained directly ex vivo with tetramers (PE) and antibodies against CCR7 and CD4 and CD19 and CD45RO. Lymphocytes were gated based on side and forward scatter and a CD4 CD19 exclusion gate. Tetramer⁺ T cells were gated and analyzed for CCR7 and CD45RO expression. To determine the cutoff of positivity, PBMC were stained for CD45RO and CCR7 without gating on tetramer⁺ cells. In the dot plots, the numbers in each quadrant indicate the percentages of tetramer⁺ cells that do or do not express the respective surface markers. In the bar graphs, hatched bars indicate the percentages of CCR7⁺ CD45RO⁺ central memory cells and solid bars indicate the percentages of CCR7⁻ CD45RO⁺ effector memory cells.

FIG. 3.

HCV-specific CD4⁺ and CD8⁺ T-cell responses of Ch1552 during HCV rechallenge. (A) Arrows, time points of intravenous inoculation. Presence (+) or absence (−) of virus below the detection limit of real-time PCR (<400 HCV RNA copies/ml) was assessed by nested RT-PCR. □, negative RIBA score (no HCV band observed); other RIBA scores are indicated as defined in the legend to Fig. 1. Only antibodies against HCV NS3 and NS4B were detected; all other antigens, including E1/E2 glycoproteins, tested negative. (B) Frequency of HCV-specific T cells that produce IFN-γ in response to HCV proteins (ELISPOT analysis). Responses are indicated as specific spot-forming cells (SFC) per 1.2 × 10⁵ peripheral blood lymphocytes (PBL) (number of SFC in the presence of antigen − number of SFC in the absence of antigen). (C) Proliferative T-cell response to HCV proteins, presented as in Fig. 1C. (D) Frequency of HCV-specific CD8⁺ T cells that produce IFN-γ in response to 15 HCV peptide epitopes (ELISPOT analysis). HCV peptides were selected according to the animal's Patr haplotype (38). Responses are indicated as in panel A.

FIG. 4.

Frequency and phenotype of tetramer⁺ T cells in the blood of Ch1552 during rechallenge. (A) Specificity of Patr tetramers was confirmed by staining peptide-specific and nonspecific T-cell lines. (B) Direct ex vivo staining of PBMC from chimpanzee Ch1552. Staining was performed with a tetramer mixture due to the limited number of available PBMC. (C) PBMC were stained directly ex vivo with tetramers (PE) and antibodies against CCR7 and CD4 and CD19 and CD45RO. Lymphocytes were gated based on side and forward scatter and a CD4 CD19 exclusion gate. Tetramer⁺ T cells were analyzed for CCR7 and CD45RO expression. Cutoffs were set as in Fig. 2C. In the dot plots, the numbers in each quadrant indicate the percentage of tetramer⁺ cells that do or do not express the respective surface markers. In the bar graphs, hatched bars indicate the percentages of CCR7⁺ CD45RO⁺ central memory cells and solid bars indicate the percentages of CCR7⁻ CD45RO⁺ effector memory cells.

FIG. 5.

HCV-specific CD4⁺ and CD8⁺ T-cell responses of Ch1605 during HCV rechallenge. (A) Arrows, time points of intravenous inoculation. Presence or absence of virus below the detection limit of real-time PCR (<400 HCV RNA copies/ml) was assessed by nested RT-PCR (+/+, positive first and positive nested PCR; −/+, negative first and positive nested PCR; −/−, negative first and negative nested PCR). RIBA scores are indicated as described in the legend to Fig. 1. Only antibodies against HCV NS3 and NS4B were detected; all other antigens, including E1/E2 glycoproteins, tested negative. (B) Frequency of HCV-specific T cells that produce IFN-γ in response to HCV proteins (ELISPOT analysis). Responses are indicated as specific spot-forming cells (SFC) per 1.2 × 10⁵ peripheral blood lymphocytes (PBL) (number of SFC in the presence of antigen − number of SFC in the absence of antigen). (C) Proliferative T-cell response to HCV proteins, presented as in Fig. 1C. (D) Frequency of HCV-specific CD8⁺ T cells that produce IFN-γ in response to five HCV peptide epitopes (ELISPOT analysis). HCV peptides were selected according to the animal's Patr haplotype (38). Responses are indicated as in panel A.

FIG. 6.

Intrahepatic immune response of Ch1552 and Ch1605 during HCV rechallenge. (A and B) Intrahepatic immune response of Ch1552 during HCV rechallenge (A) and HCV recrudescence (B). Presence (+) or absence (−) of virus below the detection limit of real-time PCR (<400 HCV RNA copies/ml) was assessed by nested RT-PCR. The numbers of intrahepatic CD3⁻ CD56⁺ conventional lymphocytes (♦), CD3⁻ CD56⁺ NK cells (□), and CD3⁺ CD56⁺ lymphocytes (▵) per cm of biopsy sample were determined by flow cytometry. Arrows, time points of intravenous inoculation. Presence or absence of virus in the serum was assessed by nested RT-PCR. (C) Intrahepatic immune response of Ch1605 during HCV rechallenge. Symbols are used as in panel A. Presence or absence of virus in the serum according to real-time PCR and first- and second-round RT-PCR is indicated as in Fig. 5A. (D) Antigen specificity of intrahepatic T cells of Ch1552 during rechallenge. Liver-derived T cells were expanded with anti-CD3 and IL-2 and tested for antigen specificity in proliferation and cytotoxicity assays. Dashed line, cutoff for a positive proliferation assay. None of the cell lines tested positive in the cytotoxicity assay.