Spontaneous recovery in acute human hepatitis C virus infection: functional T-cell thresholds and relative importance of CD4 help

Abstract

The mechanisms mediating protective immunity to hepatitis C virus (HCV) infection are incompletely understood because early infection in humans is rarely identified, particularly in those individuals who subsequently demonstrate spontaneous virus eradication. We have established a large national network of patients with acute HCV infection. Here, we comprehensively examined total HCV-specific CD4(+) and CD8(+) T-cell responses and identified functional T-cell thresholds that predict recovery. Interestingly, we found that the presence of HCV-specific cytotoxic T lymphocytes (CTLs) that can proliferate, exhibit cytotoxicity, and produce gamma interferon does not ensure recovery, but whether these CTLs were primed in the presence or absence of CD4(+) T-cell help (HCV-specific interleukin-2 production) is a critical determinant. These results have important implications for early prediction of the virologic outcome following acute HCV and for the development of novel immunotherapeutic approaches.

FIG. 1.

Peptide pools used for stimulation in ELISPOT assays. The hepatitis C virus polyprotein (genotype 1a; 3,010 amino acids; 750 peptides in total) is composed of four structural proteins (Core to p7) and six nonstructural proteins (NS2 to NS5B). This polyprotein was divided into 33 peptide pools (with 18 to 25 overlapping peptides in each pool), as described in Materials and Methods.

FIG. 2.

Summary of the HCV peptide pool specificities recognized by each individual with acute-chronic and acute-resolving HCV infections. All 31 study patients are shown. The HCV pools are grouped according to HCV protein. The grey-shaded boxes indicate qualitatively positive CD4⁺ T-cell responses (A) or CD8⁺ T-cell responses (B). The black boxes represent the strongest CD4⁺ or CD8⁺ T-cell responses in an individual subject. The first column indicates the patient identifier, and the far-right column shows the total number of pools recognized for each individual patient. The bottom row represents the number of patients in the cohort who demonstrated qualitative responses to individual peptide pools. Among the 14 patients with acute-resolving infections, 29 of the 33 distinct HCV pools elicited responses from CD4⁺ T cells and 23 distinct pools elicited responses from CD8⁺ T cells.

FIG. 3.

T-cell thresholds associated with spontaneous recovery following acute HCV infection. Using 33 subgenomic pools spanning the entire HCV polyprotein, total T-cell responses were assessed by IFN-γ ELISPOT assays. (A) For CD4⁺ T cells, qualitative responses to five or more pools at the earliest time point (enrollment) were associated with a statistically higher rate of recovery (P = 0.02 by logistic regression analysis). (B) More than 80% of patients demonstrating more than 390 CD4⁺ T-cell ELISPOTs per 2.5 × 10⁵ CD4⁺ T cells spontaneously recovered from HCV infection, whereas less than 40% not meeting this quantitative threshold did so. The total sum of ELISPOTs for 33 peptide pools was added for each individual subject. (C) For CD8⁺ T cells, qualitative responses to two or more HCV peptide pools was associated with a statistically higher rate of recovery (P = 0.04; logistic regression analysis).

FIG. 4.

Representative examples of different immunologic patterns according to whole HCV genome analyses of CD4⁺ and CD8⁺ IFN-γ ELISPOTs (the asterisks indicate significant responses compared to negative control wells). Group A demonstrated both CD4⁺ and CD8⁺ T-cell responses. Group B demonstrated CD8⁺ T-cell responses but no CD4⁺ T-cell responses, and group C demonstrated no significant CD4⁺ or CD8⁺ T-cell responses. Both representative patients for group A resolved HCV infection. In group B, HS125 developed chronic infection and PD115 resolved. The majority of patients (five of six) in group C developed chronic infection, including both patients depicted here. Table 1 shows the demographic and clinical features of the three immunologic groups. The error bars indicate standard deviations.

FIG. 5.

The HCV RNA and ALT kinetics in the three immunologic pattern groups. Data points for each individual subject are represented by different symbols at the indicated time points. The shaded area represents the threshold of sensitivity of the HCV RNA assay and the normal range of values for ALT. Of note, only one patient (the previously described [26] PD101) received antiviral therapy.

FIG. 6.

Secretion of IL-2 and IL-10 by HCV-specific CD4⁺ T cells in patients who demonstrated HCV-specific CTLs by IFN-γ ELISPOT. Briefly, bead-purified CD4⁺ cells (2.5 ×10⁵) were cultured in duplicate wells with 1 × 10⁴ autologous LCLs that had been pulsed with either HCV core and NS3 (Mikrogen), HCV NS4 and NS5 (Chiron), or relevant negative controls. After 48 h, the cell supernatants were examined by Luminex for cytokine production. Each point represents the amount of cytokine produced in response to HCV proteins after the subtraction of negative controls. Shown are the results for 6 patients in the pattern A group (+CD4+CD8 ELISPOTs) and 7 patients in the pattern B group (no CD4 ELISPOTs, +CD8 ELISPOTs); each point represents cytokine values following stimulation with either the core/NS3 or NS4/NS5 combination of HCV antigens (thus, 26 values for 13 patients). Patients with HCV-specific CTLs who spontaneously resolved HCV infection demonstrated significantly greater HCV-specific, CD4-derived IL-2 secretion (A) but no difference in the secretion of IL-10 (B).

FIG. 7.

Antigen-specific IFN-γ production and degranulation. CD4-depleted PBMCs were stimulated with CEF peptides or specific HCV pools as described in Materials and Methods in 10 patients (shown are 3 representative patients). (A) Gating strategy used to identify antigen-specific CTLs and CEF- and HCV-specific responses for one patient (TN105). CTLs were identified by initially gating on lymphocytes (P1) and then on CD8⁺ CD3⁺ T cells within the lymphocyte population (P2). Antigen-specific CTLs (HCV/CEF) were identified as IFN-γ-positive cells within the CTL population. (B and C) CD107a expression on CEF-specific responses for three representative patients (B) and HCV-specific responses for the same patients (C). Interval gates were set using FMO and isotype controls. CD107a positivity was defined as the percentage of the population staining positive for PE-CY5 above negative controls. Upregulation of CD107a did not correlate with the outcome of infection. The HCV peptide pools used were as follows: for TN105, NS3-1P, NS3-4H, and NS3-5H; for HS105, E2C, NS3-4H, NS3-7H, NS4-B2, and NS5-B1; for HS102, NS3-1P.

FIG. 8.

Proliferative capacities and phenotypes of HCV₁₀₇₃-specific CTLs in two representative patients: HS125, who developed chronicity, and PD115, who spontaneously cleared HCV. CFSE-stained PBMCs depleted of CD4 T cells were stimulated with NS3₁₀₇₃ as described in Materials and Methods. The cells were gated on CD8 and A2-1073 pentamer double positivity and analyzed for loss of CFSE, as well as expression of PD-1, CD62L, and CD127. (A) The levels of CTL proliferation were comparable in the two patients at the earliest time point (enrollment). A higher percentage of total pentamer-positive CTLs in HS125 expressed PD-1 and CD62L and fewer expressed CD127 than in PD115. (B) Four months later, the proliferative capacity of HCV1073-reactive CTLs was decreased, markedly so for patient HS125, compared to baseline responses. PD-1 and CD62L expression on pentamer-positive CTLs remained higher for patient HS125 at that time.