Although recognized as a curable disease, the persistence of hepatitis C virus (HCV) in chronically infected patients remains a great burden for public health. T cell immune responses serve a key role in anti-HCV infection; however, the features of T cell immunity in patients after a long-term infection are not well explored. We recruited a special cohort of patients with similar genetic background and natural developing progression of disease who were infected with HCV through blood donation 35 y ago. We found that self-resolved individuals had higher levels of cytokine-secreting T cells than individuals with chronic infections, indicating HCV-specific T cell immunity could be sustained for >35 y. Meanwhile, virus-specific CD8+ T cells in chronic patients were characterized by programmed cell death-1high, TIM-3high expression, which was related to liver injury characterized by aspartate transaminase/alanine aminotransferase levels and morphopathological changes. Unexpectedly, the expression of Lymphocyte-activation gene 3 on CD8+ T cells was lower in chronic patients and negatively correlated with alanine aminotransferase/aspartate transaminase. Our findings provided new insights into HCV-specific T cell responses and may shed light on a way to figure out novel effector targets and explore a way to reverse chronic infections.

Hepatitis C virus (HCV) is a single-strand RNA virus belonging to the Flaviviridae family. It was estimated that HCV caused at least 71 million infections worldwide up to 2015. This number continues to rise, posing a great threat to global health (1). There are seven known genotypes of HCV, i.e., genotype 1–7, which are distributed in varying regions (2). In East Asia, HCV genotype 1, as the most prevalent genotype, causes nearly 50% of all infections, while genotypes 2, 3, and 6 are responsible for the other half of the infections (1, 3). After acute HCV infection, 15–25% of the patients recover spontaneously, with sustained undetectable HCV RNA levels and normalization of alanine aminotransferase (ALT) levels in the serum (4). However, >75% of HCV patients carry the virus persistently and have a high risk of developing chronic liver diseases or serious hepatic complications, such as hepatocellular carcinoma (5).

T cell immunity in HCV patients was determined to induce protective antiviral responses (6, 7). Only individuals who developed sufficient HCV-specific CD4+ and CD8+ T cell responses were more likely to clear the virus transiently or permanently during acute HCV infection phase, while those failing to generate effective T cell responses tended to develop chronic symptoms (810). Rosen et al. (11) also determined a key role of virus-specific activated memory CD4+ T cells in self-resolved individuals for 19 y postinfection. The levels of memory (CD45RO+CD69+) CD4+ T cells in self-resolved cases were more than those in chronic patients (7), while the CD127hi programmed cell death-1low (PD-1low) effector memory CD8+ T cells were associated with protection against viral persistence in self-resolved HCV infection (6, 12). Further analysis indicated that activated CD4+ or CD8+ T cells had impaired effector functions and would become exhausted in chronic HCV infections, although Ag-specific T cell immunity existed (13). In chronic infections, HCV Core/NS3-specific tetramer+ CD8+ T cells had impaired proliferation capacity and less specific cytotoxicity compared with those in recovered individuals, although the amount of tetramer+ T cells was higher than the latter (13). This failure of intrahepatic virus-specific CD8+ T cells to sufficiently control the virus occurs despite the presence of virus-specific CD4+ T cells at the site of disease (14). Moreover, HCV-specific CD4+ T cells were also deficient in chronic phase, with a higher frequency of regulatory T cells (15, 16). In patients with normal ALT levels, HCV-specific CD4+CD25+high T cells in PBMCs secreted higher levels of TGF-β than that in patients with abnormal ALT levels (17); however, the ability to produce IFN-γ in patients with abnormal ALT values was stronger than that in intrahepatic HCV-specific CD4+ T cells (18).

Multiple mechanisms contributed to the exhaustion of HCV-specific T cells. In addition to the regulation of regulatory T cells, previous studies revealed that the function of virus-specific T cells in chronic infections was regulated by costimulatory and inhibitory TCRs (1921). The solo-expression and coexpression of PD-1/T cell Ig domain and mucin domain-3 (TIM-3)/CTLA-4/2B4/CD160/Lymphocyte-activation gene 3 (LAG-3)/CD200R might be associated with the CD8+ T cell exhaustion (2126). Blocking of inhibitory TCRs could restore the function of HCV-specific T cells by increasing their Ag-specific proliferation and cytokine secretion (21, 2527). Moreover, the restoration was dependent on the expression pattern and compartmentalization of these markers (28). During the disease progression, the frequency of TIM-3+PD-1+ HCV-specific CTLs was associated with the viral load (25). However, the patterns of disease progression and divergent expression of inhibitory molecules in chronic and resolved individuals were still vague.

In this study, we investigated a special cohort who were infected with HCV via blood donation ∼35 y ago in the same village without any antiviral treatment. However, different disease progressions were observed among these individuals, including self-resolved with undetectable HCV RNA and chronic infection with persistent virus RNA in the blood. We analyzed the features of Ag-specific T cell immunity and its correlation with the clinical indexes of these patients. After 35 y of the infection, the self-resolved group still possessed significantly higher levels of cytokine-secreting CD8+ and CD4+ T cell responses compared with the chronic subjects. The abnormally higher expression of inhibitory molecule, especially PD-1 and TIM-3, but lower expression of LAG-3 in CD8+ T cells was observed in the chronic patients. What is more, expressions of PD-1 and TIM-3 were positively correlated with the levels of ALT and AST, respectively. The data revealed the relationship between T cell immune status and liver damage among HCV patients with long-term infection.

During 1970s–1990s, blood/plasma donation with nondisposable supplies was manipulated among the donors from a village in Zhao Town in China. Fifty people from the village were recruited at a hospital in Shijiazhuang, China in 2015 and were divided into three groups: chronic group (HCV-seropositive, NS3 1b RNA-positive patients, n = 15; HCV-seropositive, NS3 2a RNA-positive patients, n = 7), resolved group (HCV-seropositive, RNA-negative subjects who had spontaneously clear virus, n = 14), and normal healthy controls (HCV-seronegative subjects, n = 14). Demographic characteristics and general inquisition were recorded by doctors (Table I). Serum and PBMCs were collected for immunological experiments, and PBMC isolation was performed (28). The Ethics Committee of National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention (China CDC) approved the study. All of the subjects signed an informed consent before the study.

Physical examinations for the individuals were completed in the hospital, including blood routine, blood biochemistry, color Doppler ultrasonography, and computed tomography. The information related to liver function was listed in Table I.

HCV RNA was extracted from 200 μl serum using QIAamp Viral RNA Mini kit (Qiagen, China) according to the manufacturer's recommendations. The quantity of HCV RNA was measured using one-step real-time PCR assay (Abbott Molecular China) following the manufacturer's recommendations. HCV RNA level of all chronic infections was shown in Supplemental Table I.

To evaluate T cell responses against HCV, we used nonstructural protein 3 (NS3) of HCV as the antigenic target for overlapping peptide design as described previously (29). Each peptide was 17–20-mer, was overlapped by 10 aa spanning the whole protein, and was synthesized (purity > 90%) by Beijing SciLight Biotechnology. NS3 peptide pools were defined as 2a or 1b according to the genotypes, and 66 peptides were included in each peptide pool. These peptides were stored in aliquots at −80°C in a lyophilized environment after synthesis and dissolved in DMSO before use.

Thawed PBMCs frozen in liquid nitrogen from patients were stimulated with HCV-specific peptide pools as described previously (29). In brief, 1 × 106 PBMCs were incubated with RPMI 1640 medium with 10% FBS for 2 h at 37°C in 5% CO2. Then peptide pool (2 μg/ml individual peptide) was added to stimulate PBMCs. Each sample was stimulated, respectively, with HCV 1b and 2a peptide pools. After 2 h, GolgiStop (BD) was added to each well to stop IFN-γ secretion and incubated for 4 h at 37°C. After stimulation, PBMCs were stained with surface fluorescence-labeled mAbs, including anti–CD4-allophycocyanin-Cy7, anti–CD8-BV510, anti–CD45RA-AF700, anti–CCR7-AF647, anti–PD-1-BV605, anti–CD200R-allophycocyanin, anti–TIM-3-PE-CF594, anti–LAG-3-PE, and anti–CTLA-4-BV421. Then PBMCs were fixed with BD fix/perm buffer and stained in BD wash buffer with intracellular markers, including IFN-γ, IL-2, TNF-α, and CTLA-4. After washing twice, PBMCs were resuspended using FACS buffer for flow cytometry analysis (BD LSR Fortessa). Cytokine-secreting (IFN-γ, TNF-α, IL-2) T cells were considered as HCV-specific T cells. All fluorescence-labeled Abs were purchased from BD. Data were analyzed with FlowJo software Version 10.

Statistical analysis was performed as described previously (30). Two-tailed Student t test was used for comparing the values of two groups. To compare the values between multiple groups, we performed one-way ANOVA analysis with Bonferroni posttest. The linear correlation test was used for correlation analyses. All tests were two-tailed with a significance level of 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001). All data analyses were performed with GraphPad Prism 6.0 (GraphPad Software, San Diego, CA).

Fifty participants living in the same village in Hebei Province were recruited to participate in this study. Thirty-eight of the fifty participants (76%) had a history of blood or plasma donation. The initial blood or plasma donation time of the subjects was in a period ranging from 1970 to 1994. The median of initial blood donation time was 1980, while the plasma donation time was later than the blood, which was concentrated around 1989 (Fig. 1A). The previous epidemiological investigation has indicated a correlation between the blood or plasma donation and the high prevalence (28.5%) of HCV infection among the villagers (31). Based on the HCV Ab test and the RNA test, subjects recruited in this study were divided into three groups: 22 chronically infected patients; 14 self-resolved subjects, which were defined as HCV Ab positive and HCV RNA negative; and 14 HCV Ab-negative individuals as healthy controls. The blood donation rates between the resolved (92.9%) and chronic group (95.5%) were on the same level (Fig. 1B). The duration of HCV infection was estimated from the date of initial exposure to the date the assay was performed, averaging 35.8 ± 6.1 y for the resolved subjects and 34.6 ± 7.2 y for the chronic patients. The average age of the participants recruited in our study was 57.8 y, with no significant differences among the three groups.

FIGURE 1.

The blood/plasma donation information and ALT (AST) level of the cohort.

(A) The initial donation time of blood/plasma for three groups (x-axis shows the years 1965–1995). The black solid dots represent the year of donation for donors in the negative control, resolved HCV, and chronic HCV. The hollow dots represent the plasma donation as above. The green line represents the median time of blood donation (1980), and the purple line represents that of plasma donation (1989). The red line means the sampling time (2015). (B) The blood/plasma donation rate of three groups, with the x-axis representing blood donation and plasma donation. For blood donation, the donation rate of negative control (green) was 4/14 (28.6%), that for resolved HCV (rose red) was 13/14 (92.6%), and that for chronic HCV (blue) was 21/22 (95.5%). The plasma donation rate of the negative control was 1/14 (7.1%), that for resolved HCV was 13/14 (92.6%), and that for chronic HCV was 21/22 (95.5%). (C) The ALT and AST levels in negative (green), resolved (rose red), and chronic (blue) groups. Statistical significance of differences between the three groups was determined by one-way ANOVA analysis with Bonferroni’s test. ***p < 0.001. (D) The linear correlation between viral load and ALT (solid triangle) or AST (hollow triangle). x-axis shows the viral load of chronic HCV patients, and y-axis shows the corresponding ALT and AST values. Gray area represents the concentration range of ALT or AST (0–40 U/L) in healthy individuals. ns, no difference between resolved HCV and chronic HCV.

FIGURE 1.

The blood/plasma donation information and ALT (AST) level of the cohort.

(A) The initial donation time of blood/plasma for three groups (x-axis shows the years 1965–1995). The black solid dots represent the year of donation for donors in the negative control, resolved HCV, and chronic HCV. The hollow dots represent the plasma donation as above. The green line represents the median time of blood donation (1980), and the purple line represents that of plasma donation (1989). The red line means the sampling time (2015). (B) The blood/plasma donation rate of three groups, with the x-axis representing blood donation and plasma donation. For blood donation, the donation rate of negative control (green) was 4/14 (28.6%), that for resolved HCV (rose red) was 13/14 (92.6%), and that for chronic HCV (blue) was 21/22 (95.5%). The plasma donation rate of the negative control was 1/14 (7.1%), that for resolved HCV was 13/14 (92.6%), and that for chronic HCV was 21/22 (95.5%). (C) The ALT and AST levels in negative (green), resolved (rose red), and chronic (blue) groups. Statistical significance of differences between the three groups was determined by one-way ANOVA analysis with Bonferroni’s test. ***p < 0.001. (D) The linear correlation between viral load and ALT (solid triangle) or AST (hollow triangle). x-axis shows the viral load of chronic HCV patients, and y-axis shows the corresponding ALT and AST values. Gray area represents the concentration range of ALT or AST (0–40 U/L) in healthy individuals. ns, no difference between resolved HCV and chronic HCV.

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Physical examination, routine blood test, blood biochemistry, color Doppler ultrasound, and computed tomography were performed for each subject in the hospital. By using the serum of all the subjects, we evaluated the biochemical indexes relating to liver function and found that levels of 9 of 20 indexes were abnormal in the chronic group, especially the levels of alanine transaminase (ALT) and aspartate aminotransferase (AST) (Fig. 1C). Meanwhile, compared with the negative group, the self-resolved group had no statistically significant difference, with abnormal ALT or AST in only one donor. This indicated that the liver indexes of the self-resolved group had returned to normal range, and the group was considered to be self-resolved. The detailed clinical characteristics of the subjects were described in Table I. In addition, the viral load of chronically infected HCV patients was performed and analyzed (Supplemental Table I). Interestingly, we found that the viral load was positively correlated with levels of ALT and AST (Fig. 1D).

Table I.

Characteristics of the study population

CharacteristicsNegativeResolved HCVChronic HCVReference Rangep Valuea
n 14 14 22  NA 
Male sex, n (%) 6 (43) 5 (36) 13 (59)  b 
Age (y), mean ± SD 54.00 ± 11.30 60.00 ± 4.30 58.70 ± 6.90  * 
Blood donation (%) 28.6 92.9 95.5  * 
Plasma donation (%) 7.1 92.9 95.5  * 
HCV Ab Negative Positive Positive  NA 
HCV RNA Negative Negative Positive  NA 
Liver function, mean ± SD      
 ALT (U/l) 25.71 ± 18.89 23.14 ± 16.53 66.77 ± 39.69 5.00–40.00 <0.0001 
 AST (U/l) 23.21 ± 5.32 23.86 ± 7.01 51.72 ± 27.52 8.00–40.00 <0.0001 
 LDH (U/l) 178.79 ± 17.88 190.14 ± 24.11 206.41 ± 31.70 114.00–240.00 * 
 ADA (U/l) 11.57 ± 2.82 13.57 ± 3.44 18.45 ± 6.81 0–20.00 <0.01 
 TBIL (μmol/l) 14.47 ± 4.57 14.00 ± 3.51 19.45 ± 7.06 1.71–17.10 <0.01 
 DBIL (μmol/l) 4.64 ± 1.45 4.50 ± 1.45 7.45 ± 3.39 1.71–7.00 <0.01 
 IBIL (μmol/l) 9.33 ± 3.29 9.50 ± 2.41 12.00 ± 4.41 1.70–13.70 <0.05 
 TBA (μmol/l) 3.86 ± 4.87 4.64 ± 3.73 5.95 ± 4.72 0.10–10.00 <0.05 
 ALP (U/l) 73.35 ± 25.68 83.86 ± 41.14 81.91 ± 17.05 30.00–90.00 * 
 GGT (U/l) 23.21 ± 10.49 18.79 ± 8.44 37.68 ± 39.40 0–40.00 <0.05 
 TP (g/l) 73.57 ± 2.79 74.29 ± 3.54 75.64 ± 4.01 60.00–80.00 * 
 ALB (g/l) 48.64 ± 1.82 47.29 ± 1.98 45.86 ± 3.75 35.00–50.00 <0.01 
 GLB (g/l) 24.93 ± 2.92 27 ± 3.35 29.77 ± 4.52 20.00–30.00 * 
 PA (mg/l) 251.43 ± 66.43 251.14 ± 50.22 172.27 ± 64.35 250.00–380.00 <0.001 
 CHE (U/l) 8991 ± 1503 10,390 ± 1861 7810 ± 2177 4300–10500 <0.05 
 CHOL (mmol/l) 5.01 ± 0.81 5.12 ± 1.07 4.62 ± 0.92 2.30–5.17 * 
 HDL-C (mmol/l) 1.49 ± 0.27 1.47 ± 0.50 1.57 ± 0.42 1.04–1.90 * 
 LDL-C (mmol/l) 3.15 ± 0.82 3.10 ± 0.72 2.68 ± 0.90 2.00–3.12 <0.05 
 APO-B (g/l) 0.91 ± 0.17 0.85 ± 0.15 0.71 ± 0.20 0.60–1.00 <0.05 
 APO-A1 (g/l) 1.60 ± 0.20 1.68 ± 0.42 1.67 ± 0.39 1.00–1.60 * 
CharacteristicsNegativeResolved HCVChronic HCVReference Rangep Valuea
n 14 14 22  NA 
Male sex, n (%) 6 (43) 5 (36) 13 (59)  b 
Age (y), mean ± SD 54.00 ± 11.30 60.00 ± 4.30 58.70 ± 6.90  * 
Blood donation (%) 28.6 92.9 95.5  * 
Plasma donation (%) 7.1 92.9 95.5  * 
HCV Ab Negative Positive Positive  NA 
HCV RNA Negative Negative Positive  NA 
Liver function, mean ± SD      
 ALT (U/l) 25.71 ± 18.89 23.14 ± 16.53 66.77 ± 39.69 5.00–40.00 <0.0001 
 AST (U/l) 23.21 ± 5.32 23.86 ± 7.01 51.72 ± 27.52 8.00–40.00 <0.0001 
 LDH (U/l) 178.79 ± 17.88 190.14 ± 24.11 206.41 ± 31.70 114.00–240.00 * 
 ADA (U/l) 11.57 ± 2.82 13.57 ± 3.44 18.45 ± 6.81 0–20.00 <0.01 
 TBIL (μmol/l) 14.47 ± 4.57 14.00 ± 3.51 19.45 ± 7.06 1.71–17.10 <0.01 
 DBIL (μmol/l) 4.64 ± 1.45 4.50 ± 1.45 7.45 ± 3.39 1.71–7.00 <0.01 
 IBIL (μmol/l) 9.33 ± 3.29 9.50 ± 2.41 12.00 ± 4.41 1.70–13.70 <0.05 
 TBA (μmol/l) 3.86 ± 4.87 4.64 ± 3.73 5.95 ± 4.72 0.10–10.00 <0.05 
 ALP (U/l) 73.35 ± 25.68 83.86 ± 41.14 81.91 ± 17.05 30.00–90.00 * 
 GGT (U/l) 23.21 ± 10.49 18.79 ± 8.44 37.68 ± 39.40 0–40.00 <0.05 
 TP (g/l) 73.57 ± 2.79 74.29 ± 3.54 75.64 ± 4.01 60.00–80.00 * 
 ALB (g/l) 48.64 ± 1.82 47.29 ± 1.98 45.86 ± 3.75 35.00–50.00 <0.01 
 GLB (g/l) 24.93 ± 2.92 27 ± 3.35 29.77 ± 4.52 20.00–30.00 * 
 PA (mg/l) 251.43 ± 66.43 251.14 ± 50.22 172.27 ± 64.35 250.00–380.00 <0.001 
 CHE (U/l) 8991 ± 1503 10,390 ± 1861 7810 ± 2177 4300–10500 <0.05 
 CHOL (mmol/l) 5.01 ± 0.81 5.12 ± 1.07 4.62 ± 0.92 2.30–5.17 * 
 HDL-C (mmol/l) 1.49 ± 0.27 1.47 ± 0.50 1.57 ± 0.42 1.04–1.90 * 
 LDL-C (mmol/l) 3.15 ± 0.82 3.10 ± 0.72 2.68 ± 0.90 2.00–3.12 <0.05 
 APO-B (g/l) 0.91 ± 0.17 0.85 ± 0.15 0.71 ± 0.20 0.60–1.00 <0.05 
 APO-A1 (g/l) 1.60 ± 0.20 1.68 ± 0.42 1.67 ± 0.39 1.00–1.60 * 
a

p value, the difference between resolved HCV and chronic HCV.

b

By the χ2 test, others by t test.

*

p > 0.05.

ADA, adenosine deaminase; ALB, albumin; ALP, alkaline phosphatase; APO-A1, apolipoprotein A1; APO-B, apolipoprotein b; CHE, cholinesterase; CHOL, cholesterol; DBIL, direct bilirubin; GGT, γ-glutamyl transpeptidase; GLB, globulin; HDL-C, high-density lipoprotein cholesterol; IBIL, indirect bilirubin; LDH, lactate dehydrogenase; LDL-C, low-density lipoprotein cholesterol; NA, not applicable; PA, prealbumin; TBA, total bile acid; TBIL, total bilirubin; TP, total protein.

To characterize the features of T cell responses among the subjects, we detected cytokine profiles in PBMCs of the three groups after stimulating with HCV NS3 overlapping peptide pool. Both self-resolved and chronic subjects had detected activated T cell responses. However, the self-resolved individuals showed a higher percentage of CD8+ T cells secreting IFN-γ, IL-2, and TNF-α than the chronic patients (0.22% versus 0.12% for IFN-γ, 0.11% versus 0.06% for IL-2, and 0.15% versus 0.09% for TNF-α) (Fig. 2A–C). Similarly, the cytokine-secreting CD4+ T cells showed the same phenomenon as the CD8+ T cells (Fig. 2D–F). The steps of gating in flow cytometric analysis using FlowJo were displayed (Supplemental Fig. 1), and all flow analysis in the study was performed according to this standard.

FIGURE 2.

CD8+ and CD4+ T cell responses in HCV patients after long-term infection.

The frequency of HCV-specific CD8+ and CD4+ T cells was tested through ex vivo intracellular cytokines IFN-γ, IL-2, and TNF-α staining of PBMCs, under the stimulation of HCV NS3 peptide pool, with isotype staining as the background. NS3 2a peptide pool was used stimulating PBMCs from HCV 2a genotype–positive patients, while NS3 1b peptide pool was used stimulating PBMCs from HCV 1b genotype–positive patients and HCV RNA–negative individuals. (AC) Percentage of HCV-specific CD8+ T cells that secreted IFN-γ, IL-2, and TNF-α, respectively. (DF) Percentage of HCV-specific CD4+ T cells that secreted IFN-γ, IL-2, and TNF-α, respectively. The charts represent the median with an interquartile range of data. Green dots represent negative control, rose squares represent resolved HCV, and blue triangles represent chronic HCV groups. Statistical significance of differences between the three groups was determined by one-way ANOVA analysis with Bonferroni’s test. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 2.

CD8+ and CD4+ T cell responses in HCV patients after long-term infection.

The frequency of HCV-specific CD8+ and CD4+ T cells was tested through ex vivo intracellular cytokines IFN-γ, IL-2, and TNF-α staining of PBMCs, under the stimulation of HCV NS3 peptide pool, with isotype staining as the background. NS3 2a peptide pool was used stimulating PBMCs from HCV 2a genotype–positive patients, while NS3 1b peptide pool was used stimulating PBMCs from HCV 1b genotype–positive patients and HCV RNA–negative individuals. (AC) Percentage of HCV-specific CD8+ T cells that secreted IFN-γ, IL-2, and TNF-α, respectively. (DF) Percentage of HCV-specific CD4+ T cells that secreted IFN-γ, IL-2, and TNF-α, respectively. The charts represent the median with an interquartile range of data. Green dots represent negative control, rose squares represent resolved HCV, and blue triangles represent chronic HCV groups. Statistical significance of differences between the three groups was determined by one-way ANOVA analysis with Bonferroni’s test. *p < 0.05, **p < 0.01, ***p < 0.001.

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We further analyzed the profiles of triple-cytokine–secreting (IFN-γ+IL-2+TNF-α+), double-cytokine–secreting (IFN-γ+IL-2+TNF-α, IFN-γ+IL-2TNF-α+, or IFN-γIL 2+TNF-α+), and single-cytokine–secreting (IFN-γ+IL-2TNF-α, IFN-γIL-2+TNF-α, or IFN-γIL-2TNF-α+) CD8+ and CD4+ T cells during self-resolved individual and chronic infections. We found that the percentage of T cells secreting IFN-γ+IL-2TNF-α+ double cytokines occupied in CD8+ T cells of resolved individuals was more than that of chronic subjects (Fig. 3A). For CD4+ T cells, IFN-γ+IL-2TNF-α and IFN-γIL-2+TNF-α T cells took the dominant role for all the subjects. However, IFN-γIL-2+TNF-α+ double-cytokine–secreting CD4+ T cells in resolved individuals occupied a larger proportion than that in chronic patients (Fig. 3B).

FIGURE 3.

The multiple cytokine-secreting T cells and the memory phenotype in resolved and chronic HCV groups.

(A and B) Percentages of single-cytokine IFN-γ+IL2TNFα– (blue), IFN-γIL-2+TNFα– (red), or IFN-γIL-2TNFα+–secreting (green) T cells; double-cytokine IFN-γ+IL-2+TNFα– (purple), IFN-γ+IL-2TNFα+– (blue-green), or IFN-γIL-2+TNFα+–secreting (orange) T cells; or triple-cytokine IFN-γ+IL-2+TNF-α+–secreting (light blue) T cells in CD8+ (A) and CD4+ (B) T cells, respectively. (C) Memory subsets of HCV-specific, IFN-γ–secreting CD8+ or CD4+ T cells in resolved and chronic groups. Naive T cell (Naive, CD45RA+ CCR7+) (gray), central memory T cells (TCM, CD45RACCR7+) (yellow), effector memory T cells (TEM, CD45RACCR7) (orange), and effector T cells (Effector, CD45RA+CCR7) (blue). (D) The representative flow cytometry charts of memory subsets of total CD8+ (CD4+) T cells and HCV-specific CD8+ (CD4+) T cells.

FIGURE 3.

The multiple cytokine-secreting T cells and the memory phenotype in resolved and chronic HCV groups.

(A and B) Percentages of single-cytokine IFN-γ+IL2TNFα– (blue), IFN-γIL-2+TNFα– (red), or IFN-γIL-2TNFα+–secreting (green) T cells; double-cytokine IFN-γ+IL-2+TNFα– (purple), IFN-γ+IL-2TNFα+– (blue-green), or IFN-γIL-2+TNFα+–secreting (orange) T cells; or triple-cytokine IFN-γ+IL-2+TNF-α+–secreting (light blue) T cells in CD8+ (A) and CD4+ (B) T cells, respectively. (C) Memory subsets of HCV-specific, IFN-γ–secreting CD8+ or CD4+ T cells in resolved and chronic groups. Naive T cell (Naive, CD45RA+ CCR7+) (gray), central memory T cells (TCM, CD45RACCR7+) (yellow), effector memory T cells (TEM, CD45RACCR7) (orange), and effector T cells (Effector, CD45RA+CCR7) (blue). (D) The representative flow cytometry charts of memory subsets of total CD8+ (CD4+) T cells and HCV-specific CD8+ (CD4+) T cells.

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To investigate the memory phenotype of T cells, we analyzed PBMCs based on the expression of CD45RA and CCR7. The results demonstrated that virus-specific CD8+ T cells were mainly composed of effector T cells and effector memory T cells, while the majority of virus-specific CD4+ T cells were effector memory T cells (Fig. 3C, 3D). No significant differences were observed between resolved and chronic group subjects in both virus-specific CD8+ and CD4+ T cells.

To further investigate the features of T cells in the antiviral process, we analyzed the expressions of different T cell inhibitory receptors, including PD-1, CTLA-4, LAG-3, TIM-3, and CD200R, among the subjects of the three groups. When stimulating with HCV NS3 peptide pool, the expressions of PD-1 and TIM-3 were significantly higher in chronic infections relative to the self-resolved and negative group in CD8+ T cells (Fig. 4A, 4B). The expression of CTLA-4 and CD200R in CD8+ T cells did not differ significantly among the three groups (Supplemental Fig. 2A, 2B). With regard to CD4+ T cells, the expression of PD-1 was much higher in chronic infections than both self-resolved and negative groups (Fig. 4D). TIM-3 expression in CD4+ T cells was also higher in chronic infections than in negative groups (Fig. 4E). CTLA-4 and CD200R expression in CD4+ T cells were similar to that in CD8+ T cells among three groups (Supplemental Fig. 2C, 2D). The representative flowcharts of PD-1, TIM-3, and LAG-3 expression on CD8+ T and CD4+ T cells were exhibited (Supplemental Fig. 2E). Interestingly, the expression of LAG-3 in CD8+ T cells of chronic infections was significantly lower than that of the negative group (Fig. 4C). The high levels of inhibitory receptor PD-1 and TIM-3 in CD8+ and CD4+ T cells in chronic infections might be vital in suppressing T cell functions.

FIGURE 4.

Expression analysis of PD-1, TIM-3, and LAG-3 in CD8+ and CD4+ T cells of the long-term HCV patients.

The analysis included 14 negative control, 14 resolved HCV, and 22 chronic HCV patients (*p < 0.05, **p < 0.01, one-way ANOVA analysis with Bonferroni test). (AC) PD-1, TIM-3, and LAG-3 expression in CD8+ T cells. (DF) PD-1, TIM-3, and LAG-3 expression in CD4+ T cells in the three groups. Green dots represent negative control, rose squares represent resolved HCV, and blue triangles represent chronic HCV groups.

FIGURE 4.

Expression analysis of PD-1, TIM-3, and LAG-3 in CD8+ and CD4+ T cells of the long-term HCV patients.

The analysis included 14 negative control, 14 resolved HCV, and 22 chronic HCV patients (*p < 0.05, **p < 0.01, one-way ANOVA analysis with Bonferroni test). (AC) PD-1, TIM-3, and LAG-3 expression in CD8+ T cells. (DF) PD-1, TIM-3, and LAG-3 expression in CD4+ T cells in the three groups. Green dots represent negative control, rose squares represent resolved HCV, and blue triangles represent chronic HCV groups.

Close modal

We further explored the suppression status of virus-specific T cells, especially the expression of PD-1, TIM-3, and LAG-3. We found that the expression of PD-1 or TIM-3 in Ag-specific CD8+ T cells was significantly higher in chronic infections than that in resolved individuals (Fig. 5A). Although PD-1+TIM3+ coexpression in CD8+ T cells was also more in the chronic group than those in the resolved group (Fig. 5A), indicating the important suppressed roles of PD-1 and TIM-3 in chronic infections.

FIGURE 5.

Immune inhibitor expressions in HCV-specific CD8+ T cells and correlation with ALT and AST levels.

(A) The expression of PD-1, TIM-3, and LAG-3 and the coexpression of PD-1+TIM-3+ in HCV-specific CD8+ T cells (IFN-γ–positive cells) in resolved and chronic groups. Statistical significance of differences between the two groups was determined using the Mann–Whitney U test. *p < 0.05. The liner correlation analysis included subjects secreting IFN-γ (>0.15%) in resolved and chronic groups and was performed using GraphPad 6.0 (n = 13). (B) Linear correlation between PD-1 expression in HCV-specific CD8+ T cells and ALT (solid triangles) or AST (hollow triangles). (C) TIM-3 expression in HCV-specific CD8+ T cells. (D) PD-1+TIM-3+ expression in HCV-specific CD8+ T cells. (E and F) LAG-3 expression in total CD8+ T cells in negative and chronic groups.

FIGURE 5.

Immune inhibitor expressions in HCV-specific CD8+ T cells and correlation with ALT and AST levels.

(A) The expression of PD-1, TIM-3, and LAG-3 and the coexpression of PD-1+TIM-3+ in HCV-specific CD8+ T cells (IFN-γ–positive cells) in resolved and chronic groups. Statistical significance of differences between the two groups was determined using the Mann–Whitney U test. *p < 0.05. The liner correlation analysis included subjects secreting IFN-γ (>0.15%) in resolved and chronic groups and was performed using GraphPad 6.0 (n = 13). (B) Linear correlation between PD-1 expression in HCV-specific CD8+ T cells and ALT (solid triangles) or AST (hollow triangles). (C) TIM-3 expression in HCV-specific CD8+ T cells. (D) PD-1+TIM-3+ expression in HCV-specific CD8+ T cells. (E and F) LAG-3 expression in total CD8+ T cells in negative and chronic groups.

Close modal

To further investigate the role of T cell immune status in the HCV infection, we performed correlation analysis between T cell response and liver injury indexes. The linear regression test demonstrated that the PD-1 and TIM-3 expression in HCV-specific CD8+ T cells positively correlated with the levels of ALT and AST, which were defined as indexes reflecting liver injury degree (Fig. 5B, 5C). What is more, the coexpression of PD-1+TIM-3+ on HCV-specific CD8+ T cells also showed positive associations with ALT and AST (Fig. 5D). Unexpectedly, LAG-3 expression on CD8+ T cells showed negative correlativity with ALT and AST using Spearman’s rank correlation coefficient in negative and chronic groups (Fig. 5E, 5F).

Using the color Doppler ultrasound examination, we analyzed the T cell response of the individuals with normal hepatobiliary function (defined as ultrasonic normal) and patients with chronic liver disease sign (defined as ultrasonic abnormal). The indicators from color ultrasound of the ultrasonic abnormal group included low homogeneous hepatic echo, high hepatic occupying lesions (Supplemental Table II, Fig. 6A). Meanwhile, compared with normal hepatobiliary individuals, the spleen size of patients in the ultrasonic abnormal group was obviously enlarged and the gallbladder wall thickened (Fig. 6A, 6B). We found that IFN-γ/IL-2/TNF-α secreting in CD8+ T cells and IL-2 secreting in CD4+ T cells were higher in patients with ultrasonic normal level than that in ultrasonic abnormal patients during HCV chronic infections (Fig. 6C). Interestingly, TIM-3 expression in CD8+ T cells of the ultrasonic abnormal group was higher than that in the ultrasonic normal group. In contrast, expression of LAG-3 in CD8+ T cells of the ultrasonic normal group was higher (Fig. 6D).

FIGURE 6.

The T cell characteristics in patients with different manifestations in ultrasonic diagnosis.

(A) The representative ultrasound reports of ultrasonic normal group and ultrasonic abnormal group with obvious chronic liver disease characterized through ultrasound diagnosis. The red lines in each picture exhibit the liver size, gallbladder size, and spleen size, respectively. (B) The differences in gallbladder wall and spleen size between ultrasonic normal and ultrasonic abnormal groups. Orange represents the ultrasonic normal group, and blue represents the ultrasonic abnormal group. (C) The analysis of cytokines secretion in CD8+ and CD4+ T cells of ultrasonic normal (orange) and abnormal (blue) groups in resolved HCV and chronic HCV. (D) TIM-3 and LAG-3 expression levels in CD8+ T cells showed differences between ultrasonic normal (orange) and positive (blue) groups in the chronic HCV group. Statistical significance of differences between the two groups was determined using the unpaired t test. *p < 0.05, **p < 0.01.

FIGURE 6.

The T cell characteristics in patients with different manifestations in ultrasonic diagnosis.

(A) The representative ultrasound reports of ultrasonic normal group and ultrasonic abnormal group with obvious chronic liver disease characterized through ultrasound diagnosis. The red lines in each picture exhibit the liver size, gallbladder size, and spleen size, respectively. (B) The differences in gallbladder wall and spleen size between ultrasonic normal and ultrasonic abnormal groups. Orange represents the ultrasonic normal group, and blue represents the ultrasonic abnormal group. (C) The analysis of cytokines secretion in CD8+ and CD4+ T cells of ultrasonic normal (orange) and abnormal (blue) groups in resolved HCV and chronic HCV. (D) TIM-3 and LAG-3 expression levels in CD8+ T cells showed differences between ultrasonic normal (orange) and positive (blue) groups in the chronic HCV group. Statistical significance of differences between the two groups was determined using the unpaired t test. *p < 0.05, **p < 0.01.

Close modal

In this study, a special cohort from a village was recruited. These patients had been exposed to HCV through blood/plasma donation about 35 y ago. Several features of this cohort made it unique and ideal for the investigation of T cell immunity: (1) based on the epidemiological investigation, blood donation was the major transmission approach for infection (31); (2) most of the donors were 20–40 y old when they began the blood donation and were 55–75 y when they took part in this study; (3) the cohort had lived in the same village for the past 35 y, with quite similar lifestyle, and most of them are genetically related; and (4) most of the donors did not receive any systematic treatment. Thus, T cell immunity and their clinical indices may reflect a naturally developed characteristic, which made the cohort an ideal model for analyzing different outcomes. In this cohort, Ag-specific T cell immunity was defective in chronic patients but showed sustained activity in self-resolved patients even 35 y postinfection. Such dysfunction in chronic patients might be associated with the abnormally high levels of inhibitory molecules PD-1 and TIM-3 in T cells, which positively associated with liver injury indices.

Resolved individuals were more likely to respond to HCV Ags (32, 33), where virus-specific CD8+ T cells could persist for ∼20 y after recovery (34, 35). In our study, we found that T cells specific for HCV could persist for >35 y postinfection or even recovery; however, limited T cell immunity was observed in chronic infections. Because disease progression was natural in this cohort, different features of T cell immunity between the two groups may have contributed to the outcomes. T cell function in chronic infections was abnormal with impaired function or exhaustion. Tetramer-positive CD8+ T cells had impaired proliferative capacity and cytotoxic ability in chronic patients, although the number of tetramer-positive CD8+ T cells was higher (13). There are multiple mechanisms underlying the dysfunction of T cells in chronic infections, including regular T cell suppression and up-regulation of inhibitory molecules. The proportion of regulatory T cells was higher in chronic patients, contributing to the suppression of T cells and high virus titers (16, 26). In addition, inhibitory markers, such as PD-1, 2B4, LAG-3, TIM-3, and CD160, were also up-regulated in chronic patients (16, 26, 3638). The frequency of specific TIM-3+ PD-1+ T cells was correlated to the viral load (24). In our study, expression of PD-1 and TIM-3 on T cells in chronic patients were higher than those in the self-resolved group. Because blockage of PD-1 or TIM-3 could restore the function of CD8+ T cells (21, 28), the abnormally high level of PD-1 and TIM-3 in CD8+ T cells contributed significantly to the suppressed immune status in the chronic group. Unexpectedly, the expression of LAG-3 on CD8+ T cells was higher in the negative group than that in the chronic group. A previous study demonstrated that the expression of LAG-3 in CD8+ intrahepatic T lymphocytes was higher in resolved HCV groups than that in the chronic HCV infection group (39). The different expression patterns of inhibitory molecules in the chronic group and resolved group might have a great impact on their immune responses to HCV Ags.

In this study, we observed that abnormal expression of inhibitor molecules was associated with T cell dysfunction and disease progression. Abnormal expression of inhibitor molecules PD-1high, TIM-3high, or LAG-3low T cells was inversely related to the levels of cytokines; this result was consistent with previous studies. In addition, we found that inhibitor markers were also associated with disease severity, such as levels of AST or ALT and morphology of liver examined using biochemical indices and imaging. PD-1+ T cells and TIM-3+ T cells were positively related with ALT (AST), which are liver injury indices, as well as PD-1+ TIM-3+ T cells. Furthermore, expression of TIM-3 in chronic infections showed a significantly higher level in ultrasonic abnormal individuals than in ultrasonic normal individuals. In our study, we found that there was a positive relationship between PD-1/TIM-3 expression on virus-specific CD8+ T cells and liver injury indices, which might explain the relationship between T cell dysfunction and the status of disease progression.

In this study, we recruited a special HCV-infected cohort with similar genetic background and medical history and determined the longevity and characteristic features of specific T cells in HCV chronic infections and self-resolved subjects. We found subsets of T cells related with levels of T cell function and liver injury via immunological and color ultrasound. Our data provide new insights into HCV-specific T cell responses and may shed light on the way to figuring out novel effector targets and exploring the way to reverse the chronic infections.

This work was supported by the National Key Research and Development Program of China (2021YFC2301400) and the National Natural Science Foundation of China (NSFC; Grant 81971501). W.J.L. was supported by the Excellent Young Scientist Program of the NSFC (Grant 81822040).

W.J. and M.Z. designed and performed experiments, analyzed data, and wrote the paper. J.Z. and H.Z. recruited the cohort and collected the samples. N.L., B.H., W.Y., and S.B. provided expertise. G.F.G., Y.Z., and W.J.L. designed the experiments and wrote the paper. G.F.G. and W.J.L. secured funding.

The online version of this article contains supplemental material.

Abbreviations used in this article:

ALT

alanine aminotransferase

AST

aspartate transaminase

HCV

hepatitis C virus

LAG-3

lymphocyte-activation gene 3

NS3

nonstructural protein 3

PD-1

programmed cell death-1

TIM-3

T cell Ig domain and mucin domain-3

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The authors have no financial conflicts of interest.

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