Home Hepatitis Decrease of T-cells exhaustion markers programmed cell death-1 and T-cell immunoglobulin and mucin domain-containing protein 3 and plasma IL-10 levels after successful treatment of chronic hepatitis C

Decrease of T-cells exhaustion markers programmed cell death-1 and T-cell immunoglobulin and mucin domain-containing protein 3 and plasma IL-10 levels after successful treatment of chronic hepatitis C

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The impact of treatment on PD-1 and Tim-3 expression phenotype of peripheral CD4+ and CD8+ T-cells

PD-1 and Tim-3 expression phenotype of CD4
+T-cells

Pretreatment frequencies of CD4+ T-cells expressing PD-1 and PD-1 + Tim-3 were found to be significantly higher in HCV-infected patients than in controls (median 22.7% (range 7.0–52.7%) vs 14.5% (7.4–37.0%), P = 0.0016 and 1.9% (0.3–9.4%) vs 1.0% (0.3–2.9%), P = 0.0007, respectively); (Fig. 1). The pretreatment frequencies of CD4+ T-cells expressing Tim-3 were also higher in HCV-positive patients, although not statistically significant (7.5% (0.2–32.3%) vs 5.1% (1.8–12.2%). In contrast, the pretreatment frequencies of CD4+ expressing neither PD-1 nor Tim-3 were significantly lower in patients than in controls (66.9% (42.6–81.1%) vs 78.8% (57.0–86.9%), P = 0.0001).

Figure 1

Peripheral blood CD4+ T-cells expression of PD-1 and Tim-3 in 76 patients before and after successful therapy of chronic hepatitis C and in 18 non-infected controls (a) and individual PD-1 and Tim-3 expression changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P values. Pre-Tx before therapy, Post-Tx after therapy, C- uninfected controls.

Treatment resulted a significant decrease of CD4+Tim-3+ T-cells frequencies from 7.5% (0.2–32.3%) to 6.4% (0.7–24.8%), P = 0.0398 and CD4+ PD-1+Tim-3+ T-cells from 1.9% (0.3–9.4%) to 1.3% (0.0–7.6%), P < 0.0001; (Fig. 1). The frequency of CD4+ cells expressing PD-1 did not change (22.7% (7.0–52.7%) vs 22.6% (6.2–43.8%)). In contrast, CD4+PD-1Tim-3 T-cells increased significantly from 66.9% (42.6–81.1%) to 68.5% (44.5–82.0%), P = 0.0217.

After therapy, with the exception of CD4+PD-1+ and CD4+PD-1Tim-3 T-cells (22.6% (6.2–43.8%) vs 14.5% (7.4–37.0%), P = 0.0009 and 68.5% (44.5–82.0%) vs 78.8% (57.0–86.9%), P = 0.0005, respectively), frequencies of all other analyzed subpopulations did not differ significantly from those in controls. A representative cytometric analysis of treatment-related changes in the expression of exhaustion markers on CD4+ T-cells in two patients and two controls is shown in Fig. 2.

Figure 2
figure2

Representative cytometric analysis of peripheral blood CD4+ T-cells expression of PD-1 and Tim-3 before and after successful therapy for chronic HCV infection in Patient 1 (Pt. 1), Patient 2 (Pt. 2) and in two uninfected controls (C.1 and C.2). Pre-Tx before therapy, Post-Tx after therapy.

PD-1 and Tim-3 expression phenotype of CD8
+T-cells

Pretreatment frequencies of CD8+ T-cells co-expressing PD-1 and Tim-3 were significantly higher in patients than in controls (median 2.7% range (0.5–16.1%) vs 1.6% (0.3–4.8%), P = 0.0031; Fig. 3). The pretreatment frequencies of CD8+ T-cells expressing PD-1 or Tim-3 were also higher in patients, although not statistically significant (20.8% (5.5–50.1%) vs 18.7% (7.5–34.8%) and 15.2% (4.3–46.7%) vs 12.7% (6.0–25.7%), respectively). In contrast, the pretreatment frequencies of CD8+ expressing neither PD-1 nor Tim-3 were significantly lower in HCV-positive patients than in controls (58.4% (33.6–75.8%) vs 63.2% (50.3–76.0%), P = 0.0069).

Figure 3
figure3

Peripheral blood CD8+ T-cells expression of PD-1 and Tim-3 in 76 patients before and after successful therapy for chronic HCV infection and in 18 non-infected controls (a) and individual PD-1 and Tim-3 expression changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy, C non-infected controls.

As shown in Fig. 3, treatment resulted in a marked decrease of CD8+ T-cells expressing Tim-3 from 15.2% (4.3–46.7%) to 12.4% (1.8–39.1%), P < 0.0001 and CD8+ T-cells expressing PD-1 + Tim-3 from 2.7% (0.5–16.1%) to 1.8% (0.2–16.9%), P < 0.0001. In contrast, the frequency of CD8+ T-cells expressing PD-1 increased after treatment from 20.8% (5.5–50.1%) to 21.6% (5.4–56.0%), P = 0.0021 and so did the frequency of CD8+ PD-1Tim-3 T-cells (from 58.4% (33.6–75.8%) to 60.8% (34.3–80.1%), P = 0.0002).

After therapy, none of the four CD8+ T-cells subpopulations significantly differed from those in controls (21.6% (5.4–56.0%) vs 18.7% (7.5–34.8%) for PD-1+, 12.4% (1.8–39.1%) vs 12.7% (6.0–25.7%) for Tim-3+, 1.8% (0.2–16.9%) vs 1.6% (0.3–4.8%) for PD-1+Tim-3+, and 60.8% (34.3–80.1%) vs 63.2% (50.3–76.0%) for PD-1Tim-3 T-cells, respectively) (Fig. 3). A representative cytometric analysis of treatment-related changes in CD8+ T-cells exhaustion markers in two patients and two controls is shown in Fig. 4.

Figure 4
figure4

Representative cytometric analysis of peripheral blood CD8+ T-cells expression of PD-1 and Tim-3 before and after successful therapy for chronic HCV infection in Patient 1 (Pt. 1) and Patient 2 (Pt. 2) and in uninfected controls (C.1 and C.2). Pre-Tx before therapy, Post-Tx after therapy.

The effect of clinical and virological parameters on the pretreatment peripheral CD4+ and CD8+ T-cells PD-1 and Tim-3 expression phenotype and IL-10 plasma levels

Since pretreatment expression of exhaustion markers turned out to be highly variable within the analyzed group of patients, we tried to determine whether they were affected by clinical and/or virological parameters. Multivariate analysis included such factors as age, sex, viral load, ALT activity levels, baseline METAVIR liver fibrosis score, weight, and prior treatment (Table 1). In our analysis male sex was associated with higher IL-10 plasma levels (regression coefficient 1.54, 95% CI 0.03 to 3.04, P = 0.045) and higher percentage of CD8+PD-1+ T-cells (regression coefficient 7.69, 95% CI 2.39 to 13.00, P = 0.005). Furthermore, older age was associated with higher percentage of CD4+PD-1+ T-cells (regression coefficient 0.18, 95% CI 0.06 to 0.29, P = 0.003) and CD8+PD-1+Tim-3+ T-cells (regression coefficient 0.05, 95% CI 0.01 to 0.09, P = 0.025) as well as lower percentage of CD4+PD-1Tim-3 T-cells (regression coefficient − 0.18, 95% CI − 0.30 to − 0.06, P = 0.003). Importantly, when compared to F0/1, F3 liver fibrosis score was associated with higher percentage of CD4+PD-1+ T-cells (regression coefficient 5.69, 95% CI 0.13 to 11.26, P = 0.045), but the opposite was true in case of CD4+Tim-3+ T-cells (regression coefficient − 3.4, 95% CI − 6.66 to − 0.30, P = 0.032 for F2 and regression coefficient − 5.20, 95% CI − 9.09 to − 1.31, P = 0.010 for F3). Furthermore, F2 stage was associated with higher percentage of CD8+PD-1Tim-3 T-cells (regression coefficient 7.14, 95% CI 1.37 to 12.91, P = 0.016).

Table 1 Clinical, laboratory and virological characteristic of patients and controls.

Liver fibrosis scores correlation with peripheral T-cell PD-1 and Tim-3 expression phenotype and its treatment-related change

Pretreatment exhaustion markers expression correlated with liver fibrosis score: the more advanced fibrosis, the higher percentage of CD4+PD-1+ T-cells (median 20.1% (range 7.0–52.7%) in F0/1 vs 25.4% (8.8–43.8%) in F2 vs 26.8% (15.0–38.4%) in F3, P = 0.0125). However, in every fibrosis stage the pretreatment percentage of CD4+PD-1+ T-cells was higher than in healthy controls (Fig. 5). In contrast, the more advanced fibrosis, the lower the frequency of CD4+Tim-3+ T-cells (9.0% (0.7–32.3%) in F0/1 vs 5.7% (2.1–23.8%) in F2 vs 5.4% (0.2–12.0%) in F3, P = 0.0274). When compared to controls, only F0/1 patients demonstrated higher pretreatment percentage of CD4+Tim-3+ T-cells (Fig. 5). While pretreatment percentages of CD4+PD-1+Tim-3+ T-cells, and CD8+PD-1+Tim-3+ T-cells in all three fibrosis groups were significantly higher than in healthy controls (Figs. 5 and 6), there were no differences between patients with different fibrosis stage. CD4+PD-1Tim-3 and CD8+PD-1Tim-3 T-cells percentages were similar in patients displaying different fibrosis scores but were significantly lower than in healthy controls with the exception of CD8+PD-1Tim-3 T-cells in F2 group which were similar to those in controls (Figs. 5 and 6). For the remaining T-cell subpopulations (CD8+PD-1+, CD8+Tim-3+) there were no statistically significant differences between different fibrosis stages, and the values were similar to those of healthy controls (Figs. 5 and 6).

Figure 5
figure5

Peripheral blood CD4+ T-cells expression of PD-1 and Tim-3 in patients with different liver fibrosis scores (F0/1, F2, F3) before and six months after successful therapy for chronic HCV infection and in 18 non-infected controls (a) and individual PD-1 and Tim-3 expression changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy, C non-infected controls.

Figure 6
figure6

Peripheral blood CD8+ T-cells expression of PD-1 and Tim-3 in patients with different liver fibrosis stage (F0/1, F2, F3) before and six months after successful therapy for chronic HCV infection and in 18 non-infected controls (a) and individual PD-1 and Tim-3 expression changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy, C non-infected controls.

Importantly, the more advanced fibrosis, the less likely it was that treatment would change the proportions of cells expressing exhaustion markers. Thus, F0/1 patients experienced increase in CD4+PD-1+ T-cells from 20.1% (7.0–52.7%) to 21.6% (10.5–39.1%), P = 0.0026 and CD8+PD-1+ T-cells from 20.9% (5.5–46.8%) to 23.7% (8.1–46.2%), P = 0.0002, and increase in CD8+PD-1Tim-3 T-cells from 55.8% (35.1–74.5%) to 59.3 (38.6–80.1%), P = 0.0470 and decrease in CD4+Tim-3+ T-cells from 9.0% (0.7–32.3%) to 6.8% (0.7–15.7%), P = 0.0006 and in CD8+Tim-3+ T-cells from 19.0% (5.0–46.7%) to 14.1% (3.3–39.1%), P < 0.0001, and decrease in CD4+PD-1+Tim-3+ T-cells from 2.1% (0.4–7.2%) to 1.3% (0.2–4.7%), P = 0.0032 and in CD8+PD-1+Tim-3+ T-cells from 2.5% (0.5–16.1%) to 1.9% (0.4–12.1%), P = 0.0014 (Figs. 5 and 6). In contrast, F3 patients displayed no significant changes in frequencies of cells expressing PD-1 and/or Tim-3 with the exception of CD4+PD-1+Tim-3+ T-cells, which decreased from 1.5% (0.3–4.1%) to 1.4% (0.6–2.9%), P = 0.0215. Patients with stage F2 fibrosis had less pronounced changes than F0/1 patients including decrease in PD-1+Tim-3+ T-cells (both CD4+ (1.6% (0.4–9.4%) vs 1.2% (0.0–7.6%), P = 0.0043) and CD8+ (2.7% (0.6–11.2%) vs 1.7% (0.2–10.5%), P =  < 0.0001) subpopulations, decrease in CD8+Tim-3+ T-cells (12.7% (4.3–36.3%) vs 11.1% (1.8–29.4%), P = 0.0018), decrease in CD4+PD-1Tim-3 T-cells (66.9% (43.0–80.8%) vs 66.7% (44.5–82.0%), P = 0.0236) and increase in CD8+PD-1Tim-3 T-cells (63.8% (39.2–75.8%) vs 64.3% (42.9–80.1%), P = 0.0005) (Figs. 5 and 6).

After treatment, none of the analyzed populations was significantly different between the subgroups of patients with different fibrosis stage.

Despite treatment-induced changes, some of the subpopulations did not reach values seen in healthy controls. These included higher CD4+PD-1+ T-cells in F0/1 (P = 0.0022), F2 (P = 0.0125) and F3 (P = 0.0042) groups as well as lower CD4+PD-1Tim-3 T-cells in F0/1 (P = 0.0014), F2 (P = 0.0038) and F3 (P = 0.0131) groups and lower CD8+PD-1Tim-3 T-cells in F0/1 (P = 0.0495) and F3 (P = 0.0203) groups (Figs. 5 and 6).

Successful treatment affects HCV-specific CD8+ T-cell frequencies but not their PD-1 and Tim-3 expression phenotype

Assessment of HCV-specific CD8+ T-cells frequencies and their exhaustion phenotype was feasible in 32 patients with HLA-A*02 allele. As shown in Fig. 7, treatment resulted in lowering the frequency of these cells from median 2.9% (range 0.1–47.9%) to 0.7% (0–57.2%), P = 0.0003. A representative cytometric analysis of treatment-related changes in HCV-specific CD8+ T-cells frequency in two patients is shown in Fig. 8.

Figure 7
figure7

Peripheral blood HCV-specific CD8+ T-cells frequency and expression of PD-1 and Tim-3 in 32 patients before and six months after successful therapy of chronic HCV infection (a) and individual PD-1 and Tim-3 expression changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy.

Figure 8
figure8

Representative cytometric analysis of peripheral blood HCV-specific CD8+ T-cells before and after successful therapy for chronic HCV infection in Patient 3 (Pt. 3) and Patient 4 (Pt. 4). Pre-Tx before therapy, Post-Tx after therapy.

While treatment resulted in some changes in the phenotype of HCV-specific cells, these differences did not reach statistical significance (Fig. 7). Before the therapeutic intervention, HCV-specific T-cells expressed PD-1 less frequently than after the treatment (30.0% (12.8–70.0%) vs 36.0% (7.5–90.3%)), were more likely to be Tim-3+ ((18.2% (0.0–35.8%) vs 13.9% (0.0–42.9%)), equally likely to be Tim-3+PD-1+ ((1.2% (0.0–11.1%) vs 1.2% (0.0–30.8%)) and more likely to be PD-1Tim-3 ((46.7% (0–65.7%) vs 44.8% (6.5–67.6%)).

The effect of treatment scheme on the peripheral HCV-specific CD8+ PD-1+ T-cells frequency

We analyzed the effect of treatment with two different protocols (i.e., ledipasvir + sofosbuvir vs ombitasvir + paritaprevir + ritonavir + dasabuvir) correcting for variables differently distributed between the two treatment groups. Patients treated with ledipasvir + sofosbuvir displayed lower liver fibrosis score (59.3% vs 27.3% with F0/1, 40.7% vs 72.7% with F2/3, P = 0.0216) and higher age (median 61 (range 25–88) vs 49.5 (29–78), P = 0.0172). We found that changes in the percentages of cells expressing exhaustion markers were not different for these two protocols, the only exception being HCV-specific CD8+PD-1+ T-cells. Thus, while treatment with ledipasvir + sofosbuvir resulted in increase in percentage of HCV-specific CD8+PD-1+ T-cells from 28.4% (12.8–70.0%) to 40.8% (15.4–90.3%), treatment with ombitasvir + paritaprevir + ritonavir + dasabuvir resulted in their decrease from 39.9% (17.6–66.7%) to 31.7% (7.5–41.7%) (regression coefficient -39.41% 95% CI -63.71 to -15.11%, P = 0.005). The opposite was found for HCV-specific CD8+Tim-3+ T-cells: treatment with ledipasvir + sofosbuvir resulted in decrease in percentage of HCV-specific CD8+Tim-3+ T-cells from 18.7% (0–35.80%) to 6.2% (0–31.2%), while treatment with ombitasvir + paritaprevir + ritonavir + dasabuvir resulted in their increase from 14.2% (0–26.5%) to 20.4% (11.1–42.9%). However, these results were not significant in multivariate analysis (P = 0.066). Similarly, treatment with ledipasvir + sofosbuvir resulted in decrease in percentage of HCV-specific CD8+PD-1Tim-3 T-cells from 46.7% (0–65.7%) to 41.6% (6.5–67.6%), while treatment with ombitasvir + paritaprevir + ritonavir + dasabuvir resulted in their increase from 43.6% (22.2–55.9%) to 49.9% (21.4–67.5%). However, these results were also not significant in multivariate analysis (P = 0.063).

Successful treatment diminishes IL-10 levels in plasma

Before therapy, patients displayed significantly higher plasma IL-10 levels than controls (median 4.0 (range 0.8–16.5) pg/mL vs 3.0 (1.3–6.4) pg/mL, P = 0.0442) (Fig. 9). Treatment resulted in decrease of IL-10 levels to 3.3 (0.8–22.4) pg/mL, P = 0.0065, which were now similar to the levels observed in controls.

Figure 9
figure9

Plasma levels of IL-10 in 76 patients before and six months after therapy for chronic HCV infection and in 18 non-infected controls (a) and individual IL-10 changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy, C non-infected controls.

Plasma IL-10 levels were not significantly different between patients displaying different fibrosis scores neither before (4.0 (1.1–16.5) pg/mL in F0/1 vs 3.9 (0.8–11.7) pg/mL in F2 vs 4.3 (0.8–6.9) pg/mL in F3), nor after treatment (3.5 (0.8–19.8) pg/mL in F0/1 vs 3.3 (1.3–22.4) pg/mL in F2 vs 3.2 (1.6–8.0) pg/mL in F3). Plasma IL-10 levels were also not significantly different between patients with different fibrosis stage and healthy controls, neither before nor after treatment. However, a significant decrease of IL-10 levels after treatment was observed but was limited to F0/1 patients (P = 0.0395) (Fig. 10).

Figure 10
figure10

Plasma levels of IL-10 in 76 patients in groups of patients displaying different liver fibrosis scores (F0/1, F2, F3) before and six months after therapy for chronic HCV infection and in 18 non-infected controls (a) and individual IL-10 changes before and after treatment (b). Horizontal lines represent median values. Numbers above each brace express P-values. Pre-Tx before therapy, Post-Tx after therapy.

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