Hepatitis C

HCV-specific CD4+ T cells of patients with acute and chronic HCV infection display high expression of TIGIT and other co-inhibitory molecules

Increased expression level of TIGIT on bulk CD4+ T cells of patients with acute and chronic HCV infection

Recently, a critical role for TIGIT in regulating virus-specific CD4+ T cell responses in chronic HIV infection41 has been described, but little is known about the role of TIGIT together with PD-1 and TIGIT’s complementary receptor CD226 on T cells of HCV patients. The aim of this study was to comprehensively analyze the expression pattern of TIGIT together with additional co-inhibitory molecules on bulk and HCV-specific CD4+ T cells of HCV patients with different disease status.

First, we assessed the ex vivo surface expression of TIGIT on total CD4+ T cells of patients with acute (n = 10), chronic (n = 11), spontaneously resolved (n = 8) HCV infection and healthy controls (n = 10) (Table 1, Suppl. 1A C). We observed a significantly higher frequency of bulk TIGIT+ CD4+ T cells of patients with HCV infection compared to healthy controls. Bulk CD4+ T cells of acutely infected HCV patients tended to have the highest frequencies followed by chronically infected HCV patients and patients with spontaneously resolved HCV infection (Suppl. 1B). Based on the differentiation markers CD45RO and CCR7, we defined naïve and memory subsets (CCR7−/CD45RO terminal effector-TEMRA; CCR7+/CD45RO naïve T cells-Tnaïve; CCR7−/CD45RO+–effector memory TEM; CCR7+/CD45RO+–central memory TCM) of bulk CD4+ T cells and assessed the TIGIT expression of each memory subset. We could detect a significant higher TIGIT expression on all CD4+ T cell memory subsets of acutely HCV infected patients while the general level of TIGIT was highest across all patient groups in the effector memory compartment and lowest in the naïve T cell compartment (Suppl. 1C).

Table 1 Clinical, virological, and immunological patient characteristics.

TIGIT, PD-1, BTLA and Tim-3 expression pattern of HCV-specific CD4+ T cells of acutely and chronically infected patients

In order to analyze TIGIT expression of HCV-specific CD4+ T cells, we stained PBMC ex vivo with a multicolour FACS panel of patients with acute (n = 10), chronic (n = 11) and spontaneously resolved (n = 8) HCV infection using HLA-DRB1*01:01, DRB1*04:01, DRB1*11:01 and DRB1*15:01-restricted tetramers (Table 2) after bead enrichment as previously described42. In accordance with previous reports, the ex vivo frequency of HCV-specific CD4+ T cells was highest in patients with acute HCV infection (ranging from 0,005%-0,15%; median 0,05%) followed by patients with spontaneously resolved HCV infection (ranging from 0,0003%-0,1%; median 0,005%) and extremely low in patients with chronic HCV infection (ranging from 0%-0,005%; median 0,0005%) (Suppl. 2A,B).

Table 2 HLA multimeric complex information.

Differentiation markers CD45RO and CCR7 were used to define naïve and memory subset (CCR7−/CD45RO terminal effector-TEMRA; CCR7+/CD45RO naïve T cells-Tnaïve; CCR7−/CD45RO+–effector memory TEM; CCR7+/CD45RO+–central memoryTCM) of HCV specific MHC class II tetramer+ CD4+ T cells. The vast majority of HCV-specific CD4+ T cells showed a TEM phenotype independent of the infection stage (Suppl. 3A,B), and there was only a minimal decrease of TEM cells of HCV-specific MHC class II tetramer+ CD4+ T cells of chronically infected HCV patients compared to spontaneously resolved HCV patients.

Next, we looked at the expression pattern of TIGIT in combination with a number of additional co-inhibitory markers of HCV specific MHC class II tetramer+ CD4+ T cells of HCV patients with different disease status (Fig. 1A C). Figure 1A depicts exemplary plots of the inhibitory expression level of TIGIT, PD-1, BTLA, and Tim-3 of MHC class II tetramer+ HCV specific CD4+ T cells of a patient with a) acute (upper panel), b) chronic (middle panel) and c) spontaneously resolved (lower panel) HCV infection. An increased inhibitory receptor expression level of TIGIT, PD-1, and BTLA, but not Tim-3 was detectable on HCV specific CD4+ T cells of patients with acute infection and chronic HCV infection. TIGIT and PD-1 expression levels showed marked differences on MHC class II tetramer+ HCV-specific CD4+ T cells between the acute and chronic phase (high expression) and spontaneous resolution (lower expression). BTLA expression levels were generally high in all three patient groups while the Tim-3 expression level of HCV-specific CD4+ T cells was generally much lower – yet highest in chronic HCV patients compared to MHC class II tetramer+ HCV-specific T cells of acutely infected HCV patients or spontaneous resolvers (Fig. 1B). In addition to the differences of the expression frequencies of the different inhibitory molecules at different HCV disease stages, we also observed an significantly increased TIGIT and PD-1 MFI on HCV specific CD4+ T cells from patients with acute (TIGIT; acute vs. resolved: p < 0,0001) (PD-1; acute vs. resolved: p < 0,0001) and chronic (TIGIT; chronic vs. resolved: p < 0,0001) (PD-1; chronic vs. resolved: p < 0,0001) HCV infection compared to patients with spontaneously resolved HCV infection (Suppl. 4A,B). In contrast, while we could not detect any difference in the frequency of BTLA on HCV specific CD4+ T cells between the HCV patients with different disease status, the BTLA MFI was significantly higher on HCV-specific CD4+ T cells from patient with acute HCV infection compared to chronic (acute vs. chronic: p < 0,0009) and spontaneously resolved (acute vs. resolved: p < 0,0001) HCV infection (Suppl. 4C).

Figure 1

(AC) Higher ex vivo expression of TIGIT and different co-inhibitory molecules on virus-specific CD4+ T cells of acutely and chronically infected HCV patients compared to patients with spontaneously resolved HCV. (A) Representative dot plots depicting the ex vivo co-inhibitory receptor expression (TIGIT, PD-1, BTLA, TIM-3) of HCV-specific MHC class II tetramer+ CD4+ T cells of patients with acute, chronic and resolved HCV infection. (B) Frequencies of the inhibitory receptors TIGIT, PD-1, BTLA, and Tim-3 on total and HCV-specific CD4+ T cells of patients with acute, chronic, and spontaneously resolved HCV infection. P-values were calculated by the Tukey’s multiple comparison test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively. (C) SPICE analysis of TIGIT, PD-1, BTLA, and Tim-3 co-expression pattern of HCV-specific CD4+ T cells of patients with acute (n = 10), chronic (n = 10), and spontaneously resolved (n = 8) HCV infection.

In order to define the detailed expression signature of different co-inhibitory molecules on HCV-specific CD4+ T cells, SPICE analysis43 on HCV-specific MHC class II tetramer+ CD4+ T cells of patients with different HCV disease status was performed analysing TIGIT, PD-1, BTLA, and Tim-3 co-expression. This analysis revealed that acutely and chronically HCV infected patients generally showed higher frequencies of HCV-specific CD4+ T cells that expressed three inhibitory receptors (TIGIT+ PD-1, BTLA+, Tim-3−) compared to HCV-specific CD4+ T cells of spontaneously resolved HCV patients (Fig. 1C).

Furthermore, the pattern of TIGIT expression of the different memory subpopulations of MHC class II tetramer+ HCV-specific CD4+ T cells of HCV patients during different stages of disease (Fig. 2A,B) was compared. Recently, it could be shown that TIGIT is mainly expressed on intermediate/transitional and effector T cells of virus-specific CD8+ T cells of patients with HIV21. In contrast, here we could detect that TIGIT was significantly higher expressed within the TEM subset of patients with acute and chronic HCV infection compared to patients with spontaneously resolved HCV infection (acute vs. resolved: p = 0,0015) (chronic vs. resolved: p = 0,0009) (Fig. 2B). In the TCM and TEMRA subpopulation, TIGIT expression was relatively stable with no statistically significant difference between patients with acute, chronic or spontaneously resolved HCV infection (Fig. 2B).

Figure 2
figure2

(A,B) Memory subset distribution of TIGIT+, HCV-specific CD4+ T cells of HCV patients with acute, chronic and spontaneously resolved infection. The differentiation markers CD45RO and CCR7 were used to analyse the ex vivo expression of TIGIT+ HCV-specific MHC class II tetramer+ CD4+ T cells within different memory T cell subsets. (A) Representative large dot plots depicting the memory subset distribution of TIGIT+ HCV-specific MHC class II tetramer+ CD4+ T cells (red) on an overlay of gated total CD4+ cells (grey) of patients with acute, chronic and spontaneously resolved HCV infection; memory subset definition: CCR7−/CD45RO terminal effector T cells-TEMRA; CCR7+/CD45RO naïve T cells-Tnaïve; CCR7−/CD45RO+–effector memory TEM; CCR7+/CD45RO+–central memory TCM. (B) Comparison of the TIGIT receptor expression of HCV-specific MHC class II tetramer+ CD4+ T cells and different memory T cell subsets in patients with acute (n = 10), chronic (n = 11), and spontaneously resolved (n = 8) infection. P values were calculated by the Tukey’s multiple comparison test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively.

High co-expression of TIGIT and PD-1 on HCV-specific MHC class II tetramer+ CD4+ T cells of patients during acute and chronic HCV infection

Previous studies have described that especially dual blockade of TIGIT and PD-1 restored anti-viral and anti-tumour T cell effector function in the mouse model17, and this led also to an invigoration of the CD8+ T cell function in HIV patients21. Therefore, we also specifically evaluated TIGIT/PD-1 co-expression of total and HCV-specific CD4+ T cells of patients with acute, chronic and spontaneously resolved HCV infection (Fig. 3A C). TIGIT/PD-1 co-expression was significantly increased on HCV-specific CD4+ T cells of acutely and chronically HCV infected patients compared to the expression of patients with spontaneously resolved HCV infection (acute vs. resolved: p < 0,0001) (chronic vs. resolved: p < 0,0001) (Fig. 3B). We further compared the TIGIT/PD-1 co-expression of HCV-specific CD4+ T cell subsets (Fig. 3C). TEM CD4+ T cells of patients with spontaneously resolved HCV infection showed a significantly lower TIGIT/PD-1 co-expression than MHC class II tetramer+ HCV-specific TEM CD4+ T cells of patients with acute (acute vs. resolved: p < 0,0001) or than TEM CD4+ T cells of patients with chronic infection (chronic vs. resolved: p = 0,0044).

Figure 3
figure3

(AE) Ex vivo TIGIT/PD-1 co-expression of HCV specific CD4+ T cells of patients with acute, chronic and spontaneously resolved HCV infection. (A) Representative large dot plots depicting the ex vivo inhibitory molecule co-expression of TIGIT/PD-1 of HCV-specific MHC class II tetramer+ CD4+ T cells of patients with acute, chronic and spontaneously resolved HCV infection. (B) TIGIT/PD-1 co-expression on total and HCV-specific CD4+ T cells (C) TIGIT/PD-1 co-expression of HCV-specific CD4+ T cells sub-stratified for different memory T cell subsets of patients with acute (n = 10), chronic (n = 11) and spontaneously resolved (n = 8) HCV infection. (D) Comparison of TIGIT/PD-1 co-expression of HCV-specific CD4+ T cells during acute infection and between patients who later on progressed to chronic versus patients who spontaneously resolved the HCV infection (E) Comparison of the TIGIT/PD-1 co-expression HCV-specific MHC class II tetramer+ CD4+ T cells of patients with chronic HCV (cHCV without therapy); chronic HCV patients during HCV treatment (Under therapy; 3 patients received a peg-interferon-based and 5 patients a DAA-based therapy); HCV treated chronic patients with sustained virologic response (SVR; 4 patients received a peg-interferon-based and 4 patients a DAA-based therapy); and patients with spontaneously resolved HCV infection (Sp.R). P values were calculated by the Tukey’s multiple comparison test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively.

Next, we investigated whether there was any difference of the TIGIT/PD-1 co-expression of HCV specific MHC class II tetramer+ CD4+ T cells during acute HCV infection between patients who progressed to chronic versus patients who spontaneously resolved the infection. During the acute HCV infection we found a slight trend of a reduction of the TIGIT/PD-1 frequency of HCV specific CD4+ T cells of the patient group that developed a chronic HCV (n = 6) infection compared to patients who later spontaneously eliminated the virus (n = 4) (Fig. 3D), however this difference did not reach statistical significance (p = 0,0933) probably due to the small cohort size and larger cohorts are needed to investigate this question.

We next wanted to explore, whether there is any difference of the TIGIT/PD-1 expression level of HCV specific CD4+ T cells during and after successful HCV therapy in patients with a sustained virologic response (SVR). Therefore, we compared the TIGIT/PD-1 frequency on MHC class II tetramer+ HCV specific CD4+ T cells of a subset of chronic HCV patients who underwent HCV therapy. PBMC samples were taken before (cHCV), during and after successful therapy with sustained virologic response (SVR24) (Fig. 3E). Interestingly, we could not detect any difference in TIGIT/PD-1 frequency between a) chronically infected, b) during treatment (cHCV vs. under therapy: p = 0,9692) or c) patients with SVR24 (cHCV vs. SVR: p = 0,9223). This pattern was not different in DAA (direct-acting agents) or PEG-interferon treated patients (data not shown). In contrast, the TIGIT/PD-1 co-expression was significantly lower on HCV specific CD4+ T cells of patients with spontaneously resolved HCV (under therapy vs. Sp.R: p = <0,0001, SVR vs. Sp.R: p = <0,0001).

Decreased expression of the co-stimulatory molecule CD226 on HCV-specific CD4+ T cells of chronically infected HCV patients compared to patients with spontaneously resolved HCV

In a next step we took a closer look at the expression level of the complementary stimulatory receptor of TIGIT: DNAX accessory molecule 1 (DNAM-1; also called CD226) expression of HCV specific MHC class II tetramer+ CD4+ T cells at different stages of HCV infection (Fig. 4A E). Previously it has been suggested that upregulation of TIGIT can potentially inhibit other co-stimulatory molecules like CD22617,44. Generally, CD226 was expressed on the majority of HCV-specific MHC class II tetramer+ CD4+ T cells (Fig. 4A). However, there were certain differences according to the T cell and HCV disease status (Fig. 4B) and memory T cell subset (Fig. 4C): CD226 expression was significantly lower on HCV-specific CD4+ T cells of patients with chronic HCV infection compared to patients with spontaneously resolved HCV infection (chronic vs. resolved: p = 0,0010). In our cohort, there were 6 patients with acute HCV infection who went on to become chronically infected and 4 patients who spontaneously resolved the acute HCV infection. Of note, the initial CD226 expression was slightly, but significantly lower in patients who had a chronically evolving disease course (Fig. 4D). Of particular interest, we also analysed CD226 expression of HCV-specific T cells before, during and after successful antiviral therapy. There was an increase of the CD226 expression of HCV-specific CD4+ T cells after HCV therapy initiation and patients with SVR showed comparably high levels to patients with spontaneously resolved HCV infection (Fig. 4E). This pattern did not differ between in DAA versus PEG-interferon treated patients (data not shown).

Figure 4
figure4

(A E) Lower ex vivo expression of the co-stimulatory molecule CD226 on HCV-specific CD4+ T cells of chronically infected HCV patients compared to patients with SVR. (A) Representative large dot plots depicting the ex vivo co-expression of TIGIT/PD-1 of HCV-specific MHC class II tetramer+ CD4+ T cells of patients with acute, chronic and spontaneously resolved HCV infection. (B) CD226 expression of bulk and HCV-specific CD4+ T cells and (C) and in different memory subsets of patients with acute (n = 10), chronic (n = 11) and spontaneously resolved (n = 8) HCV infection. (D) Comparison of the CD226 expression of HCV-specific CD4+ T cells during the acute HCV phase between patients who later progressed to chronic infection versus patients who spontaneously resolved the HCV infection (E) Comparison of the CD226 expression HCV-specific MHC class II tetramer+ CD4+ T cells of patients with chronic HCV (cHCV without therapy); chronic HCV patients during HCV treatment (Under therapy; 3 patients received a peg-interferon-based and 5 patients a DAA-based therapy); HCV treated chronic patients with sustained virologic response (SVR; 4 patients received a peg-interferon-based and 4 patients a DAA-based therapy); and patients with spontaneously resolved HCV infection (Sp.R). P values were calculated by tukey’s multiple comparison test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively.

TIGIT MFI on HCV-specific CD4+ T cells decreased after HCV-therapy initiation in chronic HCV infection

One major finding of the current study is that the frequency of TIGIT alone or together with PD-1 on HCV specific CD4+ T cells of patients with chronic HCV infected who were treated remains high, -even after successful therapy and resulting SVR. Nevertheless, we further determined the inhibitory expression level of MHC class II tetramer+ HCV-specific CD4+ T cells longitudinally of 4 chronic HCV infected patients who received DAA therapy. We could not detect any difference of the inhibitory expression level percentage-wise at any time point after DAA initiation (data not shown). However, we observed a large and significant drop of the PD-1 and TIGIT mean fluorescence intensity (MFI) on HCV specific CD4+ T cells of all patients after DAA therapy initiation (Suppl. 4A C). Furthermore, we investigated the MFI of TIGIT, PD-1, and CD226 on HCV specific MHC class II tetramer+ CD4+ T cells in longitudinal samples from the acute patients aHCV1 and aHCV6 who later progressed to chronic infection and were eventually antivirally treated with PEG-IFN during the acute phase, as well as of patient aHCV4 who spontaneously eliminated the virus (Suppl. 5A,B). As expected, we could detect a decrease of the PD-1 MFI after HCV therapy initiation while the TIGIT MFI slightly decreased. In patient aHCV4 who spontaneously resolved the acute HCV infection, an even more profound parallel drop of PD-1 and TIGIT could be observed (Suppl. 5A,B).

HCV specific MHC class II tetramer+ CD4+ T cells are predominantly TIGIThigh in patients with acute and chronic HCV infection

Since it was previously reported that HIV-specific TIGIThigh CD4+ T cells (that is TIGIT+ T cells with high MFI) were especially negatively correlated with polyfunctionality and displayed a diminished expression of CD22645. We next investigated the MFI of TIGIT expression on CD4+ T cells of HCV patients with different disease stage (Fig. 5A C). To this aim, we divided the MHC class II tetramer+ HCV-specific CD4+ T cells into subpopulations (hi/int/neg) based on the intensity of TIGIT expression (Fig. 5A). Here, we observed that TIGIThigh cells were significantly more frequent in patients with acute and chronic compared to patients with spontaneously resolved HCV infection (acute vs. resolved: p = <0,0001, chronic vs. resolved: p = <0,0001) (Fig. 5B). In contrast, only a small fraction of HCV-specific CD4+ T cells of acutely and chronically infected patients were TIGITint or TIGITneg45. Subsequently, we investigated whether there was any difference in the frequency of TIGIThigh CD4+ T cells in longitudinal samples during DAA therapy of four chronic HCV infected patients (Fig. 5C). The frequency of TIGIThigh HCV-specific MHC class II tetramer+ CD4+ T cells decreased after DAA initiation in all four patients, but remained at a higher level compared to the TIGIThigh frequency of spontaneously resolved patients. The results might indicate that not only the overall frequency of TIGIT cells, but rather the intensity of the expression of this co-inhibitory molecule influences the T cell functionality and a similar observation has been made in HIV infection45.

Figure 5
figure5

(A C) Ex vivo frequency of HCV specific TIGIThigh and TIGITint CD4+ T cells of HCV patients with acute, chronic and spontaneously resolved infection and longitudinally during DAA therapy. (A) Depicting the gating strategy of HCV-specific MHC class II tetramer+ CD4+ T cells divided into subpopulations (TIGIThigh/int/neg) based on the intensity of TIGIT expression and (B) the frequency of TIGIThigh and TIGITint T cells of patients with acute, chronic and spontaneously resolved HCV infection. (C) The TIGIThigh frequency of HCV specific CD4+ T cells was analysed in longitudinal samples of chronic HCV patients before, during and after DAA therapy. P values were calculated by Tukey’s multiple comparison test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively.

Definition of the expression pattern of the TIGIT/CD226 axis of HCV-specific CD8+ T cells at different stages of infection

TIGIT has previously been described as an exhaustion marker of antigen-specific CD8+ T cells18,21. The co-inhibitory phenotype of MHC class I + II tetramer+ virus specific CD4+ and CD8+ T cells in HCV infection has rarely been studied side-by-side. We conducted additional preliminary experiments to also understand the expression pattern of our panel of co-inhibitory markers and TIGIT of HCV-specific CD8+ T cells (Fig. 6A C, Suppl. 6 and data not shown). Of note, the expression pattern differed between HCV-specific MHC class I/II tetramer+ CD8+ and CD4+ T cells: TIGIT expression level of MHC class I HCV-specific CD8+ T cells was generally lower in acute and spontaneously resolved HCV infection but much higher in patients with chronic (chronic vs acute: p = 0,0162, chronic vs resolved: p = 0,0056) HCV infection (Fig. 6A,B). The expression of CD226 on MHC class I tetramer+ HCV-specific CD8+ T cells was variable in chronic HCV infection (Fig. 6C). Three chronically infected patients expressed CD226 at an intermediate level, however two patients showed a complete downregulation of this molecule. Of note, the patients who expressed CD226 at an intermediate level were all HLA-A*02.01 and the other HLA-A*24:02. However, sequence of the circulating virus was not available and viral escape might be a possible explanation for this finding.

Figure 6
figure6

(A,B) Ex vivo TIGIT and CD226 expression of HCV specific CD8+ T cells of patients with acute, chronic and spontaneously resolved HCV infection. Representative dot plots depicting the inhibitory receptor expression of HCV-specific MHC class I tetramer+ CD8+ T cells of patients with acute, chronic and spontaneously resolved HCV infection. Comparison of the TIGIT and CD226 frequency of total and HCV-specific CD8+ T cells of HCV patients with acute, chronic and spontaneously resolved HCV infection. P-values were calculated using one-way ANOWA, followed by Tukey’s multiply comparisons test. P-values smaller than 0.05 were considered significant, where *,** and *** indicate p-values between 0.01 to 0.05, 0.001 to 0.01 and 0.0001 to 0.001 respectively.

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