Liver environment and HCV replication affect human T-cell phenotype and expression of inhibitory receptors

Abstract

Background & aims: There is an unclear relationship between inhibitory receptor expression on T cells and their ability to control viral infections. Studies of human immune cells have been mostly limited to T cells from blood, which is often not the site of infection. We investigated the relationship between T-cell location, expression of inhibitory receptors, maturation, and viral control using blood and liver T cells from patients with hepatitis C virus (HCV) and other viral infections.

Methods: We analyzed 36 liver samples from HCV antibody-positive patients (30 from patients with chronic HCV infection, 5 from patients with sustained virological responses to treatment, and 1 from a patient with spontaneous clearance) with 19 paired blood samples and 51 liver samples from HCV-negative patients with 17 paired blood samples. Intrahepatic and circulating lymphocytes were extracted; T-cell markers and inhibitory receptors were quantified for total and virus-specific T cells by flow cytometry.

Results: Levels of the markers PD-1 and 2B4 (but not CD160, TIM-3, or LAG-3) were increased on intrahepatic T cells from healthy and diseased liver tissues compared with T cells from blood. HCV-specific intrahepatic CD8(+) T cells from patients with chronic HCV infection were distinct in that they expressed TIM-3 along with PD-1 and 2B4. In comparison, HCV-specific CD8(+) T cells from patients with sustained virological responses and T cells that recognized cytomegalovirus lacked TIM-3 but expressed higher levels of LAG-3; these cells also had different memory phenotypes and proliferative capacity.

Conclusions: T cells from liver express different inhibitory receptors than T cells from blood, independent of liver disease. HCV-specific and cytomegalovirus-specific CD8(+) T cells can be differentiated based on their expression of inhibitory receptors; these correlate with their memory phenotype and levels of proliferation and viral control.

Keywords: CMV; Costimulation; HCV; IHL; Immune Regulation; Inflammation; PBMC; T-Cell Exhaustion; cytomegalovirus; hepatitis C virus; intrahepatic lymphocyte; peripheral blood mononuclear cell.

Figure 1

Expression analysis of PD-1, 2B4, CD160, TIM-3 and LAG-3 on CD4+ (two left graphs) and CD8+ T-cells (two right graphs) for patient-matched PBMC and IHL. The analysis includes 19 chronically HCV infected and 14 HCV negative patients (*p<0.05, **p<0.01, ***p<0.001, paired t-test). A) PD-1, B) 2B4, C) CD160, D) TIM-3, E) LAG-3 expression on CD4+ and CD8+ T-cells on PBMC versus IHL in [%].

Figure 2

Inhibitory receptor expression on CD4+ and CD8+ IHL in 4 different subgroups: HCV positive (30), HBV positive (7), virus-negative with other liver disease (36) and benign liver lesions (8). PD1 (A), 2B4 (B), CD160 (C), TIM-3 (D) and LAG-3 (E) on CD4+ (left graphs) and CD8+ IHL (right graphs). P values <0.008 were considered positive using unpaired T-tests with Bonferroni's correction.

Figure 3

Representative flow plots for analysis of virus-specific IHL(red dots) and PD1, 2B4, CD160, TIM-3 and LAG-3 in comparison to bulk CD8+ IHL for a chronically infected HCV patient (A), a patient with resolved HCV (B) as well as for an intrahepatic CMV-specific response (C). D) Co-expression patterns of T-cell inhibitory receptors in different T-cell populations by SPICE analysis (each Spice diagram represents all experiments performed in this subgroup): Total intrahepatic CD8 T-cell population (left panel), intrahepatic HCV-specific CD8 T-cell responses (middle panels, separated into all responses combined, responses in resolved HCV infection and responses in chronic HCV infection) and intrahepatic CMV responses. Green pie sections represent T-cells with co-expression of 2 or less inhibitory receptors, red pie pie sections co-expression of 3 or more inhibitory receptors.

Figure 4

Expression of T-cell inhibitory receptors on bulk CD8+ IHL (blue bars) compared to HCV specific CD8+ IHL (red bars). Patients with chronic HCV infection are shown in the left graphs A-E, patients with resolved HCV are depicted in the middle graphs A-E and the right graphs A-E display results for CMV specific IHL. F) summarizes the IHL results from the left for A) PD-1, B) 2B4, C) CD160, D) TIM-3 and E) LAG-3. (*p<0.01, ***p<0.0001 in paired and unpaired T-tests with Bonferroni's correction).

Figure 5

SPICE co-expression analysis of T cell inhibitory receptors (PD1, 2B4, CD160, TIM-3 and LAG-3) on HCV specific IHL of 4 chronically infected individuals, 3 patients with resolved infection and 3 patients with CMV infection (IgG positive). A) Co-expression of inhibitory receptors on HCV specific CD8+ IHL compared to patients with resolved infection. B) Predominant receptor combination on HCV specific CD8+ IHL in chronically infected patients is the combination of PD1, 2B4 and TIM-3. C) Combination of PD1 with 2B4 and LAG-3 is the dominating receptor combination on HCV specific CD8+ IHL in patients with resolved HCV infection. D) CMV specific IHL display co-expression of PD-1 with 2B4 and CD160.

Figure 6

Memory phenotype and proliferation marker for IHL in chronic and resolved infection. We defined central memory (CM, CD45RA-/CCR7+), effector memory (EM, CD45RA-/CCR7−), effector memory RA (TEMRA, CD45RA+/CCR7−) and naïve (CD45RA+/CCR7+) CD8+ T-cell subsets for A) total intrahepatic CD8 lymphocytes in patients with chronic HCV infection (red dots, n=5), resolved HCV infection (green dots, n=3), HCV negative liver disease (blue dots, n=5) and in healthy liver tissue (light blue dots, n=4) and B) intrahepatic virus-specific CD8 T-cells in patients with chronic HCV infection (red dots, n=4), resolved HCV infection (green dots, n=5) and on intrahepatic CMV-specific populations (blue dots, n=7). *=p<0.01 in unpaired T-test with Bonferroni's correction. Ki-67 expression. Examples of Ki-67 analysis for intrahepatic HCV-specific CD8 responses in C) a patient with chronic and D) a patient with resolved HCV infection. E) summarizes Ki-67 expression for virus-specific intrahepatic CD8 responses in chronic and resolved HCV infection and for CMV-specific populations. *=p<0.01 in unpaired T-test with Bonferroni's correction