Longitudinal fluctuations in PD1 and PD-L1 expression in association with changes in anti-viral immune response in chronic hepatitis B

Abstract

Background: Controversy exists regarding the role of PD1 and its ligand PD-L1 in chronic hepatitis B infection. In some studies, persistent HBV infection has been attributed to high levels of PD-1 and PD-L1 expression on HBV-specific T-cells and antigen-presenting cells (APCs) respectively. Other studies revealed that the up-regulation of PD-1 and PD-L1 during an acute inflammation phase is required to offset increasing positive co-stimulatory signals to avoid severe damage by an over-vigorous immune response.

Methods: Fifteen chronic hepatitis B patients, with inflammatory flare episode, were recruited prospectively. Based on serum HBV-DNA, HBsAg load, and ALT values, inflammatory flare episode were divided into initial, climax, decline and regression phase. Blood sample and liver biopsy tissues from each individual were taken in these 4 phases respectively. Circulating and intra-hepatic PD1 and PD-L1 expression levels were monitored throughout the inflammatory flare episode by flow cytometry and immunostaining and these expression levels were related to the HBV-specific T-cell changes, expression of pro-inflammatory cytokines, HBV-DNA replication and HBV antigen load.

Results: ]The levels of PD-1 and PD-L1 expressions were significantly up-regulated in the inflammation ascending phase, initial and climax period and in parallel with HBV-specific colon expansion. It showed increasing the level of serum ALT and decreasing the HBV-DNA loads. As the level of inflammation reduced, the circulating and intra-hepatic PD1 and circulating PD-L1 decreased progressively in concordance with serum ALT, HBV-DNA and HBsAg loads decreased except intra-hepatic PD-1 expression. Intra-hepatic PD-L1 expression did not decrease significantly during the regression phase of inflammation compared to that in prior period. The intra-hepatic PD-L1 expression remained relatively on higher level when serum HBV-DNA load and ALT decreased to approximately normal range.

Conclusion: The relatively high level of intra-hepatic PD-L1 expression during the inflammatory regression period may contribute to constitute an immunosuppressive microenvironment, which facilitate persistent HBV infection via the inhibition of HBV-specific T cell clonal expansion.

Figures

Figure 1

Representative dot plots of pentamer staining for patients in T1, T2, T3 and T4 sequence. Fresh heparinized peripheral blood samples were lysed with FACS lysing solution and then incubated with antibodies against pentamers and CD8-FITC for 20 min at 4°C. The numbers in the upper right quadrants indicate the frequency of pentamers in total circulating CD8+ cells.

Figure 2

The numbers of core-, env- and pol-PE positive cells in 50000 CD8+ cells were compared betweemT1,T2,T3 and T4. The number of core, env and pol increased significantly from T1 to T2, and decreased progressively from T2 to T4. core, env and pol, n = 15, P T1 vs T2 <0.01, P T2 vs T3 <0.05, P T3 vs T4 < 0.01.

Figure 3

Correlation between PD1, PD-L1 expression and serum pentamer frequency, ALT level, HBsAg and HBV-DNA load. (A) Percentage of core, even and pol from T1 to T4. The frequency of pentamers including core 18–27, even 335–343 and pol 575–583 increased upon initial of inflammation (T1), reached peak level during climax of inflammation (T2) and decreased gradually during regression of inflammation (T4). (core, even and pol, n = 15, P T1 vs T2 <0.01, P T2 vs T3 <0.05, P T3 vs T4 < 0.01) (B) Fluctuation of sALT, HBsAg and HBV-DNA from T1 to T4. From T1 to T2, serum ALT level increased significantly, in parallel with pentamer cells clonal expansion, and decreased significantly in concordance with pentamer cells clonal contraction from T3 to T4. ( sALT, n = 15, P T1 vs T2 <0.01, P T2 vs T3 <0.05, P T3 vs T4 <0.05) Serum HBsAg and HBV-DNA load decreased during inflammation flare up period. (HBsAg and HBV-DNA copies, n = 15, P T1 vs T2 <0.01, P T2 vs T3 <0.01, P T3 vs T4 < 0.01) (C) Percentage of circulating PD1 and PD-L1 from T1 to T4. From T1 to T2 period, in parallel with pentamer cells clonal expansion, circulating PD1 and PD-L1 expression increased significantly. From T3 to T4 period, parallel with pentamer cells clonal contraction, circulating PD1 and PD-L1 expression decreased significantly. (P T1vsT2 <0.01, P T2 vs T3 <0.05, P T3 vs T4 < 0.01 n = 5) (D) The number of intra hepatic CD8, PD1 and PD-L1 positive cells from T2 to T4. DuringT2 period, the number of intra-hepatic PD1, PD-L1 and CD8 reached the highest level and they were decreased significantly in T3 period. From T3 to T4 period, number of intra-hepatic PD-1 and CD8 positive cells further decreased significantly, however PD-L1 did not. (PD1,PD-L1 and CD8, PT2 vs T3 < 0.01; PD1 and CD8, P T3 vs T4 < 0.01; PD-L1, P T3 vs T4 > 0.05; n = 5).

Figure 4

(A) Representative dot plots of PD1 and CD8 double staining for patients in T1,T2,T3 and T4 sequence. Fresh heparinized peripheral blood samples were lysed with FACS lysing solution and then incubated with antibodies against PD1-APC and CD8-FITC for 20 min at 4°C. The numbers in the upper right quadrants indicate the frequency of PD1 in total circulating CD8+ cells. (B) Representative dot plots of PD1 and pentamer double staining for patients in T2 period. Fresh heparinized peripheral blood samples were lysed with FACS lysing solution and then incubated with antibodies against pentamer-PE and PD1-APC for 20 min at 4°C. The numbers in the upper right quadrants indicate the frequency of PD1-APC in circulating core18-27, enve335-343 and pol575-583 cells. (C) Representative dot plots of PD-L1 and CD11c double staining for patients in T1, T2, T3 and T4 sequence. Fresh heparinized peripheral blood samples were lysed with FACS lysing solution to remove RBCs and then incubated with antibodies against PD-L1-PE and CD11c-PEcy7 for 20 min at 4°C. The numbers in the upper right quadrants indicate the frequency of PD-L1 and CD11c dual positive cells.

Figure 5

The frequency of PD1 expression on pentamers in T2 compared with which expressed on circulating CD8. During T2 period, the frequency of PD1 expression on pentamers were higher significantly than that of expressed on total circulating CD8 ( P core vs CD8 <0.05, Penv vs CD8 <0.05, P pol vs CD8 < 0.05 ).

Figure 6

Immunohistochemical staining for intrahepatic CD8-positive, PD-1-positive and PD-L1-positive cells in patients with chronic hepatitis B. Representative figures for patients in T2, T3 and T4 sequence. n = 5, High-power field, original magnification 400×. During T2 period, CD8-positive, PD-1-positive or PD-L1-positive cells infiltrated extensively in liver tissue and they were decreased significantly in T3. In T4, the CD8-positive and PD-1-positive cells were less observed in lobular areas, while PD-L1 positive cells were also frequently observed in liver lobular region.

Figure 7

(A) Co-localization of PD-L1 with CD31 and CD68 is shown by immunofluorescence double staining in liver biopsy specimens of patients with hepatitis B in T4. PD-L1 (red) is co-localized with CD31 (green) positive endothelia cells or CD68 (green) positive Kupffer cells. The 2-color merged panels show co-localization visible (yellow). Original magnification × 200. (B) Co-localization of PD-L1 and CD11c in liver tissue from patients during T3. PD-L1 (red) is co-localized with CD11c (green). The 2-color merged panels show co-localization (yellow). Most CD11c + cells expressed PD-L1. Original magnification × 200. (C) Co-localization of PD-L1 and CD11c in liver tissue from patients in T4. PD-L1 (red) is co-localized with CD11c (green). The 2-color merged panels show co-localization (yellow). Most CD11c + cells did not express PD-L1. Only CD11c + cells in the circle expressed PD-L1. Original magnification × 200.

Figure 8

Detection of TNF-α and IFN-γ mRNA expression by QT-PCR (n = 5). The levels of TNF-α and IFN-γ mRNA expression significantly increased from T1 to T2, and decreased from T2 to T4. TNF-α expression level in T4 period was still significantly higher than that in T1. TNF-α mRNA was 2.5-fold higher in T4 than that in T1.