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School of Nursing, Nanchang University, Nanchang, China; Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
Correspondence Correspondence to: Xiufang Weng, PhD, Department of Immunology, School of Basic Medicine, Tongji Medical College, Huazhong University of Science and Technology, 13# Hankong Rd, Wuhan 430030, China.
CD161-expressing CD8+ T cells consist of mucosal-associated invariant T cells with semi-invariant T-cell receptor (TCR) use and non–mucosal-associated invariant T CD161+CD8+ T cells with polyclonal TCR repertoire. Although CD161+CD8+ T cells are enriched in liver and embrace hepatitis B virus (HBV)-specific T cells in chronic hepatitis B (CHB) patients, their roles in disease progression remain poorly understood. This study aimed to decipher their profiling and dynamic changes during chronic HBV infection.
Methods
Blood samples from 257 CHB patients and nontumor liver specimens from 73 HBV-positive patients were analyzed for CD161+CD8+ T-cell characterization by flow cytometry, TCR repertoire determination, transcriptomic analyses, and cell experiments.
Results
CD161+CD8+ T cells were increased and hyperactivated in patients, while positive correlation between the CD161+CD8+ T-cell ratio and HBV-DNA level suggested this was insufficient to control HBV replication. The overlap of complementarity determining region 3 sequences supported the switch between CD161-CD8+ and CD161+CD8+ populations. Although CD161+CD8+ T cells were endowed with innateness phenotype and enhanced antiviral capacity, the population from patients had impaired type I cytokine production, and increased interleukin 17 and granzyme B secretion. The increased CD161+CD8+ T cells and their increased granzyme B secretion correlated positively with inflammation-associated liver injury. Hepatic CD161+CD8+ T cells showed neutrophil-related pathogenic potential because they had increased transcript signatures and proinflammatory cytokine production in neutrophil recruitment- and response-related pathways that changed consistently in the injured liver.
Conclusions
Our results highlight the reduced antiviral potency but increased pathogenic potential of CD161+CD8+ T cells in CHB patients, supporting CD161 expression as a marker of pathogenic CD8+ T subset and the intervention target for liver injury.
The increased non–mucosal-associated invariant T CD161+CD8+ cells show reduced antiviral potency but increased pathogenic potential in chronic hepatitis B virus–infected patients. This supports CD161 expression as a marker of pathogenic CD8+ T subset and provides an intervention target for liver injury.
Chronic hepatitis B (CHB) remains a major health problem worldwide with high morbidity and mortality.
Model to predict on-treatment restoration of functional HBV-specific CD8(+) cell response foresees off-treatment HBV control in eAg-negative chronic hepatitis B.
CHB patients fail to mount an efficient antiviral immune response to the virus, but could develop cirrhosis and liver failure as a result of inflammatory injury.
This suggests the separation of antivirus immune response and immune-mediated liver injury. It raises the possibility of the existence of dysregulated CD8+ T-cell subsets, which drop antivirus effects but maintain undesirable tissue injury potency during chronic infection. However, biomarkers distinguishing antiviral CD8+ T cells from the dysregulated pathogenic ones remain poorly understood.
CD161+CD8+ T cells with a polyclonal T-cell receptor (TCR) repertoire have been shown previously to represent highly functional effector memory T cells.
They are selectively recruited during inflammation and enriched in liver in response to hepatic infection, suggesting their participation in local immunity.
However, the distinct properties of CD161+ T-cell populations have yet to be fully determined. In CD8+ T-cell populations, CD161-expressing T cells are categorized as mucosal-associated invariant T (MAIT) cells with a semi-invariant TCR use and non-MAIT cells with a polyclonal TCR repertoire.
yet the role of non-MAIT CD161+CD8+ T cells in either disease control or immune-mediated damage is largely unknown.
Previous studies have shown that CD161 is expressed significantly on HBV- and hepatitis C virus–specific CD8+ T cells, but not on those specific for human immunodeficiency virus, cytomegalovirus, or influenza virus.
Expression of CD161 on CD8+ T cells therefore may help to identify a functionally differentiated population responding to viral infection within specific organs such as the liver.
Previous studies have shown that lectin-like transcript-1 (LLT1), the ligand of CD161, suppresses activation of tumor-infiltrating T cells after LLT1–CD161 interaction, suggesting an inhibitory role of CD161 signaling.
In addition, LLT1–CD161 interaction has been shown to promote interleukin (IL)17 expression and CD161-bearing CD8+ T cells also have been reported to exhibit high level of cytotoxicity against targets.
The multifaceted function of CD161+CD8+ T cells suggests the complicated role of this population in disease settings. An increased LLT1 protein level has been reported in liver tissue in HBV-infected patients.
Elucidation of the functional status of CD161+CD8+ T cells in CHB patients helps to uncover their roles in disease progression and provides a new intervention target for HBV-specific CD8+ T cells.
In the current study, 257 chronic HBV–infected patients including 61 acute-on-chronic liver failure (ACLF) patients receiving a continuous artificial liver system support, were enrolled to analyze the phenotype and functionality of circulating and/or hepatic CD161+CD8+ T cells. The ratio of CD161+CD8+ T cells increased in the patients, and increased to a greater degree in patients with liver injury. The increased CD161+CD8+ T cells probably resulted from a proportional shift from CD161-negative counterparts and correlated to inflammation-related liver injury in the patients. Of particular interest, CD161+CD8+ T cells from the patients showed reduced antiviral cytokine production but increased pathogenic potential, supporting CD161 expression as a marker of pathogenic CD8+ T cells in CHB.
Results
Circulating Non-MAIT CD161+CD8+ Cells Are Hyperactivated Similarly as MAIT Cells but Show Differential Fate With an Increased Ratio in HBV-Infected Patients
Non-MAIT CD161+CD8+ cells, hereinafter referred to as CD161+CD8+T cells, were identified by the TCR Vα7.2−CD161+CD8+ phenotype in the CD3+ T-cell population and predominantly αβ T cells (Figure 1A). The prevalence of circulating and hepatic CD161+CD8+ T cells in total CD3+ T cells ranged from 0.5% to 10.5% and from 5% to 55%, respectively (Figure 1B). The frequency of circulating CD161+CD8+ T cells increased significantly in CHB patients, although the frequencies of CD161−CD8+ T cells, MAIT cells, and total CD8+ T cells decreased in CHB patients (Figure 1C). The frequencies of circulating CD161+CD8+ T cells correlated positively with HBV-DNA copies (Figure 1D). This suggested that the increased CD161+CD8+ T cells were insufficient to control virus replication.
Figure 1Circulating non-MAIT CD161+CD8+T cells increase in ratio and are hyperactivated in chronic HBV-infected patients. (A) Representative plots for the gating strategy of circulating and hepatic CD3+ TCRVα7.2+CD161+ MAIT cells, non-MAIT CD3+TCRVα7.2−CD161+CD8+ T cells (CD161+CD8+ T cells), non-MAIT CD3+TCRVα7.2−CD161−CD8+ T cells (CD161−CD8+ T cells), and αβ T cells or γδ T cells in CD161+ CD8+ T cells. (B) Frequency distribution of CD161+CD8+ T cells within circulating (n = 243) or hepatic (n = 133) T cells. (C) Summarized scatter graphs for ratio of circulating CD161+CD8+ T cells, CD161−CD8+ T cells, MAIT cells, and total CD8+ T cells in HDs (n = 243) and HBV-infected patients (HBV, n = 170). (D) Spearman correlation between the frequencies of circulating CD161+CD8+ T cells and serum HBV-DNA copies. (E) Representative scatter plots and summary bar graphs display CD69, HLA-DR, and CD38 levels on circulating CD161+CD8+ T cells in the indicated groups (n ≥ 10/group). (F) Summary bar graphs display CD69, HLA-DR, and CD38 levels on circulating MAIT cells in the indicated groups (n ≥ 10/group). FSC, forward scatter; MFI, mean channel fluorescence intensity; SSC, side scatter. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001
Consistently, we found both CD161+CD8+ T cells and MAIT cells from patients had increased expression levels of activation markers including CD69, HLA-DR, and CD38 (Figure 1E and F), albeit CD161+CD8+ T cells had lower CD161 levels compared with MAIT cells (Figure 2A). This indicated that both populations were hyperactivated in patients. In addition, CD161+CD8+ T cells and MAIT cells from patients unanimously showed an enhanced apoptosis level and an increased proliferation rate as revealed by Annexin V staining and Ki67 staining, respectively (Figure 2B and C). This suggests that the apoptosis rate or proliferation capacity was not the key factor accounting for the increase of CD161+CD8+ T cells and the decrease of MAIT cells.
Figure 2Circulating CD161+CD8+T cells and MAIT cells have similar increases in apoptosis and proliferation. (A) Representative histogram and summary bar graph display CD161 levels on circulating CD161−CD8+ T cells (CD161N), CD161+CD8+ T cells (CD161P), and MAIT cells in healthy donors. Bar graphs are shown with means ± SEM (n ≥ 10/group). Representative scatter plots and summary bar graphs display frequencies of (B) Annexin-V+ and (C) Ki67+ cells of circulating CD161P and MAIT cells in the indicated groups (n ≥ 10/group). MFI, mean channel fluorescence intensity. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001
CD161+CD8+ T Cells From HBV-Infected Patients Show Reduced TCR Diversity With Partially Enriched Complementarity Determining Region 3 Sequences Identical to Those From CD161−CD8+ Counterparts
To characterize the CD161+CD8+ T cell (CD161P) profile and investigate their changes in CHB patients, we enriched circulating CD161P from healthy donors (CD161P-HD) for RNA sequencing, compared with CD161−CD8+ T cells (CD161N) and MAIT cells from the same individual (Figure 3A). Analyses of variable (V)/diversity (D)/joining (J) segments showed the diverse TCR use of CD161P, which mainly were αβ T cells (Figure 3B). The antigen specificity of TCR lies mainly in the complementarity determining region 3 (CDR3) region.
Further analyses of sequences of CDR3α and CDR3β showed lower TCR diversity in CD161P-HD than in CD161N in healthy donors (CD161N-HD), which decreased to a greater extent in CD161P from patients (CD161P-HBV) (Figure 4A and B). There was little overlap in the top 10 abundant T Cell Receptor alpha variable region (TRAV)-T Cell Receptor alpha joining region (TRAJ) or T Cell Receptor beta variable region (TRBV)-T Cell Receptor variable beta region (TRBJ) pairs between CD161P-HD and CD161P-HBV (Figure 4C), supporting the polyclonal TCR use of CD161P.
Figure 3The biased TCR use in circulating CD161+CD8+T cells. (A) The purity of the indicated cell groups from 1 healthy donor. (B) TRAV (upper panel) and TRBV (lower panel) use of CD161P and CD161N in healthy donors (CD161P-HD and CD161N-HD, n = 3) and in chronic HBV-infected patients (CD161P-HBV and CD161N-HBV, n = 3).
Figure 4Circulating CD161+CD8+T cells from chronic HBV-infected patients display reduced TCR diversity and possess overlapped CDR3 sequences with CD161−CD8+T cells. (A) The ratio of distinct T Cell Receptor alpha chain (TRA) and T Cell Receptor beta chain (TRB) CDR3 sequences of circulating CD161P and CD161N from healthy donors (n = 3). (B) The ratio of distinct TRAV and TRBV use of CD161N and CD161P in HDs (n = 3) and in HBV-infected patients (HBV, n = 3). (C) Circos plots show the most frequent TRAV–TRAJ and TRBV–TRBJ pairs in CD161P-HD (n = 3) and CD161P-HBV (n = 3). (D) The ratios of the same and different CDR3α and CDR3β sequences between CD161P and CD161N in indicated individuals. The abundant (E) CDR3α sequences and (F) CDR3β sequences in CD161P-HBV that overlap with those in CD161N-HBV. (G) Representative histograms and bar graph show the expression of CD161 in sorted circulating CD161N-HD stimulated by autologous monocytes (Mono) and supernatant from HBV pseudovirus particle-producing HepG2.2.15 cells with or without anti-CD28 antibody for 7-day treatment (n = 4/group). differ, different; MFI, mean channel fluorescence intensity; Sup), supernatant. ∗P < .05; ∗∗∗P < .001
We compared the TCR repertoire between CD161P and CD161N from the same subject. Overlapped CDR3 sequences between CD161N and CD161P increased in patients and was enriched markedly in CD161P-HBV (Figure 4D–F), suggesting the possible switch between CD161N from the patients (CD161N-HBV) and CD161P-HBV populations. Supernatant from HBV pseudovirus particle-producing HepG2.2.15 cells,
which had increased hepatitis B surface antigen (HBsAg) levels mimicking HBV-infected hepatocytes, modestly up-regulated the CD161 level in CD161N-HD (Figure 4G). Collectively, these analyses indicated that the observed increase of CD161+CD8+ T cells in patients likely derived from a proportional shift from CD161−CD8+ T cells after persistent HBV stimulation.
Transcriptomic Analysis of CD161+CD8+ T Cells Reveals They Are Endowed With Innateness Phenotype, Tissue Homing Tendency, and Enhanced Antiviral Capacity
Through principle component analysis, we found CD161P, CD161N, and MAIT cells from healthy donors distributed in different regions with clear boundaries, suggesting they were heterogeneous in genetic traits (Figure 5A). There were 244 genes distinctly changed in CD161P that were different from both CD161N and MAIT cells from healthy donors (Figure 5B). CD161P-HD expressed a higher level of innate-like immune genes and tissue homing genes than CD161N-HD (Figure 5C), supporting the common preprogramed phenotype shared with MAIT cells. The up-regulated genes included innate-related transcriptional factors highly expressed on MAIT cells, such as zinc finger and BTB domain containing 16 (ZBTB16), SH2 domain containing 1B (SH2D1B), PR/SET domain 1 (PRDM1) (BLIMP1), RAR related orphan receptor C (RORC), and T-box transcription factor 21 (TBX21) (Figure 5D, left panel). In addition, up-regulated natural killer (NK) cell receptors, including natural cytotoxicity triggering receptor 3 (NCR3), CD160, killer cell lectin like receptor D1 (KLRD1)(CD94), and killer cell lectin like receptor G1 (KLRG1), were observed in CD161P-HD, which may endow them with an enhanced innate ability to respond quickly (Figure 5D, middle panel). The higher expression of chemokine receptors such as C-C motif chemokine receptor 6 (CCR6) and C-X-C motif chemokine receptor 6 (CXCR6) accompanied with lower expression of C-C motif chemokine receptor 7 (CCR7) suggested enhanced tissue chemotaxis of circulating CD161+CD8+ T cells (Figure 5D, right panel).
Figure 5Differential gene expression reveals the distinct transcriptional profiles of CD161+CD8+T cells and functional changes of the population in chronic HBV-infected patients. MAIT cells, CD161N, and CD161P were sorted from HD and HBV groups for RNA sequencing and DEG analysis. (A) Principal component analysis of transcriptional profiling of circulating CD161P, CD161N, and MAIT cells in healthy donors. (B) Venn diagram of overlapping gene counts among the indicated cell groups. (C) Volcano plots display down-regulated genes (green dots) and up-regulated genes (red dots) in CD161P-HD compared with CD161N-HD. (D) Heatmap of relative transcript levels of selected genes of transcriptional factors, NK cell receptors, and chemokines and chemokine receptors in CD161N-HD and CD161P-HD. (E) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of up-regulated genes in CD161P-HD vs CD161N-HD and in CD161P-HD vs MAIT cells. (F) Polar heatmap of normalized gene read counts of NK cell–mediated cytotoxicity and cytokine-cytokine–receptor interaction genes in circulating CD161P, CD161N, and MAIT cells from healthy donors. (G) The up-regulated (red) and down-regulated (blue) genes in the indicated pathways in circulating CD161P-HBV vs CD161P-HD.
The Kyoto encyclopedia of genes and genomes pathway enrichment of up-regulated differentially expressed genes (DEGs) showed that cytokine-cytokine–receptor interaction and NK cell–mediated cytotoxicity pathways were up-regulated significantly in the CD161P-HD compared with CD161N-HD and MAIT cells from healthy donors (Figure 5E). CD161P-HD tended to have higher transcript levels of proinflammatory cytokines and NK cell–related cytotoxic molecules (Figure 5F). It is worth noting that CD161P-HD showed the highest transcript levels of interferon (IFN)-γ, Fas ligand, and granzyme B among the 3 populations (Figure 5F). This defined an enhanced antiviral and cytotoxicity transcriptional phenotype of CD161+CD8+ T cells.
Circulating CD161+CD8+ T Cells From HBV-Infected Patients Are Functionally Dysregulated With Biased Cytokine Production and Increased Lytic Capacity
Next, we compared the functional status of CD161P-HBV with CD161P-HD to investigate their roles in patients. It has been shown that the cytotoxicity of CD161+CD8+ T cells is mediated primarily through granzyme pathways.
Here, we found increased transcript levels of granzymes (A/B/H) and perforin 1 in CD161P-HBV (Figure 5G, left panel). With regard to cytokine-producing capacity, CD161P-HBV increased IL10 messenger RNA levels but showed a downward trend in IL6, IL1A, and IL18 secretion (Figure 5G, middle panel). In addition, the inhibitory and exhausted markers, such as killer cell lectin like receptor 1 (KLRC1) natural killer group 2A (NKG2A), T cell immunoreceptor with Ig and ITIM domains (TIGIT), cytotoxic T-lymphocyte associated protein 4 (CTLA4), and ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), and CD39, were up-regulated in CD161P-HBV, indicating their exhausted status in patients (Figure 5G, right panel).
The functional status of CD161P-HBV was evaluated further in vitro by intracellular staining for granzyme B, IL17A, IFN-γ, and tumor necrosis factor (TNF)-α. Compared with CD161N-HD and MAIT cells from healthy donors, CD161P-HD had the highest granzyme B level ex vivo (Figure 6A). Even higher granzyme B secretion was observed in CD161P-HBV compared with that in CD161P-HD (Figure 6A), further supporting the increased lytic potential of CD161P-HBV in protein levels. The increased granzyme B secretion by T cells has been suggested to have an off-target effect and mediate chronic inflammatory diseases and autoimmunity.
The high level of granzyme B secretion and positive correlation between percentages of granzyme B-expressing CD161P-HBV and serum aspartate aminotransferase (AST) levels suggests the involvement of CD161P-HBV in liver injury (Figure 6B). Moreover, CD161P-HBV showed a higher IL17A-producing but lower IFN-γ–/TNF-α–producing capacity (Figure 6C). In consistent with transcriptional upregulation of inhibitory and exhausted genes such as programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), T cell immunoglobulin and mucin domain 3 (Tim-3), and CD39 also were found in CD161P-HBV (Figure 6D and E). CD39 is a new marker of T-cell later-phase exhaustion and it has been suggested that it is involved in driving helper T 17 (Th17)-biased response through cooperation with CD161.
Therefore, we proposed the circulating CD161+CD8+ T-cell population in patients was exhausted and dysfunctional with enhanced cytotoxicity, dampened Th1-biased immunity, and increased IL17 production.
Figure 6Circulating CD161+CD8+T cells show increased granzyme B and IL17A secretion, but impaired Th1 cytokine secretion in chronic HBV-infected patients. (A) Representative dot plots and summarized scatter plots show the ratio of granzyme B (GrzB)-secreting cells and GrzB levels in MAIT cells, CD161N, and CD161P from HD (n = 46) and HBV (n = 48) groups. (B) Spearman correlation between the frequencies of circulating GrzB-secreting CD161+CD8+ T cells and serum AST levels. (C) Representative dot plots and summary scatter graphs display the frequencies of IL17A/IFN-γ/TNF-α–producing CD161+CD8+ T cells from HD (n = 43) and HBV (n = 46) groups after phorbol 12-myristate 13-acetate/ionomycin stimulation. (D) Representative graphs and summary bar graphs display PD-1, CTLA-4, and Tim-3 levels in circulating CD161+CD8+ T cells in the indicated groups (n ≥ 5/group). (E) Representative dot plots and summary bar graph display the ratio of CD39-expressing CD161+CD8+ T cells from HD (n = 9) and HBV (n = 7) groups. MFI, mean fluorescence intensity. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001
Of note, CD161P-HBV from ACLF patients with extensive liver injury and high HBV loading showed even higher expression of granzyme B and IL17A secretion, less Th1 cytokine secretion, and a higher exhaustion level (Figure 7). Although it was hard to draw conclusions about the direct effect of CD161+CD8+ T cells on disease progression, the consistent functional changes and clinical relevance supported the pathogenic potential of the population in HBV-infected patients.
Figure 7Circulating CD161+CD8+T cells from acute-on-chronic liver failure patients are dysregulated to a greater extent. (A) Summary histogram displays the level of total bilirubin (TBIL), alanine aminotransferase (ALT), AST, and HBV-DNA copies in the CHB patient group (chronic, n = 138) and acute-on-chronic liver failure HBV-infected patient group (ACLF, n = 61). (B) Representative dot plots and summary scatter graphs display the frequencies of IL17A/IFN-γ/TNF-α–producing CD161+CD8+ T cells from CHB (n ≥ 27) and ACLF (n ≥ 14) groups after phorbol 12-myristate 13-acetate/ionomycin stimulation. (C) Representative dot plots, histograms, and summary scatter graphs show the ratio of granzyme B (GrzB)-secreting cells and GrzB levels in CD161+CD8+ T cells from the CHB (n = 29) and ACLF (n = 17) groups. (D) Representative graphs and summary bar graphs display PD-1 and CTLA-4 levels in circulating CD161+CD8+ T cells in the indicated groups (n ≥ 5/group). FSC, forward scatter; MFI, mean channel fluorescence intensity. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001
Hepatic CD161+CD8+ T Cells Are Increased and Functionally Dysregulated in HBV-Infected Patients
To investigate whether the ratio and functional status of hepatic CD161+CD8+ T cells changed as circulating CD161+CD8+ T cells, T-cell subsets in nontumor liver tissue from HBV-infected patients were analyzed. The frequency of hepatic CD161+CD8+ T cells increased significantly in patients, while hepatic MAIT cells decreased (Figure 8A). This change coincided with the ratio change of their circulating counterparts. The frequency of hepatic CD161+CD8+ T cells also correlated positively with HBV-DNA levels (Figure 8B), further supporting the dispensable role of CD161+CD8+ T cells in HBV control.
Figure 8Hepatic CD161+CD8+T cells increase but are dysregulated in nontumor liver tissue from HBV-infected patients. (A) The ratio of hepatic CD161+CD8+ T cells, CD161−CD8+ T cells, MAIT cells, and total CD8+ T cells in nontumor liver tissue from HBV-negative (HBV−, n = 59) and HBV-infected individuals (HBV+, n = 73). (B) Spearman correlation between the frequencies of hepatic CD161+CD8+ T cells and serum HBV-DNA copies. (C) Representative scatter plots and summarized scatter graph depict the proliferation rates shown by Ki67 staining of circulating and corresponding hepatic CD161+CD8+ T cells in HBV-infected patients (n = 13). (D) Summarized proliferation rates of hepatic CD161+CD8+ T cells in the indicated groups (n ≥ 8/group). (E) The ratio of the same and different CDR3α and CDR3β sequences between hepatic CD161+CD8+ T cells and CD161−CD8+ T cells from HBV-infected patients (n = 4). (F) Summarized dot plots show the levels of CD69, HLA-DR, CD38, PD-1, and Tim-3 in hepatic CD161+CD8+ T cells in the indicated groups (n ≥ 5/group). (G) Representative dot plots and summarized scatter graphs depict the frequencies of IL17A/IFN-γ/TNF-α–producing hepatic CD161+CD8+ T cells upon phorbol 12-myristate 13-acetate/ionomycin stimulation in the indicated groups (n ≥ 7/group). (H) Representative graph and summarized scatter graphs display the ratio of granzyme B (GrzB)-producing and GrzB levels in hepatic CD161+CD8+ T cells in the indicated groups (n ≥ 6/group). (I) Spearman correlation between the frequencies of hepatic GrzB-secreting CD161+CD8+ T cells and serum AST levels. differ, different; FSC, forward scatter; MFI, mean fluorescence intensity; TRA, T Cell Receptor Alpha; TRB, T Cell Receptor Beta. ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001
Although hepatic CD161+CD8+ T cells were more proliferative than circulating ones (Figure 8C), they did not proliferate greater in HBV-infected patient compared with those in HBV-negative hepatic carcinoid individuals (Figure 8D). The overlap of CDR3 sequences in hepatic CD161+CD8+ T cells and CD161−CD8+ T cells from patients further supported the switch between the 2 populations in the liver (Figure 8E). Similar to circulating ones, hepatic CD161+CD8+ T cells from patients had a higher level of activation and exhaustion markers, reduced Th1-biased cytokines, but increased IL17 production, and also had an increased granzyme B level that was associated positively with serum AST level (Figure 8F–I). Collectively, hepatic CD161+CD8+ T cells similarly were increased and functionally dysregulated as their circulating counterparts in HBV-infected individuals.
Enrichment of CD161+CD8+ T Cells Correlates With Liver Fibrosis and Injury in HBV-Infected Patients
The frequencies of circulating or hepatic CD161+CD8+ T cells correlated positively with Fibrosis-4 (Figure 9A), an index for inflammatory injury–associated liver fibrosis.
To further investigate the role of CD161+CD8+ T cells in liver injury, chronic HBV-infected patients were divided into 2 groups according to serum AST levels. The ratio of circulating CD161+CD8+ T cells was higher in the abnormal AST group (AST level, >40 U/L) than those in the normal AST group (AST level, <40 U/L) (Figure 9B, upper panel). Although the ratio of hepatic CD161+CD8+ T cells changed little (Figure 9B, lower panel), the increased number of hepatic CD161+CD8+ T cells could be expected because infiltrated T cells increased significantly in the injured liver (Figure 9C). Along with greater functional alteration in circulating CD161+CD8+ T cells from patients with severe liver injury (Figure 7), even lower IFN-γ and TNF-α–producing capacity as well as a greater increase in IL17A and granzyme B secretion were found in hepatic CD161+CD8+ T cells from the abnormal AST group (Figure 9D). Similar dysfunctional changes were found in hepatic CD161+CD8+ T cells from patients with an increased bilirubin level, another indicator for liver injury
(Figure 9E). These results suggest typical injury-promoting traits of CD161+CD8+ T cells from patients with a liver injury.
Figure 9Hepatic CD161+CD8+T cells from patients with abnormal AST and bilirubin levels show lower antiviral-related cytokines but higher IL17A and granzyme B production. (A) Spearman correlation between the frequencies of circulating (upper panel) and hepatic (lower panel) CD161+CD8+ T cells with liver inflammation Fibrosis-4 (FIB-4) index. (B) Summarized scatter graphs for the ratio of circulating (upper panel) and hepatic (lower panel) CD161+CD8+ T cells in patients with a normal AST level (AST < 40 U/L; circulating, n = 81; hepatic, n = 45) and an abnormal AST level (AST > 40 U/L; circulating, n = 64; hepatic, n = 28). (C) Representative H&E staining and immunofluorescence staining of CD3+ T cells (green) in the indicated sections. (D and E) Summarized scatter graphs depict the frequencies of IL17A/ IFN-γ/TNF-α/granzyme B (GrzB)-producing cells as well as GrzB levels in hepatic CD161+CD8+ T cells in HBV-infected patients with normal and abnormal (D) AST or (E) total bilirubin (TBIL) levels upon phorbol 12-myristate 13-acetate (PMA)/ionomycin stimulation (n ≥ 6/group). MFI, mean channel fluorescence intensity. ∗P < .05; ∗∗P < .01;
Hepatic CD161+CD8+ T Cells From Patients With Liver Injury Display Strong Potential to Initiate Local Neutrophil Infiltration and Response
Although circulating CD161+CD8+ T cells from CHB patients secrete reduced proinflammatory cytokines and increased inhibitory cytokines (Figure 5G), hepatic CD161+CD8+ T cells from the abnormal AST group had a markedly increased transcript level of IL1B (IL1β) and CXCL8 (IL8) compared with those from the normal AST group (Figure 10A and B). IL1β and IL8 play important roles in chemotaxis and activation of neutrophils, which could be enhanced by IL17 and is associated with a poor outcome of acute-on-chronic liver failure.
The enhanced IL17 production and increased transcripts of IL1B and CXCL8 endowed hepatic CD161+CD8+ T cells with potent capacity in activating neutrophil responses to exacerbate local inflammation and injury. Consistently, gene set enrichment analysis of the transcriptional signature showed up-regulation of the neutrophil chemotaxis-relating gene clusters and increased genes mediating neutrophil responses in the injured liver in the abnormal AST group (Figure 10C–E). In addition, hepatic CD161+CD8+ T cells and liver tissue from the abnormal AST group consistently up-regulated T-cell and neutrophil response-related pathways (Figure 10F and G). In addition, increased TCR-induced IL8, IL6, and Monocyte chemoattractant protein-1 (MCP-1) production was found in CD161P-HBV compared with CD161-HD, supporting the proinflammatory and pathogenic potential of the population in HBV-infected patients (Figure 10H). Together, these data support the pathogenic potential of hepatic CD161+CD8+ T cells through enhancing neutrophil infiltration and response in HBV-infected patients.
Figure 10Proinflammatory profiles of hepatic CD161+CD8+T cells coincides with enhanced neutrophil responses in injured liver from chronic HBV-infected patients. (A) Volcano plots display down-regulated genes (green dots) and up-regulated genes (blue dots) of hepatic CD161+CD8+ T cells from HBV-infected patients with abnormal AST levels (AST > 40) compared with those with normal AST levels (AST < 40). (B) The up-regulated (red) and downregulated (blue) genes in hepatic CD161+CD8+ T cells from the indicated groups. (C) Gene set enrichment analysis of neutrophil chemotaxis and migration-related gene clusters in nontumor liver tissue of patients with an abnormal AST level vs patients with a normal AST level. (D) Volcano plots display down-regulated genes (green) and up-regulated genes (blue) in nontumor liver tissue of patients with abnormal AST levels compared with those with normal AST levels. (E) Heatmap shows normalized levels of neutrophil response–related genes in nontumor liver tissue in patients with abnormal AST levels compared with those with normal AST levels. Gene Ontology enriched pathway analysis of up-regulated DEGs in (F) hepatic CD161+CD8+ T cells or in (G) nontumor liver tissue in HBV-infected patients with an abnormal AST level compared with those with a normal AST level. (H) Sorted circulating CD161+CD8+ T cells (CD161P) and CD161−CD8+ T cells (CD161N) from HDs (n = 3) and chronic HBV-infected patients (HBV, n = 3) were stimulated by microspheres coated with anti-CD3/CD28 antibody in a 1:1 ratio. Cytokines in supernatant were detected by Cytometric Bead Array Flex Sets 3 days after stimulation. FACS, fluorescence-activated cell sorting; p.adjust, adjusted P value. ∗∗∗P < .001; ∗∗∗∗P < .0001
Several studies have indicated that CD161 expression defines a transcriptional and functional phenotype across distinct T-cell lineages that are critically involved in pathogen immunity and tumor immunology.
Our data show that non-MAIT CD161+CD8+ cells increased in CHB patients, which is related to liver inflammation and injury. We propose that the increased CD161+CD8+ T cells result from a proportional shift from CD161−CD8+ T cells because of the overlapped TCR sequences in both populations. Traditional antigen-specific CD8+ T-cell identification methods, including major histocompatibility complex class I tetramer,
can identify only a proportion of antigen-specific CD8+ T cells in patients. CD161+CD8+ T cells specific for hepatitis C virus and HBV have been reported previously.
The abundant TCR sequences in CD161+CD8+ T cells that overlap with CD161−CD8+ T cells suggest a possible TCR repertoire of HBV antigen–specific T cells. In-depth study of CD161+CD8+ T cells with overlapped TCR could propel the analysis of HBV-specific TCR, and could become an effective supplement to traditional identification methods.
The importance of IFN-γ secretion as a mediator of control over intrahepatic pathogens is well accepted.
Although CD161+CD8+ T cells display more enhanced IFN-γ–producing capacity than CD161−CD8+ T cells (Figure 5F), they have reduced IFN-γ–producing capacity but increased IL17 production in CHB patients (Figures 6C and 8G). These changes extend to a greater degree in patients with liver injury (Figures 7 and 9), accompanied with increased IL1B and CXCL8 transcripts in the population (Figure 10). IL8, encoded by CXCL8, has been shown to impair the antiviral actions of IFNs
Consistently, transcriptional analysis of liver tissue from patients with abnormal serum AST levels showed increased neutrophil migration and neutrophil responses (Figure 10). On the other hand, IL8 plays an additional proviral role by inhibiting IFN effects.
Therefore, dysregulated CD161+CD8+ T cells are supposed to promote the IL17/ILβ/IL8-based pathogenic inflammatory network, which contributes to attenuate antivirus response and intensify inflammation-mediated tissue damage, thus favoring virus persistence.
In human beings, the cytotoxic potential of CD161-expressing CD8+ T cells has not been clearly defined, with reports of both high and low cytotoxic potential.
After comparison among CD161+CD8+ T cells, CD161−CD8+ T cells, and MAIT cells, the granzyme B level was found to be highest in CD161+CD8+ T cells and lowest in CD161-expressing MAIT cells. This suggests that CD161 is not the key factor for controlling granzyme B expression. Cytotoxicity of CD161+CD8+ T cells is mediated primarily by granzyme B.
Here, the ratio of granzyme B–secreting CD161+CD8+ T cells is correlated positively with serum AST level (Figures 6B and 8I), although positive correlation also was found between the ratio of CD161+CD8+ T cells and HBV-DNA level (Figures 1D and 8B). This suggests their possible effect in liver damage and dispensable role in viral control. In addition, current studies have reported CD161 signaling involves the activation of acid sphigomyelinase,
and shown further interaction with CD39 to amplify acid sphigomyelinase–mediated mechanistic target of rapamycin (mTOR) and signal transducer and activator of transcription 3 (STAT3) signaling driving Th17 expansion.
These suggest CD39 co-expression and the CD161–acid sphigomyelinase–mTOR–STAT3 signaling pathway are mechanisms in dysfunction of CD161-bearing cells. Consistently, increased CD161P cells in CHB patients showed increased CD39 and IL17 expression, suggesting the CD161 signaling involved in the increased IL17 production of dysregulated CD161+CD8+ T cells.
In summary, CD161+CD8+ T cells increase and are dysregulated in CHB patients, showing impaired Th1-biased cytokine secretion, increased IL17/IL1β/IL8 proinflammatory cytokine–producing capacity, and enhanced granzyme B secretion. These changes polarize CD161+CD8+ T cells toward pathogenesis by reducing antiviral function and promoting augmented inflammation and cytotoxicity. Therefore, CD161+CD8+ T cells and IL17/IL1β/IL8 can serve as targets for reducing liver injury in CHB.
Materials and Methods
Patient Populations
Peripheral blood samples of 257 chronic HBV-infected patients (serum HBsAg-positive for >6 mo) were collected from Tongji Hospital, Wuhan, China, among which there were 61 ACLF patients who needed continuous artificial liver system support. Nontumor liver specimens were collected from 73 HBV-positive patients with hepatic carcinoid and hepatocellular carcinoma or liver failure, and 59 HBV-negative hepatic carcinoid patients undergoing liver puncture or hepatectomy at Tongji Hospital. Individuals with other types or concurrent types of viral hepatitis, human immunodeficiency virus, autoimmune liver disease, or alcohol-associated liver disease were excluded.
Patient characteristics are listed in Table 1. All aspects of this study were granted approval by the ethics committee of Tongji Medical College, Huazhong University of Science and Technology. Signed informed consent was obtained from all enrolled individuals.
Table 1Clinical Characteristics of Patients Enrolled in This Study
ALT, alanine aminotransferase; DBIL, ______; HBeAb, hepatitis B virus e antibody; HBeAg, hepatitis B virus e antigen; HCC, hepatocellular carcinoma; NA, not available; ND, not determined.
Detection Reagents and Antibodies for Flow Cytometry
Allophycocyanin (APC) or BV421-labeled MHC class I-related molecule 1 (MR1) tetramer loaded with vitamin B derivate 5-(2-oxopropylideneamino)-6-D-ribitylaminouracil (5-OP-RU/MR1 tetramer) was provided by the National Institutes of Health tetramer facility. Fluorescein isothiocyanate–Annexin V Apoptosis Detection Kit I (556547; BD Biosciences, Franklin Lakes, NJ) and Ki67 (350520; BioLegend, San Diego, CA) were used for cell apoptosis or proliferation analysis. Fluorescence-conjugated monoclonal antibodies against the molecules listed in Table 2 were purchased from BioLegend or BD Biosciences.
Table 2Various Cell Surface, Intracellular, and Intranuclear Markers in This Study
Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation, and intrahepatic lymphocytes (IHLs) were isolated by modified enzymatic dispersal protocol followed by Percoll density purification.
5-OP-RU-MR1 tetramer+ CD3+ MAIT cells, 5-OP-RU-MR1 tetramer− CD3+CD161+CD8+ T cells, 5-OP-RU-MR1 tetramer− CD3+CD161−CD8+ T cells, and CD14+ monocytes were sorted from PBMCs and/or IHL cells by fluorescence-activated cell sorting (FACSAria III; Becton, Dickinson and Company).
The supernatant of HBV genome-integrated stable cell HepG2.2.15,12 containing HBV pseudovirus particle, was collected. HBsAg in the supernatant was tested with a HBsAg enzyme-linked immunosorbent assay kit (YBS00192010; Kehua Bio-engineering Co, Ltd, Shanghai, China). The sorted circulating 5-OP-RU-MR1 tetramer− CD161−CD8+ T cells from healthy donors were co-cultured with monocytes in the presence of anti-CD28 antibody (302901, 0.5 μg/mL; BioLegend) and supernatant containing HBV pseudovirus particles from HepG2.2.15 for 7 days. Sorted CD161-CD8+ T cells and CD161+CD8+ T cells from healthy subjects and chronic HBV-infected patients were stimulated with anti-CD3 and anti-CD28 in a 96-well plate. Cells were cultured for 3 days with fresh 10% fetal bovine serum 1640 and the supernatant was collected for cytokine detection by a Cytometric Bead Array Flex Set (BioLegend). Data were collected using the FACSVerse (Becton, Dickinson and Company) and analyzed by FlowJo software (Tree Star).
Flow Cytometry
Cells were surface stained with corresponding antibodies and/or detection reagents. The forkhead/winged helix transcriptional factor P3 (Foxp3) staining buffer set (00-5523-00; eBioscience, San Diego, CA) was used for intracellular staining. Ex vivo unstimulated cells were stained with cell surface markers, fixed, and further stained for isotype control, granzyme B, and Ki67. For intracellular cytokine staining, PBMCs or IHLs were stimulated with phorbol 12-myristate 13-acetate (79346, 25 ng/mL)/calcium ionophore ionomycin (ionomycin, 407951, 500 ng/mL; all from Sigma-Aldrich, St Louis, MO) for 30 minutes and a 3.5-hour incubation with brefeldin A (00-4506-51; eBioscience), followed by surface staining with antibodies against surface molecules, fixation/permeabilization, and intracellular staining with antibodies against IFN-γ, TNF-α, and IL17A. Data were collected using FACSVerse or a LSR II cytometer (BD Biosciences) and analyzed by FlowJo software.
RNA Sequencing and Data Analysis
RNA from sorted cells and liver specimens was extracted using TRIzol (Invitrogen, Norcross, GA) for RNA sequencing library preparation by the KC Stranded Messenger RNA Library Prep Kit for Illumina (Wuhan Seqhealth, Co, Ltd, Wuhan, China). A unique molecular identifier of 8 random bases was used to label the pre-amplified complementary DNA molecules. Qualified reads were mapped to the reference genome using STAR software (version 2.5.3a;https://github.com/alexdobin/STAR) and counted by featureCounts (Subread-1.5.1; Bioconductor). DEGs among different groups were analyzed by DESeq2 packages on R (Bioconductor). The Kyoto encyclopedia of genes and genomes and Gene Ontology enrichment analyses, as well as gene set enrichment analysis for DEGs, were performed on R, with a corrected P value cut-off of .05 to judge statistically significant enrichment. TCR use of sorted cells was analyzed from RNA sequencing data by taking advantage of MIXCR software (MiLaboratories) to identify V, D, and J segments, as well as CDR3 sequences.
Histology and Immunofluorescence Staining
Liver specimens were fixed in 4% paraformaldehyde and embedded in paraffin, followed by sectioning and H&E staining. Fluorescent staining of CD3 in sections was performed with fluorescein isothiocyanate–conjugated anti-CD3 antibody (GB11014, 1:100; Servicebio, Wuhan, China). The whole sections of H&E staining or CD3 immunofluorescence staining were scanned by Pannoramic MIDI (3D HISTECH, Budapest, Hungary).
Statistical Analysis
Data were analyzed by GraphPad Prism software (version 7.0; GraphPad Software). The Student t test and analysis of variance were performed to analyze data with normal distribution and equal variance for comparisons of 2 or more groups, respectively. Non-normally distributed data were analyzed by the Mann–Whitney U test or the Kruskal–Wallis test. The Spearman rank correlation was used to assess the correlation between different parameters. P values <.05 were considered statistically significant.
All authors had access to the study data and reviewed and approved the final manuscript.
Model to predict on-treatment restoration of functional HBV-specific CD8(+) cell response foresees off-treatment HBV control in eAg-negative chronic hepatitis B.
Conflicts of interest The authors disclose no conflicts.
Funding Supported by National Natural Science Foundation of China grants 81871235 (X.W.) and 82101863 (Y.L.), Hubei National Science Foundation for Distinguished Young Scholars grant 2020CFA074 (X.W.), and Jiangxi Provincial Natural Science Foundation grant 20212BAB206088 (Y.L.).
Data Availability Statement Data are available in a public, open-access repository. Data are publicly available and deposited in the online repository Gene Expression Omnibus Database (GEO, GSE208535; and GEO, GSE208637).
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