Microbial Pathogenesis 74 (2014) 59e62
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Review
Cellular immune response in patients with chronic hepatitis B virus infection Xuefen Li, Yiyin Wang, Yu Chen* State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2014 Received in revised form 30 July 2014 Accepted 31 July 2014 Available online 12 August 2014
Hepatitis, cirrhosis and hepatocellular carcinoma caused by hepatitis B virus (HBV) infection has threatening human health seriously. As HBV is a kind of non-cytotoxic virus, host immune response plays a vital role in pathogenesis and clinical outcomes of hepatitis B. Multiple immune cells (e.g., including cytotoxic T lymphocytes, regulatory T cells, natural killer cells, dendritic cells) are important in the immune regulation of HBV infection. Therefore, focusing on the activation states of immune cells may provide new evidences and strategies for determining immune status of HBV infectors, monitoring progression of diseases and predicting efficacy of antiviral treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Hepatitis B Cellular immune response Clinical implications
1. Introduction Although effective vaccine against hepatitis B virus (HBV) infection has been developed in the 1980s, around 1/3 of the population worldwide still show a previous or existing HBV infection in serological tests [1]. Different types of clinical outcomes may appear after infection with HBV: for some people, virus can be cleared quickly, no symptoms or only short-term self-limited acute inflammation of liver can be presented; while more can be seen clinically is that patient himself cannot clear the virus effectively and progress to chronic hepatitis. Currently, it is widely recognized that HBV does not damage liver cells directly [2]. Actually, liver inflammation caused by HBV infection is a kind of immune pathological response mainly mediated by multiple immune cells, during which virus cleared and liver cells damaged meanwhile. The status of immune response plays an important role in pathogenesis and clinical outcomes of hepatitis B. At the same time, host immune responses in patients with hepatitis B affect the effectiveness of antiviral therapy greatly [3e5]. The various clinical presentations of HBV infection as well as the pathogenesis of liver diseases are immune-mediated and thus determined by the virusehost interaction [6e8]. It has been established that innate immune responses (involve macrophages and dendritic cells (DC) as well as natural killer (NK)
* Corresponding author. Tel.: þ86 571 87236382; fax: þ86 571 87236383. E-mail addresses: [email protected], [email protected] (Y. Chen). http://dx.doi.org/10.1016/j.micpath.2014.07.010 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
cells) are the host's first defense mechanism against viral infections, and favor a timely and efficient induction of virus-specific adaptive responses in a self-limited, transient liver disease [6,9]. However, a study performed on chimpanzees showed that HBV does not seem to induce a strong innate immune response in the liver, and HBV clearance was associated with a strong adaptive immune response [10]. In human, previous studies also appears that patients with self-resolving hepatitis B have confirmed an induction of NK and NKT cell responses before the onset of adaptive T cell responses [11]. Subsequently, a robust activation of HBVspecific adaptive immune responses, including CD4þ T helper cells, cytotoxic T lymphocyte (CTL) and B cells, especially HBVspecific CTL response, play central roles to clear the virus [6,12]. Acute hepatitis B have shown vigorous immune response against HBV infection, and there is a relative clear correlation between timing, strength and specificity of the cellular immune response and the outcome of self-limited infection [7,12]. In contrast to acute hepatitis B, studies on patients with chronic HBV infection indicated that HBV-specific T-cell reactivity is weak and hard to detect and showed poor immune response to viral control [6,13,14]. It is still unclear why the human immune system fails during chronic HBV infection and how can early antiviral therapy affects cellular immune responses during sustained treatment. In this review, we will mainly addressed the most recent information regarding the cellular immune status in patients with chronic HBV infection and its significance in clinical monitoring, from the perspective of changes in multiple active immune cells of hepatitis B.
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2. Characteristics of active immune cells in patients with HBV infection 2.1. HBV-specific cytotoxic T lymphocyte (CTLs) responses After infected with HBV, anti-HBV specific cellular immune response can be induced through activation of CD4þ and CD8þ T cells. It is widely accepted that there are three kinds of ways for antigen specific T cells to stay in liver: (1) As there is no further stimulation of specific antigen, T cells are cleared quickly in the liver, with no significant liver damage, such as late stage of acute HBV infection; (2) T cells are stranded in the liver while functions are inhibited, causing low immune response or tolerance, such as chronic hepatitis B; (3) T cells are activated excessively in the liver and cause further activation of natural killer (NK) cells and other nonspecific immune cells, resulting in liver cells damage, such as chronic active hepatitis B and severe hepatitis. During the process of clearing HBV in the liver by immune system, HBV specific cytotoxic T lymphocytes (CTLs) play a key role in virus clearance and liver damage. Specific CD8þ T cells are the primary effector cells to clear the virus and CTLs are effective stage as well as main subsets of CD8þ T cell. Specific CD8þ T cells recognize molecular antigen through major histocompatibility complex I (MHC-I) and clear the virus through cytotoxic and non-cytotoxic mechanism: CD8þ T cells recognize HBV short peptide presented by HLA I class molecule in target cell surface specifically, which can induce apoptosis of hepatocytes infected with HBV after being activated; release perforin and crack target cells; secrete cytokines, such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), etc., and inhibit HBV replication through non-cytolytic mechanism or attract inflammatory cells without antigen specificity, leading to liver cells necrosis. From the study of transgenic mouse model, it is found that antiviral effect of CTL is mainly mediated by non-cytotoxic mechanism, e.g., clear the virus without damaging liver cells infected with HBV by secreting IFNg, TNF-a and other cytokines, among which IFN-g plays a crucial role [4,5,13e15]. CD4þ T cells are not directly involved in viral clearance and tissue damage, but promote CD8þ T cells to release IFN-g and thereby control HBV replication indirectly through secretion of cytokines interleukin-2 (IL-2). At the same time, CD4þ T cells are able to assist B cells to produce antibodies. Lack of CD4þ T cells would weaken activity of CD8þ T cells and inhibit the production of antibodies [13,14,16]. The control of HBV infection is closely related to specific T cells response. Study has shown that in patients with acute selflimited viral hepatitis, HBV-specific CTL in peripheral blood always present to be polyclonal and multispecific, with strong response and high frequency, and identify a wide range of antigen targets. HBV-specific CD8þ T cells and CD4þ T cells emerge in early incubation period and begin to inhibit viral replication, during which specific cellular immune plays a major role. Furthermore, a large number of CD8þ T cells exist in peripheral circulation and are able to complement the consumption of specific CD8þ T cells in the liver, and thereby clear HBV strongly, causing no or only minor liver damage. On the contrary, CTL response is weak or even absent in patients with chronic HBV infection, characterized by monoclonal and monospecific. HBVspecific helper T cells (Th) and CTL response were significantly lower in patients with persistent HBV infection than that in recovering patients of acute hepatitis B [14]. Therefore, the outcome of hepatitis B infection mainly depends on cellular immune response. Low number and function defects of HBV specific CTL may be the key factor to HBV persistent infection and the development of chronic hepatitis B.
2.2. Activation status of CTL in HBV infection CTL activation is of great importance in HBV infection. The activation of CTL requires not only the first signal delivered by binding antigen MHC molecule complex and T cell receptor (TCR), but also combination of CD28 in T cell surface and CD80/86 from B7 molecule family of antigen presenting cell (APC) to provide the second signal. Therefore, CD28 molecule plays an important role in the activation of T cell immune response. CD28, expressed on the surface of T cells, is an important moleculer marker of T cells. CD8þ T cells can be divided into CD8þCD28þ and CD8þCD28 T cells, according to the expression of CD28. Under the stimulation of antigen, CD8þCD28þ T cells can be differentiated into specific CTLs, participating in viral clearance; while for CD8þCD28 T cells, a kind of cells with the function of immune inhibition, may suppress antigen presenting function of APC and inhibit the activation and amplification of T cells indirectly. Chronic viral infection is often accompanied by CD8þCD28þ T cells declined and CD8þCD28 T cells increased [17]. CD8þ T cells with no expression of CD28 cannot be differentiated into HBV-specific CTL under the stimulation of HBV antigen, resulting in failure of HBV clearance [18,19]. Activation of CD28/CD80 pathway can regulate the function of NKT cells and also inhibite HBV replication [20]. Recent findings characterized that inactivation of T-cells showed sustained expression of inhibitory immunoreceptor [21]. Cellintrinsic negative regulatory pathways, e.g., programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and CD244, have negative impact on Tcell activation, proliferation, and cytokine production, thus promoting viral persistence [22e25]. 2.3. Changes of regulatory T cells in HBV infection CD4þCD25þ regulatory T cell (Treg), a kind of T cell subset with immunosuppressive activity discovered in recent years, is able to inhibit activation and proliferation of regulatory CD4þ or CD8þ T cell. Previous investigation appears that there are four possible mechanisms for Treg to play the role of immunosuppression: (1) Produce inhibitory cytokines, such as transforming growth factor-b (TGF-b), IL-10, IL-35; (2) Cytolytic effect; (3) block metabolism; (4) Regulate maturation and function of dendritic cells (DC) [26]. Additionally, it has also been demonstrated that PD-1 plays a critical role in regulating induced regulatory T (iTreg) cells development and sustaining iTreg cells function. The pathway, consisting of the receptor PD-1 and its ligands, PD-L1/PD-L2, acts as a possible mechanism for maintaining and enhancing the suppressive function of Treg cells [27]. Clinical studies discovered that the number of Treg cells in peripheral blood was higher in patients with chronic HBV infection than that in healthy control or self-limited hepatitis B groups, and higher in HBeAg-positive group than in HBeAg-negative group, suggesting that the increase of Treg cells may be associated with HBV replication and chronic hepatitis B [28e30]. In patients with HBV infection who received antiviral therapy, the number of Treg cells in peripheral blood declined, accompanied by restoration of effector T cell response, which can predict a more satisfactory therapeutic effect [31,32]. In vitro experiments, Treg cells could suppress the amplification of HBV-specific CTL and secretion of cytokines [30,33]. However, the mechanism of Treg on effector cells to inhibit immune response remains to be elucidated. A deep study of biological effects on Treg cells contributes to the understanding of immune regulation and pathogenesis of inflammation, and ultimately developing effective and feasible therapeutic approach for the diseases.
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2.4. Subsets of NK cell and NK-related receptors in HBV infection Large numbers of NK cells exist in normal human liver, taking up approximately 1/3 of hepatic lymphocytes, and play an important role in the initial immune response. After virus infection, NK cell response appears first and activates immune response of the whole cell by interacting with other lymphocytes, such as T cells, B cells, APC, etc. Meanwhile, NK cells can exert antiviral function through natural cytotoxicity and cytokine secretion, not requiring specific antigen stimulation [34]. According to different expression of CD56 and CD16 in cell surface, NK cells can be divided into two vital subsets with different functions. The majority (90%) of NK cells are CD56dim with high expression of CD16, while the remaining 10% are CD56bright and CD16dim/neg. CD56bright cells are capable of regulating immune response and secrete numerous cytokines, such as IFN-g, TNF-a, granulocyte-macrophage colony stimulating factor (GM-CSF), TGFb, IL-3, etc.; CD56dim cells are cytotoxic and exert functions by acting as effector of lysis in natural and antibody-dependent target cells [35]. In acute hepatitis B infection, cytotoxicity of NK cells increases, but in patients with chronic hepatitis B and hepatocellular carcinoma, the number of NK cells declines and activity is inhibited [36]. Two kinds of recognition receptors with opposite function are expressed by NK cells: activation receptors (e.g., NKG2C, NKG2D, NKp30, NKp44, NKp46) and inhibition receptors (e.g., NKG2A, KIR2DL3, KIR3DL1, CD158a, CD158b) [36]. The function of NK cells is regulated by NK cell receptors (NKR) and inhibition receptors can weaken or even block the activation of NK cells. The activation receptor NKG2D is expressed by NK cells and CD8þ T cells, ligand of which can activate NK cells directly and acts as co-stimulator of CD8þ T cells, promoting immune response of HBV-specific CD8þ T cells. It has been shown in study that compared with healthy controls, CD3CD56þNKG2Dþ, CD3CD56þNKp30þ NK cells and the proportion of their activation receptors were significantly increased in patients with HBV infection [36], the proportion of NKG2Cþ NK cells increased [37], the number of CD56þNKG2Aþ NK cells was significantly declined, and the counts of CD3CD56þ or CD3CD56dimNKG2Aþ NK cells in liver were negatively correlated with viral roads [38]. Therefore, subsets of NK cells and NK cell receptor expression may reflect host immune status in patients with HBV infection, disease progression and predict effectiveness of antiviral therapy. 2.5. Dendritic cells, subsets and activation in HBV infection Dendritic cells (DC) are considered as “professional” antigen presenting cells to initiate primary immune responses, combining innate and adaptive immunity. DC plays an important role in activation of CD8þ CTL and CD4þ Th cells. Bioactive cytokines released by DC can stimulate NK cells, induce Th1 to produce cytokines and promote the proliferation of CTL. At least two DC subsets have been found in human and mouse peripheral blood, named precursor DC1 and DC2 (pDC1 and pDC2). pDC1, also called myeloid DC (mDC) or antigen presenting cells type 1, express CD11c and CD1a, mainly correlated with antigen ingestion and activation of naive T cells. pDC2, also known as plasma celllike DC or interferon producing cell (IPC), express CD123, CD4, MHC-Ⅱ molecules and BDCA-2 antigen. During different stages of infection in patients with hepatitis B, the frequency and quantity of DC were declined significantly, with maturation disorder and function defects, resulting in inadequate effective immune response to clear virus [39,40]. pDC2 is more sensitive than pDC1 when evaluating disease progress in patients with hepatitis B, with pDC2 declined in the early stage of infection and pDC1 declined later. After receiving adefovir therapy, peripheral pDC1 in patients
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with HBV infection increased, indicating a sign of effective response [41,42]. Effective T cell activation requires not only antigen specific signals mediated by TCR, but also co-stimulatory signals provided by APC (e.g., CD28-B7). The B7 family provides important signals both for stimulation and inhibition of T cell activation. B7-1 (also called CD80) and B7-2 (also called CD86), expressed in APC, deliver co-stimulatory signals to naive T cells by CD28 and deliver co-inhibitory signals by cytotoxic T lymphocyte associated antigen 4 (CTLA-4) expressed in activated T cells. CD80 signals promote the maturation of Th1 and CD86 signals promote the maturation of Th2. Immature DC hardly express CD40, CD80 and CD86, while co-stimulatory molecules (CD80 and CD86) are highly expressed on surface of mature DC. Compared with healthy control, the expression of CD80 and CD86 in patients with hepatitis B declined significantly [41], suggesting that chronic HBV infection could lead to DC function descended and T cell activation inhibited. Therefore, the quantity and dynamic changes of DC are closely related to immune status and reactivity of antiviral therapy. DC may become a valid symbol in assessment of disease progress and efficacy of antiviral therapy. 3. Importance of clinically monitoring changes of host cellular immunity in patients with hepatitis B The pathogenisis and immune mechanism of HBV infection have been widely researched in recent years. It has been shown in study that, compared with healthy control, cellular immunity in patients with hepatitis B was disordered, with T cell subsets imbalanced, percentage of CD4þ T cells decreased, CD8þ T cells increased, CD4/ CD8 declined, number of Treg cells expanded, and DC function impaired [3,28,40], all of which may be associated with chronic HBV infection. Clinical study showed that HBV-specific cellular immunity was closely related to virus clearance and disease modification. Antiviral therapy can activate virus specific CTL in peripheral blood in patients with hepatitis B, with its number increased [43e45]. During the period of lamivudine therapy in patients with chronic hepatitis B, accompanied by HBV DNA and HBeAg declined in different levels, the number and ability to kill target cells of specific CTL in peripheral blood were significantly increased than that before treatment, with HBV DNA declined synchronously. Similarly, adefovir therapy can also lead to Treg cells decreased by virus inhibition, proliferation of HBV-specific T cells, production of IFN-g, and HBV-specific CD4þ T cells increased [31,32,46e48], suggesting that effective antiviral therapy can promote function recovery of effector T cells. Our study discovered that, with rapid decline of serum virus load, CD4þ T cells and the ratio of CD4þ/CD8þ in peripheral blood increased in complete response group of telbivudine antiviral treatment, and the ability of HBV-specific CTL to secrete IFN-g enhanced, indicating a significant correlation between them. The number of specific CTL in peripheral blood and the ability to secrete IFN-g were significantly higher in complete response group than that in partial response and no response group [49]. Although antiviral therapy is currently accepted as the key to treatment of chronic hepatitis B, virus clearance depends on immune status ultimately. To restore and rebuild immune function after antiviral therapy in patients with hepatitis B is critical in obtaining satisfactory results. Therefore, it is of great importance to explore cellular immune response and apply in disease progress monitoring and antiviral efficacy prediction. 4. Summary and perspective With deep research of immune mechanism in hepatitis B, the immune regulatory network composed by a variety of active
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immune cells will be more clearly. In the future, the application of immune cells activation in clinical will be more popular, providing new evidence and strategies in judging immune status, monitoring disease progression, predicting antiviral efficacy for patients with HBV infection, and thus to strengthen prevention and treatment of hepatitis B. Acknowledgments
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This paper was supported in part by a grant from the National Natural Science Foundation of China (No. 81171565), Zhejiang Provincial Natural Science Foundation of China (No. LY14H030001) and the Major national S&T Projects for infectious diseases (2012ZX10002002).
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Contents lists available at ScienceDirect
Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Review
Cellular immune response in patients with chronic hepatitis B virus infection Xuefen Li, Yiyin Wang, Yu Chen* State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, School of Medicine, Zhejiang University, 79 Qingchun Road, Hangzhou 310003, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 18 April 2014 Received in revised form 30 July 2014 Accepted 31 July 2014 Available online 12 August 2014
Hepatitis, cirrhosis and hepatocellular carcinoma caused by hepatitis B virus (HBV) infection has threatening human health seriously. As HBV is a kind of non-cytotoxic virus, host immune response plays a vital role in pathogenesis and clinical outcomes of hepatitis B. Multiple immune cells (e.g., including cytotoxic T lymphocytes, regulatory T cells, natural killer cells, dendritic cells) are important in the immune regulation of HBV infection. Therefore, focusing on the activation states of immune cells may provide new evidences and strategies for determining immune status of HBV infectors, monitoring progression of diseases and predicting efficacy of antiviral treatment. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Hepatitis B Cellular immune response Clinical implications
1. Introduction Although effective vaccine against hepatitis B virus (HBV) infection has been developed in the 1980s, around 1/3 of the population worldwide still show a previous or existing HBV infection in serological tests [1]. Different types of clinical outcomes may appear after infection with HBV: for some people, virus can be cleared quickly, no symptoms or only short-term self-limited acute inflammation of liver can be presented; while more can be seen clinically is that patient himself cannot clear the virus effectively and progress to chronic hepatitis. Currently, it is widely recognized that HBV does not damage liver cells directly [2]. Actually, liver inflammation caused by HBV infection is a kind of immune pathological response mainly mediated by multiple immune cells, during which virus cleared and liver cells damaged meanwhile. The status of immune response plays an important role in pathogenesis and clinical outcomes of hepatitis B. At the same time, host immune responses in patients with hepatitis B affect the effectiveness of antiviral therapy greatly [3e5]. The various clinical presentations of HBV infection as well as the pathogenesis of liver diseases are immune-mediated and thus determined by the virusehost interaction [6e8]. It has been established that innate immune responses (involve macrophages and dendritic cells (DC) as well as natural killer (NK)
* Corresponding author. Tel.: þ86 571 87236382; fax: þ86 571 87236383. E-mail addresses: [email protected], [email protected] (Y. Chen). http://dx.doi.org/10.1016/j.micpath.2014.07.010 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
cells) are the host's first defense mechanism against viral infections, and favor a timely and efficient induction of virus-specific adaptive responses in a self-limited, transient liver disease [6,9]. However, a study performed on chimpanzees showed that HBV does not seem to induce a strong innate immune response in the liver, and HBV clearance was associated with a strong adaptive immune response [10]. In human, previous studies also appears that patients with self-resolving hepatitis B have confirmed an induction of NK and NKT cell responses before the onset of adaptive T cell responses [11]. Subsequently, a robust activation of HBVspecific adaptive immune responses, including CD4þ T helper cells, cytotoxic T lymphocyte (CTL) and B cells, especially HBVspecific CTL response, play central roles to clear the virus [6,12]. Acute hepatitis B have shown vigorous immune response against HBV infection, and there is a relative clear correlation between timing, strength and specificity of the cellular immune response and the outcome of self-limited infection [7,12]. In contrast to acute hepatitis B, studies on patients with chronic HBV infection indicated that HBV-specific T-cell reactivity is weak and hard to detect and showed poor immune response to viral control [6,13,14]. It is still unclear why the human immune system fails during chronic HBV infection and how can early antiviral therapy affects cellular immune responses during sustained treatment. In this review, we will mainly addressed the most recent information regarding the cellular immune status in patients with chronic HBV infection and its significance in clinical monitoring, from the perspective of changes in multiple active immune cells of hepatitis B.
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2. Characteristics of active immune cells in patients with HBV infection 2.1. HBV-specific cytotoxic T lymphocyte (CTLs) responses After infected with HBV, anti-HBV specific cellular immune response can be induced through activation of CD4þ and CD8þ T cells. It is widely accepted that there are three kinds of ways for antigen specific T cells to stay in liver: (1) As there is no further stimulation of specific antigen, T cells are cleared quickly in the liver, with no significant liver damage, such as late stage of acute HBV infection; (2) T cells are stranded in the liver while functions are inhibited, causing low immune response or tolerance, such as chronic hepatitis B; (3) T cells are activated excessively in the liver and cause further activation of natural killer (NK) cells and other nonspecific immune cells, resulting in liver cells damage, such as chronic active hepatitis B and severe hepatitis. During the process of clearing HBV in the liver by immune system, HBV specific cytotoxic T lymphocytes (CTLs) play a key role in virus clearance and liver damage. Specific CD8þ T cells are the primary effector cells to clear the virus and CTLs are effective stage as well as main subsets of CD8þ T cell. Specific CD8þ T cells recognize molecular antigen through major histocompatibility complex I (MHC-I) and clear the virus through cytotoxic and non-cytotoxic mechanism: CD8þ T cells recognize HBV short peptide presented by HLA I class molecule in target cell surface specifically, which can induce apoptosis of hepatocytes infected with HBV after being activated; release perforin and crack target cells; secrete cytokines, such as interferon-g (IFN-g), tumor necrosis factor-a (TNF-a), etc., and inhibit HBV replication through non-cytolytic mechanism or attract inflammatory cells without antigen specificity, leading to liver cells necrosis. From the study of transgenic mouse model, it is found that antiviral effect of CTL is mainly mediated by non-cytotoxic mechanism, e.g., clear the virus without damaging liver cells infected with HBV by secreting IFNg, TNF-a and other cytokines, among which IFN-g plays a crucial role [4,5,13e15]. CD4þ T cells are not directly involved in viral clearance and tissue damage, but promote CD8þ T cells to release IFN-g and thereby control HBV replication indirectly through secretion of cytokines interleukin-2 (IL-2). At the same time, CD4þ T cells are able to assist B cells to produce antibodies. Lack of CD4þ T cells would weaken activity of CD8þ T cells and inhibit the production of antibodies [13,14,16]. The control of HBV infection is closely related to specific T cells response. Study has shown that in patients with acute selflimited viral hepatitis, HBV-specific CTL in peripheral blood always present to be polyclonal and multispecific, with strong response and high frequency, and identify a wide range of antigen targets. HBV-specific CD8þ T cells and CD4þ T cells emerge in early incubation period and begin to inhibit viral replication, during which specific cellular immune plays a major role. Furthermore, a large number of CD8þ T cells exist in peripheral circulation and are able to complement the consumption of specific CD8þ T cells in the liver, and thereby clear HBV strongly, causing no or only minor liver damage. On the contrary, CTL response is weak or even absent in patients with chronic HBV infection, characterized by monoclonal and monospecific. HBVspecific helper T cells (Th) and CTL response were significantly lower in patients with persistent HBV infection than that in recovering patients of acute hepatitis B [14]. Therefore, the outcome of hepatitis B infection mainly depends on cellular immune response. Low number and function defects of HBV specific CTL may be the key factor to HBV persistent infection and the development of chronic hepatitis B.
2.2. Activation status of CTL in HBV infection CTL activation is of great importance in HBV infection. The activation of CTL requires not only the first signal delivered by binding antigen MHC molecule complex and T cell receptor (TCR), but also combination of CD28 in T cell surface and CD80/86 from B7 molecule family of antigen presenting cell (APC) to provide the second signal. Therefore, CD28 molecule plays an important role in the activation of T cell immune response. CD28, expressed on the surface of T cells, is an important moleculer marker of T cells. CD8þ T cells can be divided into CD8þCD28þ and CD8þCD28 T cells, according to the expression of CD28. Under the stimulation of antigen, CD8þCD28þ T cells can be differentiated into specific CTLs, participating in viral clearance; while for CD8þCD28 T cells, a kind of cells with the function of immune inhibition, may suppress antigen presenting function of APC and inhibit the activation and amplification of T cells indirectly. Chronic viral infection is often accompanied by CD8þCD28þ T cells declined and CD8þCD28 T cells increased [17]. CD8þ T cells with no expression of CD28 cannot be differentiated into HBV-specific CTL under the stimulation of HBV antigen, resulting in failure of HBV clearance [18,19]. Activation of CD28/CD80 pathway can regulate the function of NKT cells and also inhibite HBV replication [20]. Recent findings characterized that inactivation of T-cells showed sustained expression of inhibitory immunoreceptor [21]. Cellintrinsic negative regulatory pathways, e.g., programmed death 1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), lymphocyte activation gene-3 (LAG-3), and CD244, have negative impact on Tcell activation, proliferation, and cytokine production, thus promoting viral persistence [22e25]. 2.3. Changes of regulatory T cells in HBV infection CD4þCD25þ regulatory T cell (Treg), a kind of T cell subset with immunosuppressive activity discovered in recent years, is able to inhibit activation and proliferation of regulatory CD4þ or CD8þ T cell. Previous investigation appears that there are four possible mechanisms for Treg to play the role of immunosuppression: (1) Produce inhibitory cytokines, such as transforming growth factor-b (TGF-b), IL-10, IL-35; (2) Cytolytic effect; (3) block metabolism; (4) Regulate maturation and function of dendritic cells (DC) [26]. Additionally, it has also been demonstrated that PD-1 plays a critical role in regulating induced regulatory T (iTreg) cells development and sustaining iTreg cells function. The pathway, consisting of the receptor PD-1 and its ligands, PD-L1/PD-L2, acts as a possible mechanism for maintaining and enhancing the suppressive function of Treg cells [27]. Clinical studies discovered that the number of Treg cells in peripheral blood was higher in patients with chronic HBV infection than that in healthy control or self-limited hepatitis B groups, and higher in HBeAg-positive group than in HBeAg-negative group, suggesting that the increase of Treg cells may be associated with HBV replication and chronic hepatitis B [28e30]. In patients with HBV infection who received antiviral therapy, the number of Treg cells in peripheral blood declined, accompanied by restoration of effector T cell response, which can predict a more satisfactory therapeutic effect [31,32]. In vitro experiments, Treg cells could suppress the amplification of HBV-specific CTL and secretion of cytokines [30,33]. However, the mechanism of Treg on effector cells to inhibit immune response remains to be elucidated. A deep study of biological effects on Treg cells contributes to the understanding of immune regulation and pathogenesis of inflammation, and ultimately developing effective and feasible therapeutic approach for the diseases.
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2.4. Subsets of NK cell and NK-related receptors in HBV infection Large numbers of NK cells exist in normal human liver, taking up approximately 1/3 of hepatic lymphocytes, and play an important role in the initial immune response. After virus infection, NK cell response appears first and activates immune response of the whole cell by interacting with other lymphocytes, such as T cells, B cells, APC, etc. Meanwhile, NK cells can exert antiviral function through natural cytotoxicity and cytokine secretion, not requiring specific antigen stimulation [34]. According to different expression of CD56 and CD16 in cell surface, NK cells can be divided into two vital subsets with different functions. The majority (90%) of NK cells are CD56dim with high expression of CD16, while the remaining 10% are CD56bright and CD16dim/neg. CD56bright cells are capable of regulating immune response and secrete numerous cytokines, such as IFN-g, TNF-a, granulocyte-macrophage colony stimulating factor (GM-CSF), TGFb, IL-3, etc.; CD56dim cells are cytotoxic and exert functions by acting as effector of lysis in natural and antibody-dependent target cells [35]. In acute hepatitis B infection, cytotoxicity of NK cells increases, but in patients with chronic hepatitis B and hepatocellular carcinoma, the number of NK cells declines and activity is inhibited [36]. Two kinds of recognition receptors with opposite function are expressed by NK cells: activation receptors (e.g., NKG2C, NKG2D, NKp30, NKp44, NKp46) and inhibition receptors (e.g., NKG2A, KIR2DL3, KIR3DL1, CD158a, CD158b) [36]. The function of NK cells is regulated by NK cell receptors (NKR) and inhibition receptors can weaken or even block the activation of NK cells. The activation receptor NKG2D is expressed by NK cells and CD8þ T cells, ligand of which can activate NK cells directly and acts as co-stimulator of CD8þ T cells, promoting immune response of HBV-specific CD8þ T cells. It has been shown in study that compared with healthy controls, CD3CD56þNKG2Dþ, CD3CD56þNKp30þ NK cells and the proportion of their activation receptors were significantly increased in patients with HBV infection [36], the proportion of NKG2Cþ NK cells increased [37], the number of CD56þNKG2Aþ NK cells was significantly declined, and the counts of CD3CD56þ or CD3CD56dimNKG2Aþ NK cells in liver were negatively correlated with viral roads [38]. Therefore, subsets of NK cells and NK cell receptor expression may reflect host immune status in patients with HBV infection, disease progression and predict effectiveness of antiviral therapy. 2.5. Dendritic cells, subsets and activation in HBV infection Dendritic cells (DC) are considered as “professional” antigen presenting cells to initiate primary immune responses, combining innate and adaptive immunity. DC plays an important role in activation of CD8þ CTL and CD4þ Th cells. Bioactive cytokines released by DC can stimulate NK cells, induce Th1 to produce cytokines and promote the proliferation of CTL. At least two DC subsets have been found in human and mouse peripheral blood, named precursor DC1 and DC2 (pDC1 and pDC2). pDC1, also called myeloid DC (mDC) or antigen presenting cells type 1, express CD11c and CD1a, mainly correlated with antigen ingestion and activation of naive T cells. pDC2, also known as plasma celllike DC or interferon producing cell (IPC), express CD123, CD4, MHC-Ⅱ molecules and BDCA-2 antigen. During different stages of infection in patients with hepatitis B, the frequency and quantity of DC were declined significantly, with maturation disorder and function defects, resulting in inadequate effective immune response to clear virus [39,40]. pDC2 is more sensitive than pDC1 when evaluating disease progress in patients with hepatitis B, with pDC2 declined in the early stage of infection and pDC1 declined later. After receiving adefovir therapy, peripheral pDC1 in patients
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with HBV infection increased, indicating a sign of effective response [41,42]. Effective T cell activation requires not only antigen specific signals mediated by TCR, but also co-stimulatory signals provided by APC (e.g., CD28-B7). The B7 family provides important signals both for stimulation and inhibition of T cell activation. B7-1 (also called CD80) and B7-2 (also called CD86), expressed in APC, deliver co-stimulatory signals to naive T cells by CD28 and deliver co-inhibitory signals by cytotoxic T lymphocyte associated antigen 4 (CTLA-4) expressed in activated T cells. CD80 signals promote the maturation of Th1 and CD86 signals promote the maturation of Th2. Immature DC hardly express CD40, CD80 and CD86, while co-stimulatory molecules (CD80 and CD86) are highly expressed on surface of mature DC. Compared with healthy control, the expression of CD80 and CD86 in patients with hepatitis B declined significantly [41], suggesting that chronic HBV infection could lead to DC function descended and T cell activation inhibited. Therefore, the quantity and dynamic changes of DC are closely related to immune status and reactivity of antiviral therapy. DC may become a valid symbol in assessment of disease progress and efficacy of antiviral therapy. 3. Importance of clinically monitoring changes of host cellular immunity in patients with hepatitis B The pathogenisis and immune mechanism of HBV infection have been widely researched in recent years. It has been shown in study that, compared with healthy control, cellular immunity in patients with hepatitis B was disordered, with T cell subsets imbalanced, percentage of CD4þ T cells decreased, CD8þ T cells increased, CD4/ CD8 declined, number of Treg cells expanded, and DC function impaired [3,28,40], all of which may be associated with chronic HBV infection. Clinical study showed that HBV-specific cellular immunity was closely related to virus clearance and disease modification. Antiviral therapy can activate virus specific CTL in peripheral blood in patients with hepatitis B, with its number increased [43e45]. During the period of lamivudine therapy in patients with chronic hepatitis B, accompanied by HBV DNA and HBeAg declined in different levels, the number and ability to kill target cells of specific CTL in peripheral blood were significantly increased than that before treatment, with HBV DNA declined synchronously. Similarly, adefovir therapy can also lead to Treg cells decreased by virus inhibition, proliferation of HBV-specific T cells, production of IFN-g, and HBV-specific CD4þ T cells increased [31,32,46e48], suggesting that effective antiviral therapy can promote function recovery of effector T cells. Our study discovered that, with rapid decline of serum virus load, CD4þ T cells and the ratio of CD4þ/CD8þ in peripheral blood increased in complete response group of telbivudine antiviral treatment, and the ability of HBV-specific CTL to secrete IFN-g enhanced, indicating a significant correlation between them. The number of specific CTL in peripheral blood and the ability to secrete IFN-g were significantly higher in complete response group than that in partial response and no response group [49]. Although antiviral therapy is currently accepted as the key to treatment of chronic hepatitis B, virus clearance depends on immune status ultimately. To restore and rebuild immune function after antiviral therapy in patients with hepatitis B is critical in obtaining satisfactory results. Therefore, it is of great importance to explore cellular immune response and apply in disease progress monitoring and antiviral efficacy prediction. 4. Summary and perspective With deep research of immune mechanism in hepatitis B, the immune regulatory network composed by a variety of active
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immune cells will be more clearly. In the future, the application of immune cells activation in clinical will be more popular, providing new evidence and strategies in judging immune status, monitoring disease progression, predicting antiviral efficacy for patients with HBV infection, and thus to strengthen prevention and treatment of hepatitis B. Acknowledgments
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This paper was supported in part by a grant from the National Natural Science Foundation of China (No. 81171565), Zhejiang Provincial Natural Science Foundation of China (No. LY14H030001) and the Major national S&T Projects for infectious diseases (2012ZX10002002).
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