To assess the levels of different immune modulators in patients with hepatocellular carcinoma (HCC), in relation to other hepatic diseases.

Eighty-eight patients were included in the current study and represented patients with HCC (20), liver cirrhosis (28) and chronic hepatitis (CH; 25), and normal controls (NC; 15). Peripheral blood was isolated for immunophenotyping of active myeloid dendritic cells (mDCs; CD1c and CD40), mature inactive myeloid cells (CD1c and HLA), active plasmacytoid cells (pDCs; CD303 and CD40), mature inactive pDCs (CD30 and HLA), active natural killer (NK) cells (CD56 and CD161), active NK cells (CD56 and CD314) and inactive NK cells (CD56 and CD158) was done by flow cytometry. Serum levels of interleukin (IL)-2, IL-10, IL-12, IL-1β, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-αR2 were assessed by ELISA.

Active mDCs (CD1C+/CD40+) and inactive mDCs (CD1c+/HLA+) were significantly decreased in HCC patients in relation to NC (P < 0.001). CD40+ expression on active pDCs was decreased in HCC patients (P < 0.001), and its level was not significantly changed among other groups. Inactive pDCs (CD303+/HLA+), inactive NKs (CD56+/CD158+) and active NKs (CD56+/CD161+) were not statistically changed among the four groups studied; however, the latter was increased in CH (P < 0.05). NKG2D was statistically decreased in HCC, CH and cirrhosis (P < 0.001), and it was not expressed in 63% (12/20) of HCC patients. There was significant decrease of IL-2, IFN-α and IFN-γ (P < 0.001), and a significant increase in IL-10, IL-1β, and TNF-αR2 (P <0.01, P < 0.001 and P < 0.001; respectively) in HCC patients. There was inverted correlation between IL-12 and IL-1β in HCC (r = -0.565, P < 0.01), with a strong correlation between pDCs (CD303+/CD40+) and NKs (CD56+/CD161+; r = 0.512, P < 0.05) as well as inactive mDCs (CD1c+/HLA+) and inactive NK cells (CD56+/CD158+; r = 0.945, P < 0.001).

NKG2D, CD40, IL-2 and IL-10 are important modulators in the development and progression of HCC.

Hepatocellular carcinoma (HCC) is a major health problem worldwide. It is the sixth most common cancer and the second leading cause of cancer-related death in the world[1]. In Egypt, HCC ranks the first among cancers in males (33.6%), and the 2^nd in females after breast cancer (13.5%)[2]. This high incidence is attributed to the high prevalence of hepatitis C virus (HCV) infection, especially of genotype IV, in Egypt[3]. Despite advances in treatment modalities, sorafenib is still the only treatment approved by the Food and Drug Administration (FDA) for HCC; however, it extends the overall survival by 3 mo only. Hence, it is crucial to understand the underlying biological and immunological changes in HCC and to develop new treatment modalities based on these data[4,5].

The body’s immune defense mechanism(s) plays an important role in the inhibition or progression of cancer. However, tumors can escape immune surveillance by producing a local and/or systemic immune-suppressive environment[6]. Although the underlying molecular mechanisms are not yet fully clear, recent studies show that the immune response in cancer patients is usually down-regulated by immunosuppressive cells, mainly T regulatory cells (Tregs) and myeloid-derived suppressor cells (MDSCs), which suppress the immune system and promote immunologic tolerance[7]. These inhibitory cells accumulate during advanced cancer stages. They are involved in chronic inflammation and tumor progression and they can inhibit many immune cells (e.g., CD4+, CD8+, natural killer (NK) cells)[8]. They also secrete many immune-suppressive cytokines, such as interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-β, creating a tolerogenic and suppressive environment[9].

The NK cells represent 25%-30% of the human liver lymphocytes, compared to 10%-20% of the total peripheral blood lymphocytes[10]. They are the main effector cells of innate immunity, which play an important role in tumor surveillance. The NK cells achieve their functions through the release of cytolytic mediators, such as perforin and granzymes, or the induction of apoptosis via the expression of tumor necrosis factor (TNF) ligands and interferon (IFN)-γ[11]. Activation of NK cells is strictly regulated by a balance between activating and inhibitory signals, whereas the inhibitory signals are mainly induced by receptors for MHC class I molecules (KIRs, CD94/NKG2A). The activating signals are mainly achieved through NKG2D, a C-type lectin-like receptor which is expressed on NK cells, γδ T cells and CD8+ T cells[12,13]. NKG2D is responsible for detection and elimination of transformed cells via binding to MICA (MHC class I–related chain), MICB and the UL16-binding proteins[14]. Activation of NKG2D provides unique costimulation to antigen-specific CD8 T cells that is non-redundant to CD28 costimulation[15].

Dendritic cells (DCs) are the most professional antigen-presenting cells which respond rapidly to microenvironment signals. Upon maturation by tumor antigen, cross-priming to T and B lymphocytes occurs to produce antitumor adaptive immune responses[16]. There are two subsets of DCs: the myeloid dendritic cells (mDCs), which are characterized by their ability to produce IL-12; and, the plasmacytoid cells (pDCs), which are responsible for the production of over 95% of type-I IFNs in response to viral infection[17]. On activation, the CD8+ cytotoxic T cells (CTLs) eliminate tumor cells by the production of IFN-γ, whereas CD4+ T helper cells stimulate B cells to support the cytotoxic and humoral immune response[18].

IL-1β is a regulatory cytokine produced by tumor and immune cells, such as MDSCs. It also induces COX-2 expression, which prevents maturation and activation of antigen presenting cells at the tumor site[19].

In the current study, we assessed the role(s) of different immune regulatory cells and cytokines in the development and progression of chronic liver disease (chronic active hepatitis, cirrhosis and HCC) compared to the normal healthy volunteers. We believe that this could allow for better understanding of different pathways implicated in the development and progression of hepatocarcinogenesis, and could also help in designing new immune-therapeutic drugs.

Identification of inflammatory mediators which promote or prevent the progression of cancer depends on the tumor microenvironment. Thus, this study was designed to assess the levels of different immune modulators and cytokines in patients with HCC, nonmalignant hepatic diseases (e.g., CH and liver cirrhosis) compared to a normal group. This would enable us to study different pathways that help in identifying immune cells or biomarkers that may be implicated in hepatocellular carcinogenesis. This may also explain why liver disease progresses more rapidly in some patients than in others and provides additional therapeutic modalities for viral infections and HCC (immunotherapy).

One of the most powerful antigen presenting cells is the DC, which plays an important role in antitumor immune response. Our data revealed that active and inactive mDCs were significantly decreased in HCC patients compared to the NC group (P < 0.001); however, the level of active mDCs (CD1C+/CD40+) was significantly decreased in cirrhosis compared to CH patients (P = 0.003). These data are consistent with Obermajer et al[20], who reported that the inhibition of DC maturation in HCC may prove to be a critical feature of tumor escape, as well as with another two recent studies which reported numerical and functional defects in the peripheral DCs in hepatitis B- and C-associated HCC patients[21,22].

The pDC (CD303+, CD40+) represents the major cell responsible for antiviral immunity, and the main source of IFN-α in the body. Our data revealed that the expression level of CD40+ on active pDCs was significantly decreased in HCC patients compared to NC (P < 0.001). Meanwhile, inactive pDCs (CD303+/HLA+) did not change significantly among the four groups studied. This was also confirmed by the significant decrease of serum IFN-α, and the increase of IL-10 in HCC patients compared to the NC group (P < 0.001 and P < 0.01 respectively). This is in concordance with previous reports showing that IL-10 inhibits IFN-α production and promotes apoptosis of human pDCs[23,24]. Moreover, Gonzalez-Carmona et al[25] demonstrated that cancer patients had low CD40 expression on DCs or CD40L on T cells, which is associated with an impaired immune response, and Shurin et al[26] reported that the tumor-derived IL-10 inhibits CD40 expression on DCs and DC precursors and suppresses their maturation and function.

NK cells play an important role in controlling viral hepatitis, liver fibrosis and carcinogenesis. Their functions are modulated by different classes of receptors present on its surface[27]. Thus, it is essential to identify the roles of these cells in different stages of HCC development. Among these, we investigated the role of the killer cell immunoglobulin-like receptors (KIR; CD158), NKR-P1A (CD161) and NKG2D (CD314) in HCV CH patients, and in liver cirrhosis and its progression to HCC.

Our results revealed that the expression of the inhibitory KIR (CD158+) receptors and the activating NKR-P1A (CD161) on NK cells was not statistically changed between HCC and NC (P = 0.827 and P = 0.788 respectively). Thus, these two pathways may not be involved in the pathogenesis of HCC. However, the latter were statistically increased in CH (P < 0.05). This result was supported by our finding of a strong statistical correlation between the expression of CD161 on NK cells and IL-12 (r = 0.77, P < 0.001) in patients with HCV chronic active hepatitis. This illustrates the role of NK cells (CD56+/CD161+) in viral hepatitis.

On the other hand, the level of active NK cells expressing NKG2D were statistically decreased in HCC, CH and cirrhosis in relation to the NC group (P < 0.001), which could indicate the implication of these cells in the progression of HCC. This was clarified by representing its expression in each patient, it was not expressed in 63% (12/20) of HCC patients, emphasizing its important role in HCC development. These data were consistent with Lanier et al[28]. Yoon et al[29] reported that direct interaction between human NK cells and HCV-infected hepatoma cells down-regulates NK cell NKG2D expression and effector function with diminished IFN-γ production. Another study carried out by Kamiya et al[30] demonstrated that NKG2D was an important mediator of antiHCC activity. On the contrary, Oliviero et al[31] found a decreased percentage of NK cells expressing the inhibitory receptor KIR3DL1 and a concomitant increase in the proportion of NKG2D⁺ NK cells in Italian patients with chronic HCV. This could be explained by racial and environmental differences with different viral genotypes, and also according to the clinical stage.

Our results regarding DCs and NK cells were supported by assessment of the cytokine serum levels in the four groups studied. We found that there was a significant increase in IL-1β (P < 0.001) in HCC patients compared to the NC group. However, there was no statistically significant change in the serum level of IL-12 (P = 0.393) among the four groups studied. Furthermore, we had a significant increase in the serum level of IL-10 in the cirrhosis and HCC groups compared to the NC group. This is consistent with Accapezzato et al[32]. IL-10 is important for allowing liver NK cells to maintain immune-tolerant states[33]. Its significant increase in HCC patients is responsible for the abnormal tolerant NK cells that cannot eliminate infected or transformed cells, which leads to immune evasion.

In the current study, there was a significant decrease of the serum levels of IFN-γ in chronic HCV hepatitis, cirrhosis and HCC patients in comparison to NC (P < 0.001). This is in agreement with Li et al[34], who found that NK cells cultured with cancer-associated fibroblasts from HCC (H-CAFs) down-regulate NKG2D and NKp46 and decrease expression of granzyme B, perforin and INF-γ. Later, another study showed that TGF-β and IL-10 in hepatic carcinoma patients induce the expression of microRNA (miR)-146a, which causes reduced IFN-γ production and cytotoxicity, resulting in a poorer prognosis[35]. Meanwhile, Zhang et al[36] found that MDSCs selectively suppressed the IFN-γ production deriving from NKT cells through membrane-bound TGF-β.

IL-2 plays a critical role in activation of the immune system, which could be used to eradicate cancer. It has been demonstrated as a monotherapy, and was approved for metastatic renal cell carcinoma and metastatic melanoma by the FDA[37]. Our results showed a significant increase in TNF-αR2 (P < 0.001) and significant decrease in IL-2 (P < 0.001) in HCC patients in relation to other groups, consistent with our previous results[38,39] that concluded the possible use of serum TNFR-II, IL-2Ra and IL-8 as combined biomarkers in HCV-infected patients at high risk of developing HCC.

Another important finding in the current study was that HCC patients had a significant decrease of active NK cells (CD56+/CD314+; P < 0.05) and active pDCs (CD303+, CD40+; P < 0.05) in comparison to inactive NK cells (CD56+/CD158+) and inactive pDCs (CD303+/HLA+) respectively. NK cells expressing CD56+/CD161+ and mDCs were not statistically affected. This revealed the importance of these two subsets of cells [active NK cells (CD56+/CD314+), and active pDCs (CD303+, CD40+)] in the pathogenesis of HCC, and allows for further research around the state of this imbalance that emerges in HCC patients.

We conclude that there are immunological changes occurring in HCC patients which could be possible candidate(s) for future immunotherapy for these patients. Among these are the NK cells expressing NKG2D and pDCs expressing CD40, IL-2 and IL-10. These factors could be implicated in the pathogenesis of HCC, and provide an attractive target for therapeutics in chronic HCV hepatitis and liver cancer.