In this study, we show that the presence of tumor-infiltrating CD4+CD25+ T cells are typical Tregs based on their CD4+CD25+FoxP3highCD45RO+CTLA-4+ phenotype and suppressive functions. We also found that FoxP3+ Tregs were concentrated within HCC tumors, and that the intratumoral prevalence of FoxP3+ Tregs was associated with disease progression and poor prognosis. More importantly, we revealed that the CCL20-CCR6 axis controls the migration of circulating Tregs into tumor, resulting in the increased frequency and quantity of Tregs in tumor tissue.
It is reported that CD4+
T cells (Tregs) constitute 5–10% of peripheral CD4+
T cells in healthy people 
. Consistent with these reports, the CD4+
T cells counted for 6.7±0.7% of CD4+
T cells in healthy donor based on our gating strategy. Importantly, we observed a substantial increase of tumor-infiltrating CD4+
Tregs in both frequency and quantity, in agreement with previous findings 
. FoxP3 is a master regulator of Tregs for their development, maintenance and function 
. Here, we found that the level of FoxP3 was higher in tumor-derived Tregs. However, Tregs from HCC patients showed similar immunosuppressive effects, as CD4+
T cells from either TIL, NIL or cPBMC similarly inhibited the proliferation and INF-γ production of autologous CD4+
T cells. These data suggested that the prevalence, rather than the superior suppressive activity, of Tregs in the tumor microenviroment results in reduced tumor-specific immunity 
. In addition, we showed that, in contrast to paired blood Tregs, tumor-infiltrating Tregs display an activated phenotype, as they expressed elevated levels of CD69 and HLA-DR. Only ~30% of Tregs expressed low level of HLA-DR in patient peripheral blood, while the majority of tumor-derived Tregs highly expressed HLA-DR, which identifies a terminally differentiated subpopulation of effector Tregs 
. Therefore, this means that most of Tregs are terminally differentiated ones in tumor microenvironment.
The high number of tumor-infiltrating in the liver tumor environment raises the question of their recruitment. Over the past decade, CCR4 and its ligands have been demonstrated to play critical role in recruiting circulating Tregs into tumor tissue. Circulating Tregs have been revealed to express high CCR4 levels and to selectively migrate in response to CCR4 ligands produced in the tumor microenvironment 
. However, we observed that, in HCC patients, circulating Tregs highly express CCR6 and migrate to CCL20 present in the tumor microenvironment. This conclusion is based on two findings. First, only the CCR6 ligand CCL20 had elevated expression at both mRNA and protein levels in tumor tissues. Moreover, the CCL20 expression was strong correlated with the number of FoxP3+
Tregs in tumor environment. Second, the circulating Tregs from HCC patients highly expressed CCR6, and selectively migrate in response to CCL20 in vitro. It is worth noting that the Tregs in tumor environment expressed low to even undetectable CCR6. We infer this may result from the strong expression of CCL20 in tumor environment, which inversely internalizes the CCR6 expression like CCR4 
. In the past, CCR6 was mainly implicated to be responsible for the inflammatory recruitment of Tregs. Fox example, CCR6 is essential for the optimal recruitment of Tregs to sites of Th17-mediated inflammation in experimental autoimmune encephalomyelitis (EAE) 
. However, recently, accumulating finding show that the CCR6 expression on Tregs also plays a critical role in tumor development 
. It is well accepted that not only the suppressor potential but also appropriate localization determines the in vivo suppressive capacity of Tregs 
. All these results, together with our data, show an important role of CCR6 in Treg-mediated immunosuppression.
Although they are critical factors to mediate Treg migration into tumors or lymph nodes, CCR4 and CCR7 are at least not essential for migration of circulating Tregs from HCC patients in this study. First, none of the ligands for CCR4 and CCR7 had enhanced expression in tumor environment. Second, although they had much higher expression of CCR4 than CD4+CD25− T cells, the circulating Tregs appeared to have significantly lower frequency of CCR4 than their counterparts in normal controls. Likewise, the expression of CCR7 between CD4+ T subsets was similar and did not fluctuate substantially among groups. Third, chemotaxis assays failed to show selective migration of circulating Tregs from HCC patients to CCL22 and CCL21.
Several studies assessed the association of increased tumor-infiltrating Tregs with clinical characteristics and revealed different results. Tang et al. found that high tumor Treg density was associated with both absence of tumor encapsulation and presence of tumor vascular invasion 
. Another studies revealed that the prevalence of Tregs was correlated with the presence of cirrhosis and later TNM stages 
. We found that increased tumor FoxP3+
Tregs was also correlated with cirrhosis background, but more importantly with poorer tumor differentiation. Though somewhat different, all these results mean Tregs contribute to tumor progression in HCC patients. Meanwhile, elevated tumor-infiltrating Treg number (>6.6/hpf) also predicted a poorer prognosis with shorter disease-free and overall survival, in line with previous reports in liver tumors 
In conclusion, we report here the frequency, phenotype, and trafficking property of Tregs and their correlation with clinicapathologic factors in HCC patients. Especially, we found that CCR6 is a liver-specific determinant for the trafficking of circulating Tregs into tumor. These results extend our understanding of the mechanism of liver carcinogenesis. Thus, apart from depleting Tregs, blocking Treg-cell trafficking into tumor represents a potential strategy for treating human HCC.