Dan-Ping Xu

Elevation of plasma soluble human leukocyte antigen–G in patients with chronic hepatitis C virus infection

The subversion of immune responses that hepatitis C virus (HCV) uses to escape immune surveillance and to establish persistent infection has been poorly understood. The immune-suppressive molecule human leukocyte antigen-G (HLA-G) has been supposed to play important roles in viral infection. In the current study, HCV genotype was analyzed in 67 chronic HCV-infected (CHC) patients. Plasma soluble sHLA-G (including sHLA-G1 and HLA-G5), interleukin-10 (IL-10), and interferon-γ (IFN-γ) levels were determined in these CHC patients and in healthy subjects by enzyme-linked immunosorbent assay, and the sHLA-G isoforms present in plasma were determined by Western blot. Data showed that HCV 1b was the predominant genotype, with a prevalence of 64.2%. sHLA-G was dramatically increased in CHC patients (median: 85.54 U/ml, range: 19.40-204.07) over that in normal controls (median: 9.13 U/ml, range: 5.07-69.56) (p < 0.001). Western blotting revealed that plasma sHLA-G was derived from sHLA-G1 and HLA-G5. IL-10 and IFN-γ levels were also significant higher in CHC patients than in normal controls (median: 16.3 pg/ml vs 1.8 pg/ml, p < 0.001, and 1025.3 pg/ml vs 858.3 pg/ml, p = 0.03, respectively). No significant association was observed for the HCV genotype and viral RNA load with the levels of sHLA-G, IL-10, and IFN-γ in CHC patients. These results indicate that elevation of sHLA-G expression in HCV patients was independent of viral genotype and viral RNA load. Given its immunotolerant property, an increase in sHLA-G may play a role in the persistency of HCV infection.

Human leukocyte antigen‐G expression is associated with a poor prognosis in patients with esophageal squamous cell carcinoma

Human leukocyte antigen (HLA)‐G inhibits functions of immune component cells and promotes malignant cells evading from antitumor immunity. We investigated the clinical relevance of HLA‐G expression in esophageal squamous cell carcinoma (ESCC). In our study, HLA‐G expression in 79 primary ESCC lesions and corresponding adjacent normal tissues were analyzed with immunohistochemistry. Soluble HLA‐G (sHLA‐G) in plasma was detected with enzyme‐linked immunosorbent assay (ELISA) in 41 ESCC patients (including 19 case‐matched lesions and plasma samples) and in 153 normal healthy controls. HLA‐G expression was observed in 65.8% (52/79) of the ESCC lesions but not in adjacent normal esophageal tissues. HLA‐G expression was more frequently observed in patients with advanced disease stage (III/IV vs. I/II, p = 0.01). Patients with HLA‐G expression had a significantly worse survival, and HLA‐G could be an independent prognostic factor. sHLA‐G levels in plasma were significantly increased in patients compared to normal controls (median: 152.4 U/ml vs. 8.9 U/ml, p < 0.001). The area under receiver‐operating characteristic (ROC) curve for sHLA‐G in plasma was 0.992. However, no significant correlation was found between sHLA‐G in plasma and clinical parameters studied. In conclusion, our findings indicated that HLA‐G expression in ESCC is associated with poor survival and could be a prognostic indicator. Furthermore, increased levels of sHLA‐G in plasma might be a useful preoperative biomarker for diagnosis.

Plasma soluble human leukocyte antigen-G expression is a potential clinical biomarker in patients with hepatitis B virus infection.

The significance of upregulated soluble human leukocyte antigen-G (sHLA-G) expression under various pathologic conditions has been discussed. In this study, we evaluated the potential significance of plasma sHLA-G expression in patients with hepatitis B virus (HBV) infection. The study included 90 acute hepatitis B patients (AHB), 131 chronic hepatitis B patients (CHB), 152 resolved hepatitis B individuals (RHB), and 129 normal controls. sHLA-G were determined using enzyme-linked immunosorbent assay. A receiver operating characteristic (ROC) curve was used to evaluate the feasibility of plasma sHLA-G as a biomarker for distinguishing patients with HBV infection. sHLA-G levels in AHB (median, 193.1 U/mL; p < 0.001), CHB (median, 324.6 U/mL; p < 0.001), and RHB (median, 14.8 U/mL; p = 0.006) patients was much higher than that in normal controls (median, 9.0 U/mL). A significant difference for sHLA-G levels was also observed between patients with HBV infection (AHB vs CHB, AHB vs RHB, and CHB vs RHB; all p < 0.001). The area under the ROC curve for sHLA-G levels was 1.000 (p < 0.001) for AHB, 0.993 (p < 0.001) for CHB, and 0.604 (p = 0.003) for RHB patients versus normal controls, respectively. Data also indicated that the percentage of CD4(+)CD25(+)FoxP3(+) T regulatory cells and HLA-G(+)CD14(+) monocytes was significantly increased in AHB and CHB patients compared with normal controls (all p < 0.001). Our findings indicated that induction of HLA-G expression may play a role in HBV immune evasion and sHLA-G levels could be a useful biomarker in HBV infection.

Analysis of the plasma soluble human leukocyte antigen–G and interleukin-10 levels in childhood atopic asthma

Human leukocyte antigen-G (HLA-G) has been hypothesized to be associated with the pathogenesis of asthma; however, results remain controversial. Furthermore, HLA-G expression could be modulated by the HLA-G 14-bp insertion (+)/deletion (-) polymorphism and by interleukin-10. In this study, the 14-bp polymorphism in exon 8 of the HLA-G gene, plasma soluble HLA-G, and interleukin-10 (IL-10) levels in untreated atopic asthmatic children, and in a group of age-, gender-, and ethnicity-matched normal controls were analyzed. Data showed that HLA-G 14-bp +/- polymorphism was not significant difference between the asthmatic patients and normal controls. Plasma soluble human leukocyte antigen (sHLA)-G in atopic asthma patients (n = 72; median, 179.28 U/ml) was dramatically higher compared with that of the normal controls (n = 76; median, 35.23 U/ml; p < 0.001). Receiver operating characteristic (ROC) curve analysis showed that the area under ROC curve for sHLA-G was 0.986 (p < 0.001) in atopic asthma patients versus normal controls. IL-10 levels in the asthmatic children (n = 50; median, 5.02 pg/ml) was significantly lower than that of the normal controls (n = 48; median, 12.82 pg/ml; p < 0.001). Both HLA-G 14-bp polymorphism and IL-10 levels were unrelated to plasma sHLA-G concentration in both groups. Our findings indicated that the HLA-G 14-bp polymorphism was not a risk factor, but that sHLA-G might be considered as a biomarker for the atopic asthmatic patients. Dramatically increased sHLA-G with decreased IL-10 levels may have implications in the pathogenesis of atopic asthma.

Alteration of HLA-F and HLA I antigen expression in the tumor is associated with survival in patients with esophageal squamous cell carcinoma.

Alteration of human leukocyte antigen (HLA) expression, such as decreased HLA I (HLA‐A, ‐B and ‐C) antigens and elevated nonclassical HLA I antigens (HLA‐E, ‐F and ‐G), was reported to have an unfavorable prognosis in various cancers. In our study, HLA‐F expression in 105 primary esophageal squamous cell carcinoma (ESCC) lesions and 62 case‐matched adjacent normal tissues, and HLA I antigens among 68 cases were analyzed by immunohistochemistry. Data revealed that HLA‐F expression was observed in 58.1% (61/105) of the ESCC lesions and in 54.8% (34/62) of the normal esophageal tissues. Among the 62 case‐matched samples, HLA‐F expression (lesion vs. normal tissue) was upregulated, unchanged and downregulated in 13 (21.0%), 6 (9.6%) and 43 (69.4%) cases, respectively. Patients with HLA‐F positive had a worse survival than those with HLA‐F negative (p = 0.040). Patients with upregulated HLA‐F expression (lesion vs. normal tissue) had significantly worse survival than those with HLA‐F unchanged and downregulated (p = 0.010). Furthermore, decreased HLA I expression was observed in 41.2% (28/68) patients and was with worse prognosis in comparison to those with preserved HLA I expression (p = 0.001). Multivariate analysis using Coxs proportional hazards model revealed that upregulated HLA‐F expression (p = 0.026) and downregulated HLA I expression (p = 0.013) could be an independent unfavorable prognostic factor. In conclusion, our study provided the evidence that alteration of HLA I and HLA‐F antigen expression was associated with survival in patients with ESCC.

HLA-G expression is associated with metastasis and poor survival in the Balb/c nu/nu murine tumor model with ovarian cancer

Aberrant HLA‐G expression is associated with tumor invasiveness and poor clinical prognosis; however, there is a lack of preclinical animal model to address whether HLA‐G plays a causal role in the unfavorable prognosis of malignancies. In the current study, ovarian carcinoma cell lines (HO‐8910 and Ovcar‐3) were transfected with HLA‐G gene. HLA‐G expression was analyzed with western blot and flow cytometry. Transwell experiment was performed to analyze the cell migration and invasion capability and/or multicellular spheroid formation was investigated with the 3D culture assay in vitro. The effects of HLA‐G expression for tumor cell organ metastasis and for mouse survival was analyzed with the Balb/c nu/nu mouse model. Our data showed that HO‐8910‐G and Ovcar‐3‐G cells are of higher invasion potential compared with the parental HO‐8910 and Ovcar‐3 cells. Multicellular spheroid formation exists only in HO‐8910‐G cells in a 3D culture assay. In Balb/c nu/nu mouse model, widespread metastasis was observed in mice xenografted with HO‐8910‐G cells, but not in the group with parental cells. Mouse survival was dramatically decreased in HO‐8910‐G and Ovcar‐3‐G xenografted mice than that with HO‐8910 and Ovcar‐3 cells, respectively. In summary, our study provided the first evidence that HLA‐G expression is associated with tumor metastasis and with poor survival in an animal model with ovarian cancer.

Multiple steps of HLA-G in ovarian carcinoma metastasis: Alter NK cytotoxicity and induce matrix metalloproteinase-15 (MMP-15) expression

Previous study showed that aberrant HLA-G expression in cancer cells plays important roles in disease progression and it was associated with tumor metastasis and with poor survival in an animal model with ovarian cancer; however, the mechanisms remain to be explored. In this study, we found that HLA-G expression could dramatically decreased the NK cytotoxicity against the ovarian carcinoma cell line (HO-8910) engineered to express HLA-G (HO-8910-G), and matrix metalloproteinase-15 (MMP-15) expression was up-regulated in HO-8910-G cells. Consistent with this, a strong correlation between HLA-G and MMP-15 expression were observed in a cohort of ovarian cancer samples. Knockdown the HLA-G induced MMP-15 expression by small interfere RNA (siRNA) significantly decreased the HO-8910-G cell migration potential and tumor metastasis. Collectively, our study indicated that HLA-G involved in tumor invasiveness or metastasis may rely on the NK cytotoxicity inhibition and induction of MMP-15 expression in ovarian cancer.

Induction of cell surface human leukocyte antigen-G expression in pandemic H1N1 2009 and seasonal H1N1 influenza virus-infected patients.

A novel H1N1 virus of swine origin (H1N1v) recently caused a pandemic; however, knowledge of immunologic aspects of the virus infection are limited. Human leukocyte antigen-G (HLA-G) was speculated to play critical roles in viral infection, although its clinical relevance in H1N1 infection remains unknown. In this study, HLA-G expression in peripheral T lymphocytes, monocytes, and CD4(+) CD25(+) FoxP3+ regulatory T (Treg) cells (in 50 H1N1v-infected and 41 seasonal H1N1-infected patients and 27 control subjects) were analyzed by flow cytometry. Plasma-soluble HLA-G (sHLA-G, in 28 H1N1v-infected, 29 seasonal H1N1-infected patients and 85 control subjects) were determined with enzyme-linked immunosorbent assay. The percentage of HLA-G-positive T lymphocytes and monocytes among patients with H1N1v and seasonal H1N1 infections was dramatically increased compared with controls (all p < 0.001). Treg was markedly increased among H1N1v- infected patients compared with normal controls (p = 0.041), but not for the seasonal H1N1-infected patients. Meanwhile, no significant difference was observed for sHLA-G levels between the groups. Together, cell surface HLA-G expression was markedly induced in H1N1v-infected and seasonal H1N1-infected patients, and increased Treg was observed only in H1N1v-infected patients. Given its immune-suppressive property, elevated cell surface HLA-G expression may help to explain the virus escaping from host immune responses.

Tumor-specific upregulation of human leukocyte antigen–G expression in bladder transitional cell carcinoma

Human leukocyte antigen-G (HLA-G) is a potent immunosuppressive molecule that induces functional silencing of both innate and adaptive immune responses. The relevance of the aberrant HLA-G expression in malignant contexts has been intensively investigated. However, its expression status and clinical significance in bladder cancer remain to be elucidated. In the current study, HLA-G expression in 75 primary bladder transitional cell carcinoma (TCC) lesions was analyzed with immunohistochemistry, and relationship between HLA-G expression and clinical parameters, including disease stage was evaluated. Plasma soluble HLA-G levels were analyzed in 15 TCC patients and 109 normal controls. Data revealed that HLA-G was expressed in 68.0% (51/75) primary TCC lesions, whereas it was undetectable in adjacent normal bladder tissues. The proportion of HLA-G expression in TCC samples varied from negative to 100%, and no significant association was observed for the HLA-G expression status with the patient age, gender, and disease stage. Furthermore, no significance for sHLA-G levels was observed between the TCC patients and normal controls (median 10.75 vs 8.69 U/ml, p = 0.578). Given its immunotolerant properties, our finding suggested that lesion HLA-G expression upregulated in bladder TCC lesions might be an additional mechanism for tumor cells evading from host immunosurveillance; however, its clinical relevance needs further investigation.

NK cytolysis is dependent on the proportion of HLA-G expression.

The suppressive functions of HLA-G to various immune cells have been well established. The proportion of HLA-G expression in malignant lesion cells was found from negative to 100%. However, effects for the different proportion of HLA-G expression on the cytolysis of NK cells remain to be explored. In this study, NK cytolysis to the various proportion of HLA-G1 expression on leukemia cell line K562 was investigated. Analysis of NK cell cytotoxicity was by detecting the NK cell surface CD107a expression. Data showed that NK cell cytolysis could be inhibited by the HLA-G1 expression and in a manner of HLA-G1 expression proportion dependent manner (r = 0.925, p = 0.008). Our study provided further understanding for the roles of HLA-G1 expression in malignant cell immune escaping from NK cells.