Soluble HLA-G: Are they clinically relevant?

HLA-G is a non-classical HLA-class Ib molecule with multiple immunoregulatory properties. Its main function in physiological conditions is to abrogate maternal NK cell activity against foetal tissue and to establish immune tolerance at maternal-foetal interface. HLA-G is expressed not only as a membrane bound molecule on the surface of cells, but also as a soluble moiety in body fluids. The major isoforms of HLA-G present in serum are soluble HLA-G1 and HLA-G5 which are generated by shedding or proteolytic cleavage of the membrane bound isoform and by secretion of a soluble isoform, respectively. Here we review the data about soluble HLA-G (sHLA-G) serum levels in different pathological conditions, including immune-mediated disorders, transplantation and malignancies. In particular, we focus on sHLA-G expression and function in human neuroblastoma, a pediatric tumor, with special emphasis on a novel potential immuno escape mechanism utilized by NB to instruct monocytes to produce and release sHLA-G. Finally, the potential clinical relevance of sHLA-G serum levels is discussed.     

Soluble human leukocyte antigen--G serum level is elevated in melanoma patients and is further increased by interferon-alpha immunotherapy

BACKGROUND: The nonclassic human major histocompatibility complex class I antigens human leukocyte antigen (HLA)--G are proposed to protect tumor cells from natural killer cell lysis. In the current study, the authors measured soluble HLA-G molecules (sHLA-G) in serum from patients with malignant melanoma. METHODS: Soluble HLA-G was determined in serum samples of 190 melanoma patients with various stages of disease, with or without current therapy including interferon (IFN)-alpha and different cytostatics in comparison to 126 healthy controls by using a two-step enzyme-linked immunoadsorbent assay. RESULTS: Serum sHLA-G was significantly (P < 0.0005) elevated in melanoma patients (mean +/- standard error of the mean [SEM] = 41.95 +/- 2.15 ng/mL) compared with healthy controls (mean +/- SEM = 22.92 +/- 1.51 ng/mL). Univariate analysis revealed a correlation of sHLA-G serum level with advanced stages of disease (P < 0.001) and tumor load (P < 0.05). Patients undergoing immunotherapy with IFN-alpha (n = 31) showed an increased serum sHLA-G (mean +/- SEM = 62.05 +/- 7.58 ng/mL; P < 0.0005), whereas other treatment regimens (n = 24) did not influence sHLA-G serum concentrations. Multivariate analysis revealed treatment with IFN-alpha as the only impact factor for elevated serum sHLA-G, lacking any correlation with stage of disease or tumor burden. Furthermore, IFN-alpha was found to upregulate HLA-G cell surface expression on circulating monocytes. sHLA-G serum level was not associated with recurrence free or overall survival. CONCLUSIONS: This study shows increased sHLA-G serum concentrations in melanoma patients and additional enhancement upon treatment with IFN-alpha. The level of serum sHLA-G, however, had no negative impact on patients' prognosis.     

Secretion of sHLA-G molecules in malignancies

Our clinical studies revealed significantly increased soluble HLA-G (sHLA-G) plasma levels in patients suffering from malignant melanoma, glioma, breast and ovarian cancer. Specific ELISpot assays demonstrate that sHLA-G molecules expressing intron-4 sequences are preferentially secreted by peripheral blood monocytes. In vitro, the sHLA-G secretion of monocytes and tumor cells was strongly enhanced by TH1 cytokines like IFN-alpha, -beta, -gamma whereas TH2 cytokines (e.g. IL-4, -10) had minor effects. As sHLA-G can inhibit the functions of T and NK cells high concentration of these molecules should systemically or at the tumor side reduce the immune surveillance and thus favour the progression of cancer.     

The significance of soluble HLA-G plasma levels as well as messenger HLA-G for B-cell chronic lymphocytic leukemia (B-CLL)

The immunosuppression accompanies B-cell chronic lymphocytic leukemia (B-CLL) but might be also responsible for disease progression by enabling CLL cells to escape from the immunosurveillance. Some particles involved in the regulation of an immune system might represent prognostic value for B-CLL. Recently we found no correlation between HLA-G on messenger and protein level, suggesting that HLA-G is released in soluble form. To confront this hypothesis we characterized soluble HLA-G (sHLA-G) by the prognostic factors in the first cohort of 34 CLL patients. No correlation was observed between sHLA-G levels in ZAP-70(+) and ZAP-70(−) CLL as well as in CD38(+) CLL and CD38(−) CLL patients. Next, we wondered whether gene expression of HLA-G, which represent the whole HLA-G pool in the cell, posses prognostic value for CLL. In the second cohort of 41 CLL patients we assessed messenger levels of HLA-G by the strongest prognostic factors in CLL including cytogenetics, IgVH mutational status, ZAP-70 as well as CD38. No changes of HLA-G expression levels were found in different CLL groups characterized by IgVH gene mutational status, ZAP-70 as well as CD38. We observed no differences in expression of HLA-G in various cytogenetic groups of CLL including del17p, del13q, del11q, +8q, +3q, del14q and del6q when compared to those with normal karyotype or with 12+. Both, mRNA expression of HLA-G and levels of its soluble form in plasma bring no additional prognostic value for B-CLL patients.     

Analysis of stemness gene expression and CD133 abnormal methylation in neuroblastoma cell lines

Neuroblastoma is the most common extracranial solid tumor in children, accounting for up to 10% of all childhood malignancies. Cellular heterogeneity is a hallmark of this embryonal cancer, as distinct neural crest lineages can be found within the same tumor sample. The aim of our study was to investigate the presence of a subpopulation of immature cells with features of cancer-like stem cells in 10 neuroblastoma cell lines. RT-PCR and flow cytometry were performed in order to analyze different kinds of 'stemness genes' such as: NESTIN (NES), CD133, SOX-2, BMI1, c-KIT, MELK1, MUSASHI-1 (MSI1), FAS, CD44 and VIMENTIN (VIM). In addition, glial and neuronal markers such as NCAM1, GFAP and B-TUBULIN III (TUBB3) were analyzed. Epigenetic changes within the CD133 (Prominin-1) gene promoter were also analyzed. Neuroblastoma cell lines showed a particular pattern of expression, suggesting the presence of an immature cancer stem cell-like subpopulation. The CD133 protein, commonly used to enrich putative cancer propagating stem cell-like populations in different kinds of solid tumors, presented a half-methylated DNA state in 7 of the 12 neuroblastoma cell lines analyzed. An increase in RNA and protein levels of CD133 was achieved following demethylation by assays using 5-aza-2'-deoxycytidine (5-Aza-dC). Since cancer stem cells are believed to be responsible for tumor metastasis, escape from anticancer therapies and disease relapse, their therapeutic targeting and analysis is crucial in neuroblastoma. Moreover, the regulation of CD133 by epigenetic changes may provide an innovative mechanism of CD133 expression as its regulation still remains unclear.     

HLA-G Expression in Tumor Tissues and Soluble HLA-G Plasma Levels in Patients with Gastrointestinal Cancer

Background: Overexpression of human leukocyte antigen G (HLA-G) and increased plasma levels of solubleHLA-G (sHLA-G) have been reported in different human malignancies, and are believed to be involved in tumor immuneevasion. Objectives: This study was designed to evaluate the expression of HLA-G in tumor tissues and the plasmalevels of sHLA-G in patients with gastrointestinal cancer, and to determine their associations with clinicopathologicalfactors. The link between Helicobacter pylori infection and increased HLA-G expression or sHLA-G levels was alsoinvestigated in patients with gastric cancer. Methods: HLA-G expression was investigated in tumor tissues from 100patients with gastric and colorectal adenocarcinoma using immunohistochemistry test, and plasma levels of sHLA-Gwere measured in 82 patients with ELISA method. The presence of H. pylori genome was investigated in tumortissues from 25 patients with gastric cancer by PCR method. Results: HLA-G expression was observed in 43% ofcolorectal cancers and 34.6% of gastric cancers, and was not related with any of the clinicopathological factors. Therewas a significant correlation between increased sHLA-G level and stage I tumors. Eight of 25 (32%) gastric cancerspecimens were positive for H. pylori, of which 3 samples were positive for HLA-G. Soluble HLA-G levels were abovethe cut-off value in all H. pylori-positive patients. Conclusion: Plasma levels of sHLA-G were significantly increasedin our patients with a sensitivity of 89% and a specificity of 62%. Soluble HLA-G level can be considered a usefulindicator for the early diagnosis of gastric and colorectal adenocarcinoma.     

CD146 is a potential immunotarget for neuroblastoma

Neuroblastoma, the most common extracranial solid tumor of childhood, is thought to arise from neural crest-derived immature cells. The prognosis of patients with high-risk or recurrent/refractory neuroblastoma remains quite poor despite intensive multimodality therapy; therefore, novel therapeutic interventions are required. We examined the expression of a cell adhesion molecule CD146 (melanoma cell adhesion molecule [MCAM]) by neuroblastoma cell lines and in clinical samples and investigated the anti-tumor effects of CD146-targeting treatment for neuroblastoma cells both in vitro and in vivo. CD146 is expressed by 4 cell lines and by most of primary tumors at any stage. Short hairpin RNA-mediated knockdown of CD146, or treatment with an anti-CD146 polyclonal antibody, effectively inhibited growth of neuroblastoma cells both in vitro and in vivo, principally due to increased apoptosis via the focal adhesion kinase and/or nuclear factor-kappa B signaling pathway. Furthermore, the anti-CD146 polyclonal antibody markedly inhibited tumor growth in immunodeficient mice inoculated with primary neuroblastoma cells. In conclusion, CD146 represents a promising therapeutic target for neuroblastoma.     

Soluble factors derived from neuroblastoma cell lines suppress dendritic cell differentiation and activation

Dendritic cells (DC) play a key role in the initiation of both antitumor immunity and immunological tolerance. It has been demonstrated that exposure to soluble factors produced by tumor cells modulates DC functions and induces tolerogenic DC differentiation. In this study, we investigated the effects of neuroblastoma cell line‐derived soluble factors on DC differentiation. Monocytes isolated from healthy volunteers were incubated with interleukin (IL)‐4 and granulocyte‐macrophage colony‐stimulating factor in the presence of culture supernatants from neuroblastoma cell lines. The culture supernatants from neuroblastoma cell lines, such as NLF and GOTO, partially blocked both downregulation of CD14 and upregulation of CD1a, and dramatically decreased IL‐12 and tumor necrosis factor (TNF)‐α production from mature DC, while no effect of SH‐SY5Y cell supernatant was noted. In addition, IL‐6 and IL‐10 production from monocytes was increased by the supernatants of NLF and GOTO cells at 24 hours after incubation. Furthermore, we evaluated DC functions through stimulation of invariant natural killer T (iNKT) cells. α‐Galactosylceramide‐pulsed DC co‐cultured with supernatants of NLF cells were unable to sufficiently stimulate iNKT cells. The decreased ability of iNKT cells to produce interferon (IFN)‐γ after stimulation with neuroblastoma cell line supernatant‐cultured DC was reversed by addition of IL‐12. CD40 expression and IL‐12 production in NLF‐sup‐treated DC were increased by addition of exogenous IFN‐γ. These results indicate that tolerogenic DC are induced in the neuroblastoma tumor microenvironment and attenuate the antitumor effects of iNKT cells. Interactions between iNKT cells and αGalCer‐pulsed DC have the potential to restore the immunosuppression of tolerogenic DC through IFN‐γ production.     

Low-dose interferon-gamma-producing human neuroblastoma cells show reduced proliferation and delayed tumorigenicity

Interferon-gamma (IFN-gamma) directs T helper-1 cell differentiation and mediates antitumour effects in preclinical models. However, high-dose IFN-gamma is toxic in vivo, and IFN-gamma-transfected neuroblastoma (NB) cells secreting high amounts of the cytokine may be lost due to cell apoptosis or differentiation. Two human NB cell lines (ACN and SK-N-BE2(c)) differing as to genetic and phenotypic features were transfected with the human IFN-gamma gene and selected on the grounds of the low concentrations of IFN-gamma produced. In both IFN-gamma-transfected cell lines, autocrine and paracrine activation of IFN-gamma-mediated pathways occurred, leading to markedly reduced proliferation rate, to increased expression of surface HLA and CD40 molecules and of functional TNF binding sites. ACN/IFN-gamma cells showed a significantly delayed tumorigenicity in nude mice as compared to parental cells. ACN/IFN-gamma tumours were smaller, with extensive necrotic area as a result of a damaged and defective microvascular network. In addition, a significant reduction in the proliferation index was observed. This is the first demonstration that IFN-gamma inhibits in vivo proliferation of NB cell by acting on the tumour cell itself. This effect adds to the immunoregulatory and antiangiogenic activities operated by IFN-gamma in syngeneic tumour-bearing hosts.     

Primitive Neuroectodermal Tumor in an Ovarian Cystic Teratoma: Natural Killer and Neuroblastoma Cell Analysis

In the present study, we report an extremely rare case of a 31-year-old woman with neuroblastoma arising in an ovarian cystic teratoma. We analyzed the expression of activating receptors on natural killer (NK) cells derived from the patient''s peripheral blood and peritoneal fluid. In addition, we investigated the presence of specific ligands recognized by different NK cell receptors on tumor cells. We show that NK cells isolated from peritoneal fluid expressed certain triggering receptors including DNAM-1 (CD226) and CD16 with lower intensity as compared to peripheral blood NK cells. Remarkably, at variance with most cases of childhood neuroblastoma, the tumor cells from this patient expressed substantial amounts of HLA class-I molecules. These molecules are known to be protective against NK cell-mediated lysis. In addition, neuroblastoma cells expressed B7-H3 (CD276), another surface molecule that inhibits NK cell function. Finally, this tumor did not express the PVR (CD155) and nectin-2 (CD112) ligands for the DNAM-1 activating NK receptor, which plays a crucial role in NK/neuroblastoma interactions. Altogether, these findings indicate that the neuroblastoma cells of this patient express an NK-resistant surface phenotype, which is at least in part similar to that previously described in a fraction of childhood neuroblastoma.Key words: Activating natural killer receptors, Natural killer cells, Neuroblastoma, Ovarian cancer     

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.     

Implication of Soluble HLA-G and HLA-G +3142G/C Polymorphism in Breast Cancer Patients Receiving Adjuvant Therapy in Tanzania

Background: During cancer growth, immunosuppressive microenvironment is created that enables tumour cells to evade an eliminative immune response and hence manage to grow into malignancy. HLA-G, existing as either membrane-bound (mHLA-G) or soluble (sHLA-G) molecule is thought to be immunosuppressive and produced more by tumor cells. The +3142G/C polymorphism in HLA-G gene affects its expression, and G allele is considered to be a protective mutant allele associated with less expression of HLA-G. The implication of HLA-G in cancer development has been reported in different cancers and populations. But, its implication in most African populations has not yet been investigated. The aim of this study was to determine the possible associations of soluble HLA-G and HLA-G +3142G/C SNP with breast cancer. Materials and Methods: 75 breast cancer patients and 84 normal controls were recruited in this study. The genotyping of HLA-G +3142G/C polymorphism was determined by LightSNiP typing assay using quantitative Real-Time PCR and sHLA-G levels were determined by ELISA. Results: The sHLA-G levels were significantly lower in breast cancer patients than in controls (p<0.001). Also, they were significantly lower in mastectomized patients compared to non-mastectomized patients (p=0.018). The ROC analysis revealed a significant ability of sHLA-G to differentiate breast cancer patients versus normal controls (AUC=0.697, 95% CI= 0.619-0.767, p<0.001) and identify mastectomized patients (AUC=0.667, 95% CI= 0.549 to 0.772, p=0.041). The assessment of +3142G/C polymorphism revealed a relatively similar distribution of frequencies of genotypes and alleles between breast cancer patients and normal controls (p>0.05) and was neither associated with sHLA-G levels. Conclusion: While the +3142G/C SNP was found not to be relevant to breast cancer, the changes of sHLA-G levels in response to medical interventions such as mastectomy may be translated into its potential prognostic utility for breast cancer. More studies are needed to provide clear evidence of sHLA-G as a diagnostic and prognostic marker of breast cancer in Tanzania.     

CD59 expressed on a tumor cell surface modulates decay-accelerating factor expression and enhances tumor growth in a rat model of human neuroblastoma

It has been hypothesized that complement inhibitors expressed on the surface of tumor cells prevent effective immune-mediated clearance. Whereas there are in vitro data to support this hypothesis, the species-selective activity of complement inhibitors has been a hindrance to investigating the role of membrane-bound complement inhibitors in rodent models of human cancer. The CD59-positive LAN-1 human neuroblastoma cell line was significantly more sensitive to lysis by rat complement than by human complement, illustrating the species selectivity of endogenously expressed complement inhibitors. Transfection of LAN-1 cells with rat CD59, an inhibitor of the terminal cytolytic membrane attack complex, effectively protected the cells from lysis by rat complement in vitro. When LAN-1 cells stably expressing rat CD59 were inoculated into immune-deficient rats, the onset of tumor growth and the rate of tumor growth were significantly enhanced compared with those of control-transfected LAN-1 cells. These data show directly that the expression of a complement inhibitor on a tumor cell promotes tumor growth. Flow cytometric analysis revealed that the endogenous expression of decay-accelerating factor (DAF), an inhibitor of complement activation, was up-regulated on the surface of cells after in vivo growth. Of further interest, higher levels of DAF were present on CD59-transfected cells than on control-transfected cells derived from tumors. Increased DAF expression correlated with decreased complement deposition on the tumor cell surface. These results show that expression of complement inhibitors on a tumor cell has functional consequences with regard to complement deposition in vivo and indicate that CD59 can indirectly effect complement activation and C3 deposition in vivo via a link between CD59 and DAF expression.     

Induction of caspase 8 by interferon gamma renders some neuroblastoma (NB) cells sensitive to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) but reveals that a lack of membrane TR1/TR2 also contributes to TRAIL resistance in NB

The resistance of neuroblastoma (NB) cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis has been attributed to a lack of caspase 8 expression. Here we demonstrate a clinically applicable molecular targeting strategy that not only increases caspase 8 expression ex vivo in NB cell lines but also in the tumor tissues of NB patients receiving IFN-gamma treatment. We identify the functional caspase 8 promoter, which is different from the methylated region reported previously, and show promoter activity is up-regulated by IFN-gamma through a IFN-gamma activation site-containing region. IFN-gamma also induces TRAIL expression in NB cell lines. However, the IFN-gamma restoration of caspase 8 in some NB cells revealed persistent TRAIL resistance in most NB cell lines examined. This additional lesion in the TRAIL path is because of a loss of cell membrane TRAIL receptors (TR1/TR2) not only in cell lines but in most of the NB tumor tissues evaluated. Restoration of TR2 expression by transfection enhances IFN-gamma-induced TRAIL sensitivity. Furthermore, we have found that we can improve TRAIL sensitivity in NB by reconstituting caspase 8 with IFN-gamma and TR2 with chemotherapeutic agents.     

Interferon-gamma-induced alterations of monocyte susceptibility to lysis by autologous lymphokine-activated killer (LAK) cells

Interleukin-2 (IL-2)-activated killer (LAK) cells specifically lyse human monocytes, which may account for some of the toxicity seen during LAK/IL-2 immunotherapy of cancer patients. In an effort to protect autologous monocytes, we treated monolayer cultures of monocytes with various doses of recombinant human interferon-gamma (IFN-gamma) and assessed their sensitivity to LAK-mediated lysis. IFN-gamma lessens the sensitivity of monocytes to lysis in a dose-dependent manner. Treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 hr incubation with IFN-gamma was sufficient for protection to occur, and that monocytes which were treated with IFN-gamma for 2 hr, washed, and then cultured in medium alone retained their resistance to lysis for at least 4 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Finally, binding studies demonstrated that there was no significant difference between the number of conjugates formed using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal.     

Angiogenesis in a human neuroblastoma xenograft model: mechanisms and inhibition by tumour-derived interferon-gamma

Tumour progression in neuroblastoma (NB) patients correlates with high vascular index. We have previously shown that the ACN NB cell line is tumorigenic and angiogenic in immunodeficient mice, and that interferon-gamma (IFN-gamma) gene transfer dampens ACN tumorigenicity. As IFN-gamma represses lymphocyte-induced tumour angiogenesis in various murine models and inhibits proliferation and migration of human endothelial cells, we have investigated the antiangiogenic activity of tumour-derived IFN-gamma and the underlying mechanism(s). In addition, we characterised the tumour vasculature of the ACN xenografts, using the chick embryo chorioallantoic membrane assay. We show that the ACN/IFN-gamma xenografts had a lower microvessel density and less in vivo angiogenic potential than the vector-transfected ACN/neo. The vascular channels of both xenografts were formed by a mixed endothelial cell population of murine and human origin, as assessed by the FICTION (fluorescence immunophenotyping and interphase cytogenetics) technique. With respect to ACN/neo, the ACN/IFN-gamma xenografts showed more terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling-positive human and murine endothelial cells, suggesting that inhibition of angiogenesis by IFN-gamma was dependent on the induction of apoptosis, likely mediated by nitric oxide. Once the dual origin of tumour vasculature is confirmed in NB patients, the xenograft model described here will prove useful in testing the efficacy of different antiangiogenic compounds.     

Increased susceptibility of IFN-gamma-treated neuroblastoma cells to lysis by lymphokine-activated killer cells: participation of ICAM-1 induction on target cells.

We have investigated the effect of interferon-gamma (IFN-gamma) treatment of neuroblastoma cells on the susceptibility to lysis by lymphokine-activated killer (LAK) cells and examined the participation of cell-adhesion molecules on the target cells in LAK cell lysis. Untreated neuroblastoma cells expressed lymphocyte-function-associated antigen 3 (LFA-3) and neural-cell-adhesion molecule (NCAM), but did not express MHC-class-I, MHC-class-II, or intercellular-adhesion molecule I (ICAM-I). IFN-gamma treatment of neuroblastoma cells induced the expression of MHC-class-I and ICAM-I antigens, but did not affect the expression of MHC-class-II, LFA-3, and NCAM. This was accompanied by an increased susceptibility to lysis by LAK cells. Anti-ICAM-I antibody inhibited partially the increased sensitivity of IFN-gamma-treated neuroblastoma cells to LAK cell lysis, and blocked completely the increase in binding of LAK cells observed after IFN-gamma treatment of the target cells. These results suggest that the increased LAK sensitivity of IFN-gamma-treated neuroblastoma cells is partially attributable to the induction of ICAM-I on neuroblastoma cells and indicate that post-binding events also play a role in the increased sensitivity to LAK cell lysis observed after IFN-gamma treatment.