The imbalance between NKG2A and NKG2D expression is involved in NK cell immunosuppression and tumor progression of patients with hepatitis B virus-related hepatocellular carcinoma

Background

Immunosuppression in a tumor microenvironment is associated with enhanced tumor progression. Natural killer group 2 (NKG2) family proteins, including inhibitory receptors and activators, can be used as attractive targets for immunotherapy of immune checkpoint inhibition. We further explore the expression level prognostic value of NKG2A and NKG2D in hepatitis B virus-related hepatocellular carcinoma (HBV-HCC).

Methods

This study was a prospective study involving 92 patients with HBV-HCC, 16 patients with HBV-related liver cirrhosis, 18 patients with CHB, and 38 healthy donors. We analyzed the expression and related functions of NKG2A, NKG2D, and the NKG2A/NKG2D ratio in the peripheral blood of patients with HBV-HCC and analyzed tumor progression. The tissue samples from patients with HBV-HCC were further used for multiple immunofluorescence and immunohistochemistry.

Results

In patients with HBV-HCC with tumor progression, the ratio of NKG2A/NKG2D is higher in NK cells and T cells. The Kaplan–Meier survival curve showed that the NKG2A/NKG2D ratio on NK cells could predict tumor progression in patients with HBV-HCC, and that an increase in this ratio was associated with inhibition of NK cell function. The Cancer Genome Atlas (TCGA) database was further used to verify that the higher the NKG2A/NKG2D ratio, the shorter the progression-free survival of patients with HCC, and the more likely the immune function was suppressed.

Conclusions

The imbalance between NKG2A and NKG2D of NK cells is involved in NK cell immunosuppression, and the increase of the NKG2A/NKG2D ratio is related to the tumor progression of HBV-HCC.     

Impact of Pegylated Interferon-alfa-2a on Perforin Level in Patients With Chronic Hepatitis B; Preliminary Study

Background

Chronic hepatitis B is one of the most common causes of cirrhosis and hepatocellular toxicity in many countries, including Iran. Cytotoxic T lymphocyte (CTL) and Natural killer (NK) cells are the two of main cell populations considered as cytotoxic cells. One of the distinct pathways CTL and NK cells exert cytotoxicity is perforin/granzyme. After the cytotoxic cell/target cell junction, perforin is released from granules by exocytosis. Once it is anchored, perforin forms cylindrical pores through which granzymes and granulysin enter and induce apoptosis.

Objectives

Large controlled trials have demonstrated the efficacy of PEG-IFN-α-2a in treatment of chronic hepatitis B. This study was aimed to examine whether the enhancement of cytotoxicity by PEG-IFN-α-2a is mainly due to the perforin pathway.

Patients and Methods

This research work was performed on 50 patients and five healthy people. Patients with chronic hepatitis B were further subdivided into two groups: patients with inactive chronic hepatitis B (carriers, n = 30), and those with active chronic hepatitis B who were under treatment with PEG-IFN-alfa-2a (n = 20) for minimum six and maximum 12 months. Serum perforin level was measured using ELISA method (CUSABIO Company), HBV viral load was assessed using COBAS Taq-man, and we used Elecsys hepatitis B surface antigen (HBs Ag) II quantitative assay method for HBs Ag determination. HBeAg was evaluated by ELISA method, and AST and ALT were measured by routine laboratorymethods.

Results

Based on the results obtained serum perforin level in healthy group was 0.64 ng/mL, the mean of serum perforin level in inactive HBs Ag carriers was 2.63ng/mL, and 4.63 ng/mL in patients with active chronic hepatitis B under treatment with PEG-IFN-α-2a. The mean of serum perforin level in patients with and without virologic response to treatment were 5.45 ng/mL,and 3.4 ng/mL respectively. Finally in patients with virologic response and seroconverted serum perforin level was 7.23 ng/mL.

Conclusions

Based on our results higher perforin level in patients under treatment with PEG-IFN-α-2a, could be an indication of elevated cytotoxicity via perforin/granzyme pathway.     

A Comprehensive Long-Term Prognosis of Chronic Hepatitis C Patients With Antiviral Therapy: A Meta-Analysis of Studies From 2008 to 2014

Context:

Attaining a sustained virological response with antiviral therapy is a sign of clinical cure for chronic hepatitis C patients. The aim of this meta-analysis was to evaluate the long-term efficiency and outcome of antiviral therapy in patients with hepatitis C who attained a sustained virological response.

Evidence Acquisition:

A literature search was performed on published articles between January 2008 and February 2014. Patients with Hepatitis C who received interferon with or without ribavirin therapy were enrolled. Relative risks were estimated using either fixed or random effect models.

Results:

Patients who attained sustained virological response had a less risk (85%) for all-cause mortality and about 63% reduced risk of hepatocellular carcinoma incidence than those who did not achieve sustained virological response. Based on deeply analysis, the stage of liver fibrosis was a risk factor at baseline for the incidence of hepatocellular carcinoma.

Conclusions:

Sustained virological response can reduce all-cause mortality and the incidence of hepatocellular carcinoma of patients with hepatitis C. Advanced liver fibrosis is still a risk factor for the incidence of hepatocellular carcinoma, in spite of hepatitis C patients attained a sustained virological response.     

The Role of Fas/Fas Ligand System in the Pathogenesis of Liver Cirrhosis and Hepatocellular Carcinoma

Background

The Fas receptor/ligand system including soluble forms is the most important apoptotic initiator in the liver. Dysregulation of this pathway may contribute to abnormal cell proliferation and cell death and is regarded as one of the mechanisms preventing the immune system from rejecting the tumor cells.

Objectives

To analyze the role of Fas system Fas/ Fas ligand (Fas/ FasL) in the multi-step process of hepatic fibrosis/carcinogenesis, and to use of the serum markers as possible candidate biomarkers for early detection of hepatocellular carcinoma (HCC).

Patients and Methods

Ninety patients were enrolled: 30 cases of chronic hepatitis C (CHC) without cirrhosis, 30 cases of CHC with liver cirrhosis, and 30 cases of HCC and hepatitis V virus (HCV) infection. Ten wedge liver biopsies, taken during laparoscopic cholecystectomy, were served as normal controls. Serum soluble Fas (sFas) levels were measured using ELISA technique; Fas and FasL proteins were detected in hepatic tissue by indirect Immuno-histochemical technique (IHC); electron microscopic (EM) and immune electron microscopic examinations were performed for detection of Fas expression on lymphocytes.

Results

Hepatic expression of both Fas and FasL as well as expression of Fas on separated lymphocytes were significantly increased in the diseased groups (P < 0. 01) compared to the control specimens. The highest expression was noticed in CHC specimens, particularly with the necro-inflammatory activity and advancement of the fibrosis. The sFas in cirrhotic patients and HCC were significantly higher than that in normal controls and CHC without cirrhosis group (P < 0.01).

Conclusions

Apoptosis and the Fas system were significantly involved in the process of converting liver cirrhosis into hepatocellular carcinoma. Down-regulation of Fas expression, up regulation of FasL expression in hepatocytes, and elevation of serum sFas levels were important in tumor evasion from immune surveillance, and in hepatic carcinogenesis.     

Blockade of NKG2D ameliorates disease in mice with collagen‐induced arthritis: A potential pathogenic role in chronic inflammatory arthritis

Objective

To assess the role of the activating receptor NKG2D in arthritis.

Methods

Levels of NKG2D and its ligands were determined by fluorescence‐activated cell sorting, real‐time polymerase chain reaction, and immunohistochemistry in rheumatoid arthritis (RA) synovial membrane tissue and in paw tissue from arthritic mice. Arthritis was induced in DBA/1 mice by immunization with type II collagen, and mice were treated intraperitoneally with a blocking anti‐NKG2D antibody (CX5) on days 1, 5, and 8 after clinical onset and were monitored for 10 days.

Results

We demonstrated expression of NKG2D and its ligands on human RA synovial cells and extended this finding to the paws of arthritic mice. Expression of messenger RNA for the NKG2D ligand Rae‐1 was up‐regulated, and NKG2D was present predominantly on natural killer (NK) and CD4+ T cells, in arthritic paw cell isolates. NKG2D was down‐modulated during the progression of collagen‐induced arthritis (CIA). NKG2D expression in arthritic paws was demonstrated by immunohistochemistry. Blockade of NKG2D ameliorated established CIA, with significant reductions in clinical scores and paw swelling. Histologic analysis of arthritic joints from anti‐NKG2D–treated mice demonstrated significant joint protection, compared with control mice. Moreover, anti‐NKG2D treatment significantly reduced both interleukin‐17 production from CD4+ T cells in arthritic paws and splenic NK cell cytotoxic effector functions in vivo and in vitro.

Conclusion

Our findings indicate that blockade of NKG2D in a murine model and in human explants has beneficial therapeutic potential that merits further investigation in RA.

     

Adiponectin and Its Receptors in Chronic Hepatitis B Patients With Steatosis in China

Background

HBV infection is a serious public health problem worldwide, which can contribute to the incidence of chronic hepatitis B (CHB), cirrhosis, and hepatocellular carcinoma (HCC).

Objectives

In the present report, we assessed the association between adiponectin, its receptors and hepatic steatosis, fibrosis, and inflammation with hepatitis B virus.

Patients and Methods

Liver biopsies from 89 patients with untreated chronic hepatitis B (34 steatosis vs. 55 without steatosis) were analyzed; liver biopsies from 50 healthy adults were used as control. The liver biopsies were subjected to routine histological examination, and stained immunohistochemically for adiponectin and adiponectin receptor2 (adipoR2).

Results

The two groups were found to be comparable with respect to demographic, biochemical, metabolic, histological, and viral characteristics. BMI, γ-GT, FPG, insulin, and insulin sensitivity estimated by the HOMA index were significantly higher in patients with steatosis. The viral load of HBV and HBeAg positivity was higher in patients with steatosis than those without steatosis. High serum adiponectin levels were significantly correlated with abnormal serum ALT level (vs. normal ALT, P = 0.000), and HBV genotype C (vs. genotype B, P = 0.018). In patients with chronic HBV, the insulin sensitizing adipokine adiponectin, and its receptor AdipoR2were associated with steatosis. While adiponectin may becorrelated with inflammation, adiponectin, and its receptors were not associated with viral factors.

Conclusions

Our results suggest that the role of adiponectin might be impaired in chronic hepatitis B with steatosis. Reduced hepatic expression of adiponectin and adipoR2 might be of pathophysiological relevance in CHB patients with steatosis. These findings indicated that reduced liver adiponectin expression may play an important role in the pathogenesis, and progression of CHB patients with steatosis. However, hepatic expression of adiponectin, and adipoR2 was not associated with various measures of HBV infection.     

Blast-derived microvesicles in sera from patients with acute myeloid leukemia suppress natural killer cell function via membrane-associated transforming growth factor-beta1

Background

Natural killer cell cytotoxicity is decreased in patients with acute myeloid leukemia in comparison to that in normal controls. Tumor-derived microvesicles present in patients’ sera exert detrimental effects on immune cells and may influence tumor progression.

Design and Methods

We investigated the microvesicle protein level, molecular profile and suppression of natural killer cell activity in patients with newly diagnosed acute myeloid leukemia.

Results

The patients’ sera contained higher levels of microvesicles compared to the levels in controls (P<0.001). Isolated microvesicles had a distinct molecular profile: in addition to conventional microvesicle markers, they contained membrane-associated transforming growth factor-β1, MICA/MICB and myeloid blasts markers, CD34, CD33 and CD117. These microvesicles decreased natural killer cell cytotoxicity (P<0.002) and down-regulated expression of NKG2D in normal natural killer cells (P<0.001). Sera from patients with acute myeloid leukemia contained elevated levels of transforming growth factor-β, and urea-mediated dissociation of microvesicles further increased the levels of this protein. Neutralizing anti-transforming growth factor-β1 antibodies inhibited microvesicle-mediated suppression of natural killer cell activity and NKG2D down-regulation. Interleukin-15 protected natural killer cells from adverse effects of tumor-derived microvesicles.

Conclusions

We provide evidence for the existence in acute myeloid leukemia of a novel mechanism of natural killer cell suppression mediated by tumor-derived microvesicles and for the ability of interleukin-15 to counteract this suppression.     

Excessive interleukin‐15 transpresentation endows NKG2D+CD4+ T cells with innate‐like capacity to lyse vascular endothelium in granulomatosis with polyangiitis (Wegener's)

Objective

Granulomatosis with polyangiitis (Wegener's) (GPA) is a rare systemic vasculitis of unknown etiology. Contribution of T cell–mediated immunity is suggested by the presence of granulomatous inflammation and T cell infiltrates in different tissues. We undertook this study to determine whether CD4+ T cells aberrantly expressing the NKG2D activating receptor might participate in the pathophysiology of the disease.

Methods

We performed a detailed phenotype and functional analysis of CD4+ T cells in a cohort of 90 GPA patients (37 with localized GPA and 53 with generalized GPA) in comparison with 39 age‐matched controls.

Results

We observed circulating innate‐like CD4+ T cells expressing an assortment of activating natural killer (NK) cell receptors (NKG2D, 2B4, DNAX‐associated molecule 1, and some killer cell Ig‐like receptors) and their signaling partners. Expansions of NKG2D+CD4+ T cells greater than a critical threshold of 3% yielded 100% specificity for generalized vasculitis versus localized granulomatosis, suggesting their participation in endothelium damage. Excessive interleukin‐15 (IL‐15) transpresentation through increased expression of IL‐15 receptor α (IL‐15Rα), together with abnormal expression of major histocompatibility complex (MHC) class I chain–related A protein on monocyte/macrophages, induced abnormal expansion of NKG2D+CD4+ T cells. These cells were primed in vivo to exert direct, MHC‐independent cytotoxicity toward microvascular endothelial cells expressing the cognate ligands of NK cell receptors.

Conclusion

Our results suggest that NK cell–like CD4+ T cells might be the driving force of the vasculitis in GPA, and point to IL‐15 as an important mediator in the progression of GPA toward generalized vasculitis. IL‐15/IL‐15Rα antagonists may thus become novel therapeutic tools to decrease the pool of NK cell receptor–positive CD4+ T cells in selected GPA patients.

     

The Expression of Programmed Death-1 in Circulating CD4+ and CD8+ T Cells during Hepatitis B Virus Infection Progression and Its Correlation with Clinical Baseline Characteristics

Background/Aims

Programmed death-1 (PD-1) expression was investigated in CD4⁺ and CD8⁺ T cells from hepatitis B virus (HBV)-infected patients at the chronic hepatitis B (CHB) infection, liver cirrhosis (LC), and hepatocellular carcinoma (HCC) stages.

Methods

PD-1 expression in circulating CD4⁺ and CD8⁺ T cells was detected by flow cytometry. The correlations between PD-1 expression and HBV viral load, alanine aminotransaminase (ALT) levels and aspartate aminotransferase (AST) levels were analyzed using GraphPad Prism 5.0.

Results

PD-1 expression in CD4⁺ and CD8⁺ T cells was significantly increased in both the CHB group and advanced-stage group (LC plus HCC). In the CHB group, PD-1 expression in both CD4⁺ and CD8⁺ T cells was positively correlated with the HBV viral load, ALT, and AST levels. However, in the LC plus HCC group, significant correlations between PD-1 expression and the clinical parameters were nearly absent.

Conclusions

PD-1 expression in peripheral CD4⁺ and CD8⁺ T cells is dynamic, changes with HBV infection progression, and is related to HBV viral load and liver function, especially in CHB. PD-1 expression could be utilized as a potential clinical indicator to determine the extent of virus replication and liver injury.     

High Expression of Aldo-Keto Reductase 1B10 Is an Independent Predictor of Favorable Prognosis in Patients with Hepatocellular Carcinoma

Background/Aims

Upregulation of aldo-keto reductase 1B10 (AKR1B10) through the mitogenic activator protein-1 signaling pathway might promote hepatocarcinogenesis and tumor progression. The goal of this study was to evaluate the prognostic significance of AKR1B10 protein expression in patients with hepatocellular carcinoma after surgery.

Methods

A tissue microarray was used to detect the expression level of AKR1B10 protein in tumors from 255 patients with hepatocellular carcinoma who underwent curative hepatectomy. The impact of AKR1B10 expression on the survival of patients was analyzed. The median follow-up period was 119.8 months.

Results

High AKR1B10 protein expression was observed in 125 of the 255 patients with hepatocellular carcinoma (49.0%). High AKR1B10 expression was significantly associated with a lack of invasion of the major portal vein (p=0.022), a lack of intrahepatic metastasis (p=0.010), lower the American Joint Committee on Cancer T stage (p=0.016), lower the Barcelona Clinic Liver Cancer stage (p=0.006), and lower α-fetoprotein levels (p=0.020). High AKR1B10 expression was also correlated with a lack of early recurrence (p=0.022). Multivariate analyses of survival revealed that intrahepatic metastases and lower albumin levels were independent predictors of both shorter recurrence-free survival and shorter disease-specific survival. High AKR1B10 expression was an independent predictor of both longer recurrence-free survival (p=0.024) and longer disease-specific survival (p=0.046).

Conclusions

High AKR1B10 protein expression might be useful as a marker of a favorable prognosis in patients with hepatocellular carcinoma after curative hepatectomy.     

Occult Hepatitis B (OBH) in Clinical Settings

Context

Occult hepatitis B (OHB), or persistent HBV DNA in patients who are hepatitis B surface antigen (HBsAg) negative, is a recently recognized entity. In an attempt to summarize the issues, this review presents an overview of the current proposed hypothesis on the clinical relevance and also updates the knowledge on the classification of OHB in different clinical settings.

Evidence Acquisition

OHB could be found in different population and clinical backgrounds including: viral co-infections (with either human immunodeficiency or hepatitis C viruses), HBV chronic carriers, dialysis patients, transplantation settings and certain clinical situations (named in here: special clinical settings) with no apparent distinguishable clinical parameters.

Results

The exact magnitude, pathogenesis, and clinical relevance of OHB are unclear. Even the possible role exerted by this cryptic infection on liver disease outcome, and hepatocellular carcinoma development remains unknown.

Conclusions

Monitoring of Individuals with positive anti-HBc, mass immunization programs and improvement in diagnostic tools seem to be important to control the probability of transmission of HBV through cryptic HBV infection.     

Prevalence of Hepatitis D Virus Infection Among Patients With Chronic Hepatitis B Attending Birjand Hepatitis Clinic (East of Iran) in 2012

Background

Hepatitis delta virus (HDV) is a defective RNA virus dependent on Hepatitis B virus (HBV) infection for its replication and expression. All patients with HBV infection should be tested for the presence of HDV infection. It is estimated that approximately 5% of hepatitis B surface antigen (HbsAg) carriers in the world are HDV infected patients. HBV-HDV co-infection may lead to more severe acute disease and higher risks of fulminant hepatitis, cirrhosis, and hepatocellular carcinoma than those having HBV infection alone. Also, HBV infected patients with HDV super-infection have a higher rate of progression to chronic disease and serious complications.

Objectives

Our aim was to determine the prevalence of HDV infection among chronic hepatitis B (CHB) patients attending Birjand Hepatitis Clinic, East of Iran.

Materials and Methods

A cross-sectional analytical study was conducted on 413 CHB patients in 2012. Serology test for anti-HDV was measured by ELISA in these patients. CHB patients had positive hepatitis B surface antigen for at least 6 months before the study entrance.

Results

The mean age of CHB patients was 38.5± 11.9 years and 55.9% of them (231 patients) were male. There were 13 cases (3.1%) with HDV infection. There was no association between positive anti-HDV serology and factors such as age, gender, carrier state, liver enzymes, and positive hepatitis B e antigen (HBeAg) serology.

Conclusions

Although HDV had a low prevalence in our area, it is important for healthcare providers and policy makers to plan preventive strategies for HDV spread as well as HBV prevention programs among high risk population.     

G1896A Precore Mutation and Association With HBeAg Status,Genotype and Clinical Status in Patients With Chronic Hepatitis B

Background:

Precore stop codon (G1896A) mutation is one of the commonest mutations found in patients with chronic hepatitis B. However, over the years, this mutation was not reported much in Malaysia.

Objectives:

We therefore investigated the presence of G1896A mutation in Malaysian population and its association with HBeAg status, clinical stage, hepatitis B virus (HBV) genotype and e-seroconversion rate.

Patients and Methods:

Serum samples from 93 patients confirmed as hepatitis B carriers were collected for molecular assay. The whole genome of HBV was amplified by polymerase chain reaction and directly sequenced. The precore and basal core promoter regions were analyzed for presence of mutations.

Results:

The most commonly observed mutation in the precore region was C1858T with 64.5% prevalence. The precore mutation of interest (G1896A) was identified in 25.8% of isolates. The basal core promoter mutations detected were A1762T-G1764A (26.9%), C1653T (8.6%), A1752G (10.8%) and C1766T (2.2%). No significant association was observed between G1896A mutation and HBeAg-negativity. Nonetheless, G1896A was highly prevalent among HBV genotype B. Clinical association revealed that subjects with G1896A mutations were mainly detected in asymptomatic chronic hepatitis B (58.3%) and liver cirrhosis (41.7%). One subject was diagnosed with fulminant hepatitis (4.2%) and 8.3% had hepatocellular carcinoma (HCC).

Conclusions:

Our data suggested an intermediate prevalence of G1896A mutation among Malaysian hepatitis B carriers. The stop codon mutation has a significant association with genotype B and patients with chronic hepatitis B and liver cirrhosis.     

The clinical and laboratory characteristics of patients with chronic hepatitis B using current or past antiviral therapy in Korea: a multi-center, nation-wide, cross-sectional epidemiologic study

Background/Aims

The proper assessment of the current disease status of patients with chronic hepatitis B would be valuable for establishing optimal management strategies.

Methods

The clinical and laboratory characteristics of 2,954 patients with current or previous antiviral treatment (46.2±10.8 years, 69.7% male) enrolled from 46 referral hospitals and 129 local hospitals or clinics throughout Korea were analyzed.

Results

The disease status included chronic hepatitis, cirrhosis, and hepatocellular carcinoma in 79.9%, 16.4%, and 3.7% of the patients, respectively. The major mode of hepatitis B virus (HBV) infection was vertical transmission. The hepatitis C virus (HCV) co-infection rate was 1.5%; however, only 50.8% of patients were evaluated for HCV. The use of herbal or complementary medicines was reported in 33.5% of the patients. The majority of patients (97.6%) were treated with oral nucleoside/nucleotide analogues. Several characteristics were different between the patients treated at referral hospitals and local hospitals/clinics, including the disease state, choice of antiviral drug, and methods of HBV DNA measurement.

Conclusions

This study provides a comprehensive picture of the clinical and laboratory characteristics of patients treated in Korea. Efforts to optimize management strategies are warranted.     

Hepatitis B Core Antigen Expression in Hepatocytes Reflects Viral Response to Entecavir in Chronic Hepatitis B Patients

Background/Aims

Hepatitis B core antigen is known to be a major target for virus-specific T cells and also reflects the progression of liver dissease and viral replication. Hepatitis B core antigen expression in hepatocytes leads to altered histological activity, viral replication, and immune response. The purpose of this study is to evaluate whether the topographical distribution of hepatitis B core antigen expression can predict the viral response to entecavir in patients with chronic hepatitis B.

Methods

We enrolled 91 patients with treatment-naïve chronic hepatitis B. All the patients underwent liver biopsy, and the existence and pattern of hepatitis B core antigen evaluated by immunohistochemistry. All patients received 0.5 mg of entecavir daily following a liver biopsy. We checked the viral response at 3, 6, and 12 months during antiviral therapy.

Results

Of the 91 patients, 64 (70.3%) had hepatitis B core antigen expression. Of the subcellular patterns, the mixed type was dominant (n=48, 75%). The viral response was significantly higher in the hepatitis B core antigen-negative group than in the hepatitis B core antigen-positive group (88.9% and 54.7%, respectively; p=0.001) after 12 months of entecavir therapy.

Conclusions

Chronic hepatitis B patients who are hepatitis B core antigen-negative have a better response to entecavir therapy than do hepatitis B core antigen-positive patients.     

Efficient lysis of rhabdomyosarcoma cells by cytokine-induced killer cells: implications for adoptive immunotherapy after allogeneic stem cell transplantation

Background

Rhabdomyosarcoma is the most common soft tissue sarcoma in childhood and has a poor prognosis. Here we assessed the capability of ex vivo expanded cytokine-induced killer cells to lyse both alveolar and embryonic rhabdomyosarcoma cell lines and investigated the mechanisms involved.

Design and Methods

Peripheral blood mononuclear cells from six healthy donors were used to generate and expand cytokine-induced killer cells. The phenotype and composition of these cells were determined by multiparameter flow cytometry, while their cytotoxic effect against rhabdomyosarcoma cells was evaluated by a europium release assay.

Results

Cytokine-induced killer cells efficiently lysed cells from both rhabdomyosarcoma cell lines. Antibody-mediated masking of either NKG2D molecule on cytokine-induced killer cells or its ligands on rhabdomyosarcoma cells (major histocompatibility antigen related chain A and B and UL16 binding protein 2) diminished this effect by 50%, suggesting a major role for the NKG2D molecule in rhabdomyosarcoma cell killing. No effect was observed after blocking CD11a, CD3 or TCRαβ molecules on cytokine-induced killer cells or CD1d on rhabdomyosar-coma cells. Remarkably, cytokine-induced killer cells used tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to activate caspase-3, as the main caspase responsible for the execution of apoptosis. Accordingly, blocking TRAIL receptors on embryonic rhabdomyosarcoma cell lines significantly reduced the anti-tumor effect of cytokine-induced killer cells. About 50% of T cells within the cytokine-induced killer population had an effector memory phenotype, 20% had a naïve phenotype and approximately 30% of the cells had a central memory phenotype. In addition, cytokine-induced killer cells expressed low levels of activation-induced markers CD69 and CD137 and demonstrated a low alloreactive potential.

Conclusions

Our data suggest that cytokine-induced killer cells may be used as a novel adoptive immunotherapy for the treatment of patients with rhabdomyosarcoma after allogeneic stem cell transplantation.