Impact of a multi-professional expert team on EOL care of children with cancer.

BACKGROUND: The quality of end-of-life (Q-EOL) care is influenced by various factors such as resources for palliative care (PC). We introduced a multi-professional expert team (MET) in 2014, which provides home-based care for children and adolescents with incurable cancer. This study investigated the impacts of the outreach activities by the MET on Q-EOL care of pediatric oncology patients. METHODS: This observational study retrospectively examined 112 patients receiving end-of-life care between 1989 and 2018 at a pediatric cancer center in Japan. Some of the indicators of Q-EOL care before and after the introduction of the outreach activities by the MET were compared. The subjects were 92 in pre-MET and 20 in post-MET periods. RESULTS: The median number of days for which the patients stayed at home during the final seven or 30 days were significantly prolonged in the post-MET period (0.0 vs 1.5 days, P = 0.020, 3.0 vs 12.0 days, P = 0.042). The change was more significant in hematologic malignancies than solid and central nervous system tumors. Patients receiving longer PC before their deaths could stay at home longer during the last 7 days. The ratio of patients receiving PC for more than 2 months was significantly increased in post-MET period (60.9 vs 90.0%, P = 0.014). More patients also greeted their deaths at home in the post-MET period (3.3 vs 25.0%, P < 0.001). CONCLUSIONS: The activities of the MET transformed the end-of-life care of children and adolescents with incurable cancer. Earlier transitions to PC from curative treatment were associated with longer home-based care and more deaths at home.

Refractory Ileal Perforations in a Cytomegalovirus-Infected Premature Neonate Resolved After Ganciclovir Therapy.

Severe neonatal gastrointestinal diseases such as necrotizing enterocolitis or spontaneous intestinal perforation are potentially lethal conditions which predominantly occur in preterm infants. Cytomegalovirus (CMV), which is known to cause congenital and acquired infections in the newborns, has also been implicated in such severe gastrointestinal diseases in premature infants. However, the pathogenic role of CMV and effect of antiviral therapy in severe gastrointestinal disease in premature neonates is currently unclear. We present an infant, born at 26-weeks' gestation, presented with progressive dyspepsia and abdominal distention after the closure of the symptomatic patent ductus arteriosus at the day of life (DOL) 4, requiring the emergent surgery for ileal perforation at the DOL8. After the surgery, abdominal symptoms persisted and the second emergent surgery was performed for the recurrent ileal perforation at DOL17. Even then the abdominal symptoms prolonged and pathological examination in the affected intestine at the second surgery showed CMV inclusion body. Immunoreactivity for CMV antigen was detected in the specimen at the first surgery on DOL8. Blood and urinary CMV-DNA were detected at DOL28. CMV-DNA was also detected in the dried umbilical cord which was obtained within a week from birth. A 6-week course of intravenous ganciclovir (12 mg/kg/day) was started at DOL34 and then symptoms resolved along with decreasing blood CMV-DNA. Pathological findings characteristic of CMV were not detected in the resection specimen at the ileostomy closure at DOL94. These observations indicate that anti-CMV therapy may be beneficial for some premature infants with severe CMV-associated gastrointestinal diseases and warrants further studies focusing on pathogenic role, diagnosis, treatment and prevention of this underrecognized etiology of severe gastrointestinal diseases particularly in premature neonates.

Close interaction with bone marrow mesenchymal stromal cells induces the development of cancer stem cell-like immunophenotype in B cell precursor acute lymphoblastic leukemia cells.

Minimal residual disease of leukemia may reside in the bone marrow (BM) microenvironment and escape the effects of chemotherapeutic agents. This study investigated interactions between B cell precursor (BCP)-acute lymphoblastic leukemia (ALL) cells and BM mesenchymal stromal cells (BM-MSCs) in vitro. Five BCP-ALL cell lines established from pediatric patients and primary samples from a BCP-ALL patient were examined by flow cytometry and immunocytochemistry for expression of specific cell surface markers and cell adhesion proteins. The cell lines developed chemoresistance to commonly used anti-leukemic agents through adhesion to MSC-TERT cells in long-term culture. The change in chemosensitivity after adhering to BM-MSCs was associated with the expression of CD34, CD133, P-glycoprotein and BCRP/ABCG2, and downregulation of CD38. Similar phenotypic changes were observed in primary samples obtained by marrow aspiration or biopsy from a BCP-ALL patient. BM-MSC-adhering leukemia cells also showed deceleration of cell proliferation and expressed proteins in the Cadherin and Integrin pathways. These results suggest that BCP-ALL cells residing in the BM microenvironment may acquire chemoresistance by altering their phenotype to resemble that of cancer stem cells. Our results indicate that cell adhesion could be potentially targeted to improve the chemosensitivity of residual BCP-ALL cells in the BM microenvironment.

A non-selective endothelin receptor antagonist bosentan modulates kinetics of bone marrow-derived cells in ameliorating pulmonary hypertension in mice.

The aim of this study was to investigate whether a dual endothelin receptor antagonist bosentan modulates the kinetics of bone marrow-derived stem cells in inhibiting the development of pulmonary hypertension. Bone marrow chimeric mice, transplanted with enhanced green fluorescent protein (eGFP)-positive bone marrow mononuclear cells, were exposed to hypobaric hypoxia or kept in the ambient air, and were daily treated with bosentan sodium salt or saline for 21 days. After the treatment period, right ventricular pressure was measured and pulmonary vascular morphometry was conducted. Incorporation of bone marrow-derived cells was analyzed by immunohistochemistry. Gene expression and protein level in the lung tissue were evaluated by quantitative real-time PCR and western blotting, respectively. The results showed that, in hypoxic mice, right ventricular pressure and the percentage of muscularized vessel were increased and pulmonary vascular density was decreased, each of which was reversed by bosentan. Bone marrow-derived endothelial cells and macrophages in lungs were increased by hypoxia. Bosentan promoted bone marrow-derived endothelial cell incorporation but inhibited macrophage infiltration into lungs. Quantitative real-time PCR analysis revealed that interleukin 6, stromal cell-derived factor-1, and monocyte chemoattractant protein-1 were upregulated by hypoxia, in which interleukin 6 and monocyte chemoattractant protein-1 were downregulated and stromal cell-derived factor-1 was upregulated by bosentan. Protein level of endothelial nitric oxide synthase (eNOS) in the whole lung was significantly upregulated by hypoxia, which was further upregulated by bosentan. Bosentan modulated kinetics of bone marrow-derived ECs and macrophages and related gene expression in lungs in ameliorating pulmonary hypertension in mice. Altered kinetics of bone marrow-derived stem cells may be a novel mechanism of the endothelin receptor blockade in vivo and confer a new understanding of the therapeutic basis for pulmonary hypertension.

Role of microRNAs in glucocorticoid­resistant B­cell precursor acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is the most common malignant disorder in children and intensive combination therapy has markedly improved patient prognosis. However, efficacy of the treatment still fails in 10­15% of patients. Glucocorticoids (GCs) such as prednisone and dexamethasone (DEX) are essential drugs used for ALL chemotherapy, and the response to GC treatment is a strong independent factor of ALL prognosis. In the present study, we examined the mechanism of GC resistance of B­cell precursor ALL (BCP­ALL). As determined by RT­qPCR and western blot analyses, GC treatment upregulated glucocorticoid receptor (GR) protein and Bcl­2­interacting mediator of cell death (BCL2L11, BIM) protein expression, resulting in apoptosis of a GC­sensitive BCP­ALL cell line, but not of a GC­resistant BCP­ALL cell line as shown by flow cytometry. GR was downregulated in a DEX­resistant BCP­ALL cell line which was induced by treatment of cells with increasing concentrations of DEX. Importantly, expression levels of miR­142­3p and miR­17~92 cluster were upregulated in the BCP­ALL cell line with acquired DEX resistance as examined by RT­qPCR. Our results suggest that interference of miR­142­3p and miR­17~92 may overcome the resistance of BCP­ALL to GCs.

Weekly Rituximab Followed by Monthly Rituximab Treatment for Autoimmune Disease Associated With RAS-associated Autoimmune Leukoproliferative Disease.

Recently, a new disease of lymphocyte homeostasis caused by somatic mosaicism for the RAS mutation has been discovered (known as RALD, RAS-associated leukoproliferative disorder). Since few cases have been reported in literature, the prognosis and standard treatment for autoimmune diseases associated with RALD remain poorly understood. Standard rituximab therapy (375 mg/m for 4 wk) is effective in patients with autoimmune diseases, but early recurrences are common. We highlight the potential for monthly administration of rituximab in a patient with autoimmune thrombocytopenia and hemolytic anemia associated with RALD. RALD was diagnosed in an 11-year-old girl following a 9-year history of severe hepatosplenomegaly and autoimmune cytopenias. Genetic analyses confirmed somatic mosaicism for the G13C KRAS mutation without an autoimmune lymphoproliferative syndrome-related gene mutation. Rituximab therapy was used because of the refractory character of the autoimmune cytopenias which failed to respond to steroids and other immunosuppressive agents. Her treatment consisted of weekly infusions of rituximab for 4 weeks followed by monthly rituximab for 11 months. She maintained her response in hematologic parameters for 2 years after monthly rituximab was ceased and her scores representing quality of life were improved. Rituximab could improve clinical responses and quality of life of the patients with RALD.

Anti-tumor effect of AZD8055 against neuroblastoma cells in vitro and in vivo.

Neuroblastoma (NB) is one of the most common solid tumors in children. High-risk NB remains lethal in about 50% of patients despite comprehensive and intensive treatments. Activation of PI3K/Akt/mTOR signaling pathway correlates with oncogenesis, poor prognosis and chemotherapy resistance in NB. Due to its central role in growth and metabolism, mTOR seems to be an important factor in NB, making it a possible target for NB. In this study, we investigated the effect of AZD8055, a potent dual mTORC1-mTORC2 inhibitor, in NB cell lines. Our data showed that mTOR signaling was extensively activated in NB cells. The activity of mTOR and downstream molecules were down-regulated in AZD8055-treated NB cells. Significantly, AZD8055 effectively inhibited cell growth and induced cell cycle arrest, autophagy and apoptosis in NB cells. Moreover, AZD8055 significantly reduced tumor growth in mice xenograft model without apparent toxicity. Taken together, our results highlight the potential of mTOR as a promising target for NB treatment. Therefore, AZD8055 may be further investigated for treatment in clinical trials for high risk NB.

Induction of MEK/ERK activity by AZD8055 confers acquired resistance in neuroblastoma.

Mammalian target of rapamycin (mTOR) complex (mTORC) is frequently activated in diverse cancers. Although dual mTORC1/2 inhibitors are currently under development to treat various malignancies, the emergence of drug resistance has proven to be a major complication. AZD8055 is a novel, potent ATP-competitive and specific inhibitor of mTOR kinase activity, which blocks both mTORC1 and mTORC2 activation. In this study, we acquired AZD8055-resistant neuroblastoma (NB) cell sublines by using prolonged stepwise escalation of AZD8055 exposure (4-12 weeks). Here we demonstrate that the AZD8055-resistant sublines (TGW-R and SMS-KAN-R) exhibited marked resistance to AZD8055 compared to the parent cells (TGW and SMS-KAN). The cell cycle G1/S transition was advanced in resistant cells. In addition, the resistance against AZD8055 correlated with over-activation of MEK/ERK signaling pathway. Furthermore, combination of AZD8055 and MEK inhibitor U0126 enhanced the growth inhibition of resistant cells significantly in vitro and in vivo. In conclusion, these data show that targeting mTOR kinase and MEK/ERK signaling simultaneously might help to overcome AZD8055 resistance in NB.

Long-term Results of the Risk-adapted Treatment for Childhood B-Cell Acute Lymphoblastic Leukemia: Report From the Japan Association of Childhood Leukemia Study ALL-97 Trial.

PURPOSE: This study was conducted as the first clinical trial by Japan Association of Childhood Leukemia Study to improve the outcome of B-cell acute lymphoblastic leukemia and explore a less toxic reinduction block. PATIENTS AND METHODS: From 1997 to 2002, 563 patients with B-cell acute lymphoblastic leukemia aged 1 to 15 years were enrolled. The patients were assigned into 4 risk groups (standard, intermediate, high, or extremely high risk) and treated with regimens intensified according to the risk. Two randomized trials were conducted to compare 2 regimens with and without a 3-week reinduction therapy in the standard-risk group, and to compare the efficacy of pirarubicin with daunorubicin in the intermediate-risk and high-risk groups. Prophylactic cranial irradiation was restricted in patients with high or extremely high risk. RESULTS: The event-free survival (EFS) rate at 10 years for all patients was 77.0%. Those in the standard-risk to extremely high-risk groups were 79.3%, 72.5%, 71.7%, and 66.3%, respectively. The 15-week induction/consolidation not followed by reinduction produced 76.4% of the EFS at 10 years comparable with the regimen with reinduction therapy. Pirarubicin at 25 mg/m administered 11 times throughout the treatment produced the EFS comparable with daunorubicin at 30 mg/m. CONCLUSION: The trial produced high survival rates in NCI-HR patients, although the outcomes in NCI-SR patients were not satisfactory possibly due to less intensive central nervous system-directed therapy.

Acute and late toxicities of pirarubicin in the treatment of childhood acute lymphoblastic leukemia: results from a clinical trial by the Japan Association of Childhood Leukemia Study.

BACKGROUND: Anthracyclines are used to treat childhood acute lymphoblastic leukemia (ALL). Even when administered at low doses, these agents are reported to cause progressive cardiac dysfunction. We conducted a clinical trial comparing the toxicities of two anthracyclines, pirarubicin (THP) and daunorubicin (DNR), in the treatment of childhood ALL. The results from our study that relate to acute and late toxicities are reported here. METHODS: 276 children with B-ALL were enrolled in the trial from April 1997 to March 2002 and were randomly assigned to receive a regimen including either THP (25 mg/m2 × 11) or DNR (30 mg/m2 × 11). Acute toxicity was prospectively assessed based on the National Cancer Institute Common Toxicity Criteria. Acute hematological toxicity was also examined via some parameters. Patients with event-free survival of >5 years were retrospectively surveyed for cardiac function at 5 and 10 years and at the most recent assessment more than 10 years from the onset of ALL. RESULTS: Acute hematological toxicity in the early phase was more significant in the THP arm. Based on ultrasound cardiography, cardiac function was impaired in both groups during the follow-up period, but there was no significant difference between the groups except for a greater decline in fractional shortening on ultrasound cardiography in the DNR arm. CONCLUSIONS: While acute hematological toxicity was more significant in the THP arm, THP also appeared to be less cardiotoxic. However, the evaluation of late cardiotoxicity was limited because only a few subjects were followed beyond 10 years after ALL onset. Considering that the THP regimen produced an EFS rate comparable with that of the DNR regimen, the efficacy and toxicity of THP at reduced doses should be studied in order to identify potentially safer regimens.

Ewing Sarcoma of the Bone With EWS/FLI1 Translocation After Successful Treatment of Primary Osteosarcoma.

Although prognosis in patients with localized osteosarcoma has been dramatically improved by the introduction of multiple chemotherapy agents known as combination chemotherapy, there is growing concern about the development of secondary malignant neoplasms. We report the case of a 13-year-old girl in whom the diagnosis of Ewing sarcoma of bone localized on the shaft of left femur was made 2 years after successful treatment without radiotherapy for osteosarcoma of right proximal femur. EWS-FLI1 fusion gene was detected by reverse transcriptase-polymerase chain reaction. To our knowledge, this is the first case with Ewing sarcoma of the bone as a secondary malignant neoplasm developed in osteosarcoma survivor. We collected 15 cases, included this case, with secondary Ewing sarcoma family of tumor by utilizing the PubMed search and might consider the causes of this secondary cancer.

Correction:Mesenchymal Stromal Cell Secretome Up-Regulates 47 kDa CXCR4 Expression, and Induce Invasiveness in Neuroblastoma Cell Lines.

[This corrects the article DOI: 10.1371/journal.pone.0120069.].

Disease Recurrence After Early Discontinuation of Eculizumab in a Patient With Atypical Hemolytic Uremic Syndrome With Complement C3 I1157T Mutation.

Eculizumab, terminal complement inhibitor, has become the frontline treatment for atypical hemolytic uremic syndrome (aHUS). However, the optimal treatment schedule has not yet been established. We describe here an aHUS patient with a mutation of C3 I1157T who achieved remission with eculizumab and suffered a recurrence after eculizumab discontinuation, a clinical situation that has not been previously described in patients with C3 mutation. A 9-year-old male experienced an onset of aHUS after viral gastroenteritis and was treated with hemodialysis. At 13 years of age he developed bacterial enterocolitis due to Campylobacter jejuni and experienced a recurrence of aHUS. Eculizumab was initiated on day 4 after disease onset resulting in recovering laboratory parameters. The patient received eculizumab for 5 months before its discontinuation. Second relapse induced by bacterial pharyngitis was confirmed 4 months after eculizumab discontinuation and prompt eculizumab reinitiation resulted in rapid remission. The patients carrying mutations in CFH or C3 have a high frequency of relapse and worse prognosis. More than 50% of aHUS relapses occurred during the first year after the onset. Therefore, long-term treatment with eculizumab is appropriate in patients with aHUS who have experienced a relapse or have mutations associated with poor prognosis.

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity.

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4-100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10(-5) and 0.0054, respectively; meta-analysis P = 4.45 × 10(-8), allelic effect size = -11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

Randomized study of granulocyte colony stimulating factor for childhood B-cell non-Hodgkin lymphoma: a report from the Japanese pediatric leukemia/lymphoma study group B-NHL03 study.

The objective of this study was to assess the impact of the primary prophylaxis of granulocyte colony-stimulating factor (G-CSF) in the management of childhood B-cell non-Hodgkin lymphoma (B-NHL). Patients with advanced-stage mature B-NHL were randomized to receive prophylactic G-CSF (G-CSF+) or not receive G-CSF (G-CSF-) based on protocols of the B-NHL03 study. The G-CSF group received 5 µg/kg/d Lenograstim from day 2 after each course of six chemotherapy courses. Fifty-eight patients were assessable, 29 G-CSF + and 29 G-CSF-. G-CSF + patients showed a positive impact on the meantime to neutrophil recovery and hospital stay. On the other hand, they had no impact in the incidences of febrile neutropenia, serious infections, stomatitis and total cost. Our study showed that administration of prophylactic G-CSF through all six chemotherapy courses for childhood B-NHL showed no clinical and economic benefits for the management of childhood B-NHL treatment.

Erratum to: PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.

[This corrects the article DOI: 10.1186/s12935-015-0239-4.].

Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: a systematic genetic study.

BACKGROUND: Hereditary predisposition is rarely suspected for childhood acute lymphoblastic leukaemia (ALL). Recent reports of germline ETV6 variations associated with substantial familial clustering of haematological malignancies indicated that this gene is a potentially important genetic determinant for ALL susceptibility. Our aims in this study were to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which they contributed to the overall risk of childhood ALL. METHODS: Whole-exome sequencing of an index family with several cases of ALL was done to identify causal variants for ALL predisposition. Targeted sequencing of ETV6 was done in children from the Children's Oncology Group and St Jude Children's Research Hospital front-line ALL trials. Patients were included in this study on the basis of their enrolment in these clinical trials and the availability of germline DNA. ETV6 variant genotypes were compared with non-ALL controls to define ALL-related germline risk variants. ETV6 variant function was characterised bioinformatically and correlated with clinical and demographic features in children with ALL. FINDINGS: We identified a novel non-sense ETV6 variant (p.Arg359X) with a high penetrance in an index family. Subsequent targeted sequencing of ETV6 in 4405 childhood ALL cases identified 31 exonic variants (four non-sense, 21 missense, one splice site, and five frameshift variants) that were potentially related to ALL risk in 35 cases (1%). 15 (48%) of 31 ALL-related ETV6 variants clustered in the erythroblast transformation specific domain and were predicted to be highly deleterious. Children with ALL-related ETV6 variants were significantly older at leukaemia diagnosis than those without (10·2 years [IQR 5·3-13·8] vs 4·7 years [3·0-8·7]; p=0·017). The hyperdiploid leukaemia karyotype was highly over-represented in ALL cases harbouring germline ETV6 risk variants compared with the wild-type group (nine [64%] of 14 cases vs 538 [27%] of 2007 cases; p=0·0050). INTERPRETATION: Our findings indicated germline ETV6 variations as the basis of a novel genetic syndrome associated with predisposition to childhood ALL. The development of recommendations for clinical interventions and surveillance for individuals harbouring ALL-related ETV6 variants are needed. FUNDING: US National Institutes of Health and American Lebanese Syrian Associated Charities.

PDK1-mTOR signaling pathway inhibitors reduce cell proliferation in MK2206 resistant neuroblastoma cells.

PURPOSE: AKT plays a pivotal role in the signal transduction of cancer cells. MK2206, an AKT inhibitor, has been shown to be an effective anti-cancer drug to a variety of cancer cell lines. However, some cancer cells acquire resistance to MK2206 and new strategies to suppress these cell lines remain to be developed. EXPERIMENTAL DESIGN: Acquired MK-2206-resistant neuroblastoma (NB) cell sublines were induced by stepwise escalation of MK-2206 exposure (4-12 weeks). MTT assay was used to validate cell proliferation. Flow cytometry was performed for cell cycle analysis. Western blot assay was used for cell signaling study. RESULTS: MK2206 (5-10 µmol) significantly suppressed cell growth of MK2206 non-resistant NB cells (LAN-1, KP-N-SIFA, NB-19 and SK-N-DZ), but is less efficient in inhibiting that of resistant sublines, even after 2-week MK2206-free incubation. MK2206 acted in mTOR-S6K dependent and independent methods. MK-2206 resistant sublines (LAN-1-MK, KP-N-SIFA-MK, and SK-N-DZ-MK) showed lower IC50 of GSK2334470 (PDK1 inhibitor). The cell growth of all sublines was prohibited by AZD8805 (mTOR inhibitor), with IC50 of AZD8805 3-10 times lower than MK2206 non-resistant cells. The signaling profiles of these resistant sublines were characterized by elevated PDK1-mTOR-S6K activity, accompanying by low phosphorylation of AKT compared with non-resistant counterparts. GSK2334470 and AZD8055 effectively inhibited phosphorylation of PDK1 and mTOR, respectively, and induced higher G0-G1 ratio in LAN-1-MK than that in LAN-1 as well. PDK1 and mTOR inhibitors effected on phosphorylation of GSK3ß in some of resistant sublines. CONCLUSION: NB cells can acquire MK2206 resistance after exposure for 4-12 weeks. Resistant cells feature reliance on PDK1-mTOR-S6K pathway and are more sensitive to PDK1 and mTOR inhibitors than the non-resistant counterparts. Thus, suppression of PDK1-mTOR-S6K signaling pathway is an effective way to overcome the MK2206 resistance, and this may be a promising strategy for targeted therapy.

Neutrophils induced licensing of natural killer cells.

Natural killer (NK) cells acquire effector function through a licensing process and exert anti-leukemia/tumor effect. However, there is no means to promote a licensing effect of allogeneic NK cells other than cytomegalovirus reactivation-induced licensing in allogeneic hematopoietic stem cell transplantation in human. In mice, a licensing process is mediated by Ly49 receptors which recognize self-major histocompatibility complex class I. The distribution of four Ly49 receptors showed similar pattern in congenic mice, B10, B10.BR, and B10.D2, which have B10 background. Forty Gy-irradiated 2 × 10(6) B10.D2 cells including splenocytes, peripheral blood mononuclear cells in untreated mice, or granulocyte colony-stimulating factor treated mice were injected intraperitoneally into B10 mice. We found that murine NK cells were effectively licensed by intraperitoneal injection of donor neutrophils with its corresponding NK receptor ligand in B10 mice as a recipient and B10.D2 as a donor. Mechanistic studies revealed that NK cells showed the upregulation of intracellular interferon-γ and CD107a expression as markers of NK cell activation. Moreover, enriched neutrophils enhanced licensing effect of NK cells; meanwhile, licensing effect was diminished by depletion of neutrophils. Collectively, injection of neutrophils induced NK cell licensing (activation) via NK receptor ligand interaction.