Nutritional immunology: function of natural killer cells and their modulation by resveratrol for cancer prevention and treatment.

Natural killer (NK) cells as part of the innate immune system represent the first line of defence against (virus-) infected and malignantly transformed cells. The emerging field of nutritional immunology focuses on compounds featuring immune-modulating activities in particular on NK cells, which e.g. can be exploited for cancer prevention and treatment. The plant-based nutrition resveratrol is a ternary hydroxylated stilbene, which is present in many foods and beverages, respectively. In humans it comprises a large variety of distinct biological activities. Interestingly, resveratrol strongly modulates the immune response including the activity of NK cells. This review will give an overview on NK cell functions and summarize the resveratrol-mediated modulation thereof.

Arabinoxylan rice bran (MGN-3/Biobran) enhances natural killer cell-mediated cytotoxicity against neuroblastoma in vitro and in vivo.

BACKGROUND AIMS: Natural killer cell (NK) cytotoxic activity plays a major role in natural immunologic defences against malignancies. NK cells are emerging as a tool for adoptive cancer immunotherapies. Arabinoxylan rice bran (MGN-3/Biobran) has been described as a biological response modifier that can enhance the cytotoxic activity of NK cells. This study evaluated the effect of MGN-3/Biobran on NK cell activation, expansion and cytotoxicity against neuroblastoma cells. METHODS: NK cells were enriched with magnetic beads and stimulated with MGN-3/Biobran. NK cell activation was evaluated via analysis of their phenotype, and their expansion capability was tracked. The in vitro cytotoxic ability of the activated NK cells was tested against K562, Jurkat, A673, NB1691, A-204, RD and RH-30 cell lines and the in vivo cytotoxic ability against the NB1691 cell line. RESULTS: MGN-3/Biobran stimulation of NK cells induced a higher expression of the activation-associated receptors CD25 and CD69 than in unstimulated cells (P < 0.05). The expression of NKG2D, DNAM, NCRs and TLRs remained unchanged. Overnight MGN-3/Biobran stimulation increased NK cell cytotoxic activity against all cell lines tested in vitro and decelerated neuroblastoma growth in vivo. The mechanism is not mediated by lipopolysaccharide contamination in MGN-3/Biobran. Furthermore, the addition of MGN-3/Biobran promoted NK cell expansion and decreased T cells in vitro. CONCLUSIONS: Our data show that MGN-3/Biobran upregulates NK cell activation markers, stimulates NK cell cytotoxic activity against neuroblastoma in vitro and in vivo and selectively augments the expansion of NK cells. These results may be useful for future NK cell therapeutic strategies of the treatment of neuroblastoma.

Cytokine serum levels during post-transplant adverse events in 61 pediatric patients after hematopoietic stem cell transplantation.

BACKGROUND: Veno-occlusive disease, Graft-versus-Host disease, invasive or localized bacterial, viral and fungal infections are known as adverse events after hematopoietic stem cell transplantation representing the major cause for morbidity and mortality. Detection and differentiation of these adverse events are based on clinical symptoms and routine measurements of laboratory parameters. METHODS: To identify the role of cytokines as a possible complication-marker for adverse events, 61 consecutive pediatric patients with a median age of 7.0 years who underwent hematopoietic stem cell transplantation were enrolled in this single-center retrospective study. Interleukin-1 beta (IL-1ß), soluble interleukin-2 receptor (sIL-2R), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and tumor necrosis factor-α serum (TNF-α) levels were regularly assessed after transplantation and during transplantation related adverse events. RESULTS: Veno-occlusive disease was accompanied by a significant increase in levels of IL-6, IL-8 and TNF-α.Graft-versus-Host disease was associated with a significant increase of IL-10, sIL-2R, IL-6 and TNF-α, depending on the respective stage or grade. Cytokine IL-6 enabled a significant differentiation between sepsis and fungemia, sepsis and viremia, and sepsis and bacteremia. Moreover, cytokine IL-8 enabled a significant differentiation between sepsis and viremia, sepsis and bacteremia, and bacteremia and viremia whereas IL-10 made a distinction between sepsis and viremia possible. CONCLUSION: The data demonstrate that proinflammatory cytokines might be putative indicators for early detection and differentiation of post-transplant adverse events and may allow prompt and adequate clinical intervention. Prospective clinical trials are needed to evaluate these findings.

Transplantation of CD3/CD19 depleted allografts from haploidentical family donors in paediatric leukaemia.

Transplantation of T- and B-cell depleted allografts from haploidentical family donors was evaluated within a prospective phase II trial in children with acute lymphoblastic leukaemia, acute myeloid leukaemia and advanced myelodysplastic syndrome (n = 46). 20 patients had active disease; 19 patients received a second or third stem cell transplantation (SCT). Toxicity-reduced conditioning regimens consisted of fludarabine or clofarabine (in active disease only), thiotepa, melphalan and serotherapy. Graft manipulation was carried out with immunomagnetic microbeads. Primary engraftment occurred in 88%, with a median time to reach >1·0 × 109/l leucocytes, >20 × 109/l platelets and >0·1 × 109/l T-cells of 10, 11 and 50 days, respectively. After retransplantation, engraftment occurred in 100%. Acute graft-versus-host disease (GvHD) grade II and III-IV occurred in 20% and 7%, chronic GvHD occurred in 21%. Both conditioning regimens had comparable toxicity. Transplant-related mortality (TRM) was 8% at one year and 20% at 5 years. Event-free survival at 3 years was: 25% (whole group), 46% (first, second or third complete remission [CR], first SCT) vs. 8% (active disease, first SCT) and 20% (second or third SCT, any disease status). This approach allows first or subsequent haploidentical SCTs to be performed with low TRM. Patients in CR may benefit from SCT, whereas the results in patients with active disease were poor.

Pediatric posttransplant relapsed/refractory B-precursor acute lymphoblastic leukemia shows durable remission by therapy with the T-cell engaging bispecific antibody blinatumomab.

We report on posttransplant relapsed pediatric patients with B-precursor acute lymphoblastic leukemia with no further standard of care therapy who were treated with the T-cell engaging CD19/CD3-bispecific single-chain antibody construct blinatumomab on a compassionate use basis. Blast load was assessed prior to, during and after blinatumomab cycle using flow cytometry to detect minimal residual disease, quantitative polymerase chain reaction for rearrangements of the immunoglobulin or T-cell receptor genes, and bcr/abl mutation detection in one patient with Philadelphia chromosome-positive acute lymphoblastic leukemia. Blinatumomab was administered as a 4-week continuous intravenous infusion at a dosage of 5 or 15 µg/m(2)/day. Nine patients received a total of 18 cycles. Four patients achieved complete remission after the first cycle of treatment; 2 patients showed a complete remission from the second cycle after previous reduction of blast load by chemotherapy. Three patients did not respond, of whom one patient proceeded to a second cycle without additional chemotherapy and again did not respond. Four patients were successfully retransplanted in molecular remission from haploidentical donors. After a median follow up of 398 days, the probability of hematologic event-free survival is 30%. Major toxicities were grade 3 seizures in one patient and grade 3 cytokine release syndrome in 2 patients. Blinatumomab can induce molecular remission in pediatric patients with posttransplant relapsed B-precursor acute lymphoblastic leukemia and facilitate subsequent allogeneic hematopoietic stem cell transplantation from haploidentical donor with subsequent long-term leukemia-free survival.

Siglec-7 tetramers characterize B-cell subpopulations and leukemic blasts.

Cell surface glycosylation has important regulatory functions in the maturation, activation, and homeostasis of lymphocytes. The family of human sialic acid-binding immunoglobulin-like lectins (siglecs) comprises inhibitory as well as activating receptors intimately involved in the regulation of immune responses. Analyses of the interaction between siglecs and glycans are hampered by the low affinity of this interaction. Therefore, we expressed siglec-7 in eukaryotic cells, allowing for glycosylation, and oligomerized the protein in analogy to MHC tetramers. Using this tool, flow cytometric analysis of lymphocytes became possible. Sialic acid-dependent binding of siglec-7 tetramers was confirmed by glycan array analysis and loss of siglec tetramer binding after neuraminidase treatment of lymphocytes. In contrast to most lymphocyte subpopulations, which showed high siglec-7 ligand expression, B-cell subpopulations could be further subdivided according to different siglec-7 ligand expression levels. We also analyzed blasts from acute lymphoblastic leukemias of the B-cell lineage as well as the T-cell lineage, since malignant transformation is often associated with aberrant cell surface glycosylation. While pediatric T-ALL blasts highly expressed siglec-7 ligands, siglec-7 ligands were barely detectable on cALL blasts. Taken together, oligomerization of recombinant soluble siglec-7 enabled flow cytometric identification of physiologic lymphocyte subpopulations and malignant blasts.

Depletion of T-cell receptor alpha/beta and CD19 positive cells from apheresis products with the CliniMACS device.

BACKGROUND AIMS: The CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany) was used for depletion of T-cell receptor alpha/beta positive (TCRαß(+)) and CD19 positive (CD19(+)) cells from apheresis products. METHODS: Investigators performed 102 separations. Apheresis products with a median 5.8 (minimum to maximum, 1.2-10.4) × 10(10) mononuclear cells were used with a median 358 (92-1432) × 10(6) CD34(+) cells. There were 24.8% (6.1-45.7%) median TCRαß(+) cells and 4.4% (1.2-11.7%) median B cells in the apheresis product. RESULTS: After depletion, a median 0.00097% (0.00025-0.0048%) of TCRαß(+) cells could be detected, and B cells, as determined as CD20(+) cells, were reduced to 0.0072% (0.0008-0.072%). TCRαß(+) cells were depleted by log 4.7 (3.8-5.5), and B cells were depleted by log 4.1 (3.0-4.7). Recovery of mononuclear cells was 55% (33-77%), and recovery of CD34(+) cells was 73% (43-98%). Recovery of CD56(+)/3(-) natural killer cells was 80% (35-142%), recovery of TCR gamma/delta positive (TCRγδ(+)) T cells was 83% (39-173%) and recovery of CD14(+) cells was 79% (22-141%). Viability of cells was 98% (93-99%) after separation (all values median). CONCLUSIONS: Profound depletion of TCRαß(+) T cells can be achieved with the CliniMACS system. Recovery of CD34(+) stem cells is in the same range than after CD34(+) enrichment and CD3/CD19 depletion. Transplantation with >4 × 10(6) CD34(+) cells/kg can be performed for every patient with 1-5 × 10(4) TCRαß(+) cells/kg and about 5-10 × 10(6) TCRγδ(+) cells/kg with two rounds of apheresis.

High proportion of leukemic stem cells at diagnosis is correlated with unfavorable prognosis in childhood acute myeloid leukemia.

In acute myeloid leukemia (AML), the leukemia-initiating cell is found within the CD34(+)/CD38(-) cell compartment. Over the last years evidence grew that AML is initiated and propagated by leukemic stem cells (LSCs). Conceivably, these most immature leukemia cells are more resistant to therapy and subsequently initiate relapse. The authors studied 17 patients with childhood AML treated according to the AML-BFM 98/04 protocol. At diagnosis, the authors determined the characteristic immunophenotype of the leukemic cells by flow cytometry and investigated the expression of CD34, CD38, and CD45 to define a population of immunophenotypically immature cells (CD34(+)/CD38(-)/CD45(-/low)) enriched for LSCs in many cases of AML. The authors compared the fraction of this population of all myeloid cells at diagnosis with event-free survival. Kaplan-Meier analysis revealed significant higher event free survival of patients with low CD34(+)/CD38(-)/CD45(-/low) cell proportion (<0.68%) compared to patients with high burden of this population (>0.83%; log-rank P < .04). This correlation was not found for the total number of CD34(+) cells. This is the first study to show that a higher proportion of immature CD34(+)/CD38(-)/CD45(-/low) blasts at diagnosis correlates with unfavorable prognosis in childhood AML. The results suggest that a large CD34(+)/CD38(-)/CD45(-/low) population reflects a higher fraction of LSCs, leading to increased chemotherapy resistance and elevated relapse rate. Thus the initial frequency of CD34(+)/CD38(-)/CD45(-/low) cells may serve as a prognostic marker in pediatric AML. Future treatment in childhood AML should specifically target this immature population as well as the mature blast population.

Fulminant Rhizomucor pusillus mucormycosis during anti-leukemic treatment with blinatumomab in a child: A case report and review of the literature.

This is the first published case report of a child with acute lymphatic leukemia developing a fatal mucormycosis during blinatumomab treatment. The patient showed multiple, systemic thromboembolic lesions with ischemia, bleeding and infarction in almost all organs. The child succumbed to increased brain pressure resulting in cerebral herniation. This case particularly illustrates the fulminant progression and huge challenges of diagnosing and treating mucormycosis in children with hemato-oncological diseases during treatment with targeted therapeutic antibodies (blinatumomab).

Reconstitution of natural killer cell receptors influences natural killer activity and relapse rate after haploidentical transplantation of T- and B-cell depleted grafts in children.

BACKGROUND: Natural killer cells have been demonstrated to exert remarkable graft-versus-leukemia effects after haploidentical transplantation. Acquisition of both, inhibiting and activating, receptors on developing natural killer cells is an important step in their functional maturation. Here, we report on the reconstitution of natural killer receptors after haploidentical transplantation of T-and B-cell (CD3/CD19) depleted grafts with co-transfusion of natural killer cells in children and its influence on natural killer cell activity and clinical outcome. DESIGN AND METHODS: We analyzed reconstitution patterns of natural killer receptors at different time intervals after haploidentical transplantation by multi-color flow cytometry. Natural killer cell activity and antibody-dependent cellular cytotoxicity was tested against cell lines and leukemic blasts in vitro. Survival was analyzed using Kaplan-Meier estimates. RESULTS: Recovery of CD56(+)/CD16(+) cells was fast with high cytolytic activity against K562 and strong antibody-dependent cellular cytotoxicity activity against neuroblastoma and leukemic blasts as early as day 14 posttransplant. KIR reconstitution showed a predominance of KIR negative natural killer cells early after transplantation and an early reconstitution of CD158b compared to CD158a and CD158e. These differences were independent of presence or absence of the corresponding KIR ligands in donors or recipients. This reconstitution pattern was associated with a higher relapse probability of patients homozygous for HLA-C1-alleles compared to patients homozygous or even heterozygous for HLA-C2-alleles. CONCLUSIONS: Our results indicate a fast recovery of functional and alloreactive natural killer cells with a constant KIR pattern after haploidentical transplantation with T- and B-cell depleted grafts. Moreover, these natural killer cells can mediate antibody-dependent cellular cytotoxicity and therefore may allow for an early use of antibodies against residual malignant cells.

The bifunctional flavokinase/flavin adenine dinucleotide synthetase from Streptomyces davawensis produces inactive flavin cofactors and is not involved in resistance to the antibiotic roseoflavin.

Streptomyces davawensis synthesizes the antibiotic roseoflavin, one of the few known natural riboflavin analogs, and is roseoflavin resistant. It is thought that the endogenous flavokinase (EC 2.7.1.26)/flavin adenine dinucleotide (FAD) synthetase (EC 2.7.7.2) activities of roseoflavin-sensitive organisms are responsible for the antibiotic effect of roseoflavin, producing the inactive cofactors roseoflavin-5'-monophosphate (RoFMN) and roseoflavin adenine dinucleotide (RoFAD) from roseoflavin. To confirm this, the FAD-dependent Sus scrofa D-amino acid oxidase (EC 1.4.3.3) was tested with RoFAD as a cofactor and found to be inactive. It was hypothesized that a flavokinase/FAD synthetase (RibC) highly specific for riboflavin may be present in S. davawensis, which would not allow the formation of toxic RoFMN/RoFAD. The gene ribC from S. davawensis was cloned. RibC from S. davawensis was overproduced in Escherichia coli and purified. Analysis of the flavokinase activity of RibC revealed that the S. davawensis enzyme is not riboflavin specific (roseoflavin, kcat/Km = 1.7 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 7.5 10(-3) microM(-1) s(-1)). Similar results were obtained for RibC from the roseoflavin-sensitive bacterium Bacillus subtilis (roseoflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1); riboflavin, kcat/Km = 1.3 10(-2) microM(-1) s(-1)). Both RibC enzymes synthesized RoFAD and RoFMN. The functional expression of S. davawensis ribC did not confer roseoflavin resistance to a ribC-defective B. subtilis strain.

Feasibility and outcome of reduced-intensity conditioning in haploidentical transplantation.

Allogeneic stem cell transplantation is for a number of patients with malignant and nonmalignant diseases the only curative approach. For those patients who do not have an HLA-identical-related or -unrelated stem cell donor, a related three-loci mismatch haploidentical stem cell transplantation with T cell-depleted stem cells is a viable option. T cell depletion either by CD34(+) positive selection or by CD3-negative depletion strategies is available and has been investigated. We have shown that reduced-intensity conditioning haploidentical transplantation using mobilized peripheral stem cells negatively depleted from T and B lymphocytes is associated with a rapid immune reconstitution, a low transplant-related mortality rate, and a favorable outcome in patients in remission at the time of transplant. For chemorefractory patients, additional posttransplant cellular and humoral immunotherapeutic strategies are needed for prevention of relapse after transplantation.

Reconstitution of an apicoplast-localised electron transfer pathway involved in the isoprenoid biosynthesis of Plasmodium falciparum.

In the malaria parasite Plasmodium falciparum isoprenoid precursors are synthesised inside a plastid-like organelle (apicoplast) by the mevalonate independent 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway. The last reaction step of the DOXP pathway is catalysed by the LytB enzyme which contains a [4Fe-4S] cluster. In this study, LytB of P. falciparum was shown to be catalytically active in the presence of an NADPH dependent electron transfer system comprising ferredoxin and ferredoxin-NADP(+) reductase. LytB and ferredoxin were found to form a stable protein complex. These data suggest that the ferredoxin/ferredoxin-NADP(+) reductase redox system serves as the physiological electron donor for LytB in the apicoplast of P. falciparum.

NKG2D Signaling Leads to NK Cell Mediated Lysis of Childhood AML.

Natural killer cells have been shown to be relevant in the recognition and lysis of acute myeloid leukemia. In childhood acute lymphoblastic leukemia, it was shown that HLA I expression and KIR receptor-ligand mismatch significantly impact ALL cytolysis. We characterized 14 different primary childhood AML blasts by flow cytometry including NKG2D ligands. Further HLA I typing of blasts was performed and HLA I on the AML blasts was quantified. In two healthy volunteer NK cell donors HLA I typing and KIR genotyping were done. Blasts with high NKG2D ligand expression had significantly higher lysis by isolated NK cells. Grouping the blasts by NKG2D ligand expression led to a significant inverse correlation of HLA I expression and cytolysis in NKG2D low blasts. Furthermore, a significant positive correlation of NKG2D ligand expression and blast cytolysis was shown. No impact of KIR ligand-ligand mismatch was found but a significantly increased lysis of homozygous C2 blasts by KIR2DL1 negative NK cells (donor B) was revealed. In conclusion, NKG2D signaling leads to NK cell mediated lysis of childhood AML despite high HLA I expression.

Influence of Histone Deacetylase Inhibitors and DNA-Methyltransferase Inhibitors on the NK Cell-Mediated Lysis of Pediatric B-Lineage Leukemia.

Epigenetic drugs like histone deacetylase inhibitors (HDACi) and DNA-methyltransferase inhibitors (DNMTi) have been shown to be effective against a variety of tumor entities. Among different molecular anticancer activities of epigenetic active substances, up-regulation of natural killer (NK) cell ligands was described to contribute to an enhanced NK cell-mediated killing of tumor cell lines. So far, no data is available on this effect in childhood acute lymphoblastic leukemia. We investigated the effect of two HDACi [vorinostat, valproic acid (VPA)] and two DNMTi (azacytidine, decitabine) on the viability, expression of NK ligands, and NK susceptibility of the pre-B-cell-ALL cell line MHH-CALL-4. Whereas vorinostat, azacytidine, and decitabine directly reduced viability of the cell line, VPA had no direct cytotoxic effect. NKG2D-ligands were expressed only at very low levels and not affected by epigenetic treatment. Higher expression was found for the DNAM-1 ligands with significant up regulation of CD112 after treatment with VPA (p = 0.02). No significant increase in lysis mediated by resting NK cells could be observed, whereas incubation of target cells with decitabine resulted in a significant increase in lysis mediated by IL-2 activated NK cells (p = 0.0051, p = 0.06 for azacytidine). Vorinostat and VPA could increase the lysis by expanded NK cells which was statistically not significant due to high inter-individual variability. Furthermore, HDACi but not DNMTi reduced the NK-mediated lysis of MHH-CALL-4 after incubation of effector cells. In conclusion, there is a synergistic effect between epigenetic drugs and NK cells against MHH-CALL-4 which is not as strong as in other tumor entities. In situations where NK-mediated control of leukemia is assumed or wanted, a sophisticated combination of single epigenetic drugs and ex vivo expanded NK cells is needed to maximize the synergistic effect of both treatment strategies and DNMTIs may be preferred based on the direct inhibitory effect of HDACi on NK cell cytotoxicity.

Evaluation of nucleocapsid and phosphoprotein p functionality as critical factors during the early phase of paramyxoviral infection.

In the beginning of a paramyxovirus infection after cell entry viral survival depends on efficient primary (1°) transcription and on the stability of only one input nucleocapsid. Here we examined the influence of the viral polymerase co-factor phosphoprotein P on the very early phase of an infection, i.e. before progeny nucleocapsids are synthesized. We used a novel set-up with Sendai virus (SeV) mutants incapable of genome replication: SeV-ΔP with the entire P ORF deleted, SeV-PΔ2-77 with the deletion of aa 2-77. These mutants allow maintaining the state of the very beginning of an infection when statistically one viral genome is present in the cell. This single genome serves as template for transcription. During SeV-ΔP infections only early 1° transcription takes place at low levels. However, when the truncated P protein is expressed in SeV-PΔ2-77 infections, 1° transcription levels rise significantly up to an 8-fold increased amount of viral mRNA. This shows that the P protein is able to support transcription and thereby mediates the transition from early to late 1° transcription. Importantly, nucleocapsids of both mutants could be shown to remain stable and functional for at least 5 days - even without de novo P protein synthesis. These results describe a novel function of the P protein: enhancing viral gene expression even before genome replication has started. Thus, the since long postulated supportive function of the P protein is not related to stabilization of the nucleocapsid but rather enhances the processivity of the viral polymerase during late 1° and secondary (2°) transcription and genome replication.

CD155 is involved in NK-cell mediated lysis of human hepatoblastoma in vitro.

NK cells are involved in the lysis of different solid tumors and leukemias. NK-activity is thereby regulated by activating and inhibitory receptors. Until now, nothing is known about the NK-activity against hepatoblastoma and the involved receptors. We tested NK cells for cytotoxicity against HB in vitro. Expression levels of activating NK ligands were analysed on 13 primary HB samples as well as on 3 HB cell lines. All HB cell lines showed low HLA-class-I-expression. CD155 expression was strong on primary HB samples and cell lines. NKG2D-ligands (MICA/B, ULBP1-3) were heterogeneous expressed in primary samples and cell cultures. There were no differences between the various histological subtypes. NK cells showed strong cytotoxicity in vitro which was significantly increased through interleukin-2 and -15 stimulation (p less than 0.001). Blockade of CD155 resulted in decreased lysis rates. Our findings show that NK cells exert high activity against hepatoblastoma in vitro and that CD155 is involved in the NK mediated killing of HB. The inclusion of a NK-based immunotherapy into novel treatment strategies might be a promising alternative especially for advanced tumors.

Natural killer cell activity influences outcome after T cell depleted stem cell transplantation from matched unrelated and haploidentical donors.

Lytic activity and recovery of natural killer (NK) cells was monitored in pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n = 18) and mismatched related (haploidentical, n = 29) donors. CD34 + selection with magnetic microbeads resulted in 8 × 10(3)/kg residual T cells. No post-transplant immune suppression was given. NK cells recovered rapidly after transplantation (300 CD56+/µL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/µL at day 90). NK activity was measured as specific lysis of K 562 targets several times (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 2 years, p < 0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 2 years, p < 0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered.

Long-term IL-2 therapy after transplantation of T cell depleted stem cells from alternative donors in children.

The aim of this pilot study was to evaluate the feasibility of long-term subcutaneous application of low-dose IL-2 in children with malignancies at very high risk of relapse who underwent highly T cell and B cell depleted HLA-identical (MUD) or full haplotype mismatched related hematopoetic stem cell transplantation. We studied 11 patients with acute leukemias / myelodysplastic syndrome and juvenile myelomonocytic leukemia (active disease and/or second stem cell transplantation, n = 8; ≥CR 2, n = 2) and relapsed or progressive Ewing's sarcoma (n = 2) who received prophylactic IL-2 treatment for a high probability of disease recurrence after allo-HSCT. Toxicities from IL-2 were transient fever, fatigue and local inflammation. In one patient GvHD grade III with no clear association to IL-2 administration occurred. IL-2 administration was started at median day 57 (range 13-154) post-transplant for a mean duration of 28 days (range 15-250). IL-2 administration clearly increased NK cell activity. 3 of 11 patients (ALL, AML, multifocal Ewings sarcoma) survived with a follow-up of ten years. In conclusion, long-term low-dose IL-2 subcutaneous application is feasible in children due to a low side effect profile even after HLA mismatched transplantation and may be a strategy to prevent relapse in pediatric malignancies with extremely high risk of relapse.

Establishment of a rhabdomyosarcoma xenograft model in human-adapted mice.

The outcome of patients with advanced stage rhabdomyosarcoma (RMS) is still sobering. This outcome has not improved through conservative treatments. Therefore, novel treatment approaches such as immunotherapy need to be evaluated in human-adapted animal models. The aim of this study was to develop a humanized mouse model of childhood RMS as a basis for the study of immunotherapeutic approaches. Therefore, NOD/LtSz-scid IL2rgammanull-mice were used for all the experiments (n=19). The animals underwent sublethal irradiation on days 1 and 2 (1 x 300 cGy). After irradiation, the transplantation of human CD34+-cells (1,000,000 cells per animal i.v.) was carried out. Five animals served as the control and did not undergo stem cell transplantation. The engraftment of human cells was assessed in peripheral blood on days 21 and 55 by FACS analysis. Eight weeks after transplantation, the subcutaneous xenotransplantation of human alveolar and embryonal RMS cell lines was carried out. Tumor growth was monitored and tumors were resected 93 days after CD34+-transplantation. The tumor specimens were evaluated histologically. The successful engraftment of human cells with the establishment of a human immune system was observed in 12 out of 14 animals. B and T cells were mostly detected in the peripheral blood. There were only a few monocytes and almost no natural killer cells. The xenotransplantation of alveolar RMS resulting in subcutaneous tumor growth was feasible in 7 animals. The xenotransplantation of embryonal RMS was performed in 5 animals and led to tumor growth in 1 animal. A histological work up showed either alveolar or embryonal RMS cells with central necrosis. This is the first time a xenotransplantation model of human RMS has been developed in a humanized mouse model. The establishment of subcutaneous tumor xenografts was more effective in the alveolar subtype. This model offers a basic tool for further analyzing novel immunotherapeutic approaches in RMS, and could possibly be used in other solid pediatric tumors.