Automated production at the curie level of no-carrier-added 6-[(18)F]fluoro-L-dopa and 2-[(18)F]fluoro-L-tyrosine on a FASTlab synthesizer.

An efficient, fully automated, enantioselective multi-step synthesis of no-carrier-added (nca) 6-[(18)F]fluoro-L-dopa ([(18)F]FDOPA) and 2-[(18)F]fluoro-L-tyrosine ([(18)F]FTYR) on a GE FASTlab synthesizer in conjunction with an additional high- performance liquid chromatography (HPLC) purification has been developed. A PTC (phase-transfer catalyst) strategy was used to synthesize these two important radiopharmaceuticals. According to recent chemistry improvements, automation of the whole process was implemented in a commercially available GE FASTlab module, with slight hardware modification using single use cassettes and stand-alone HPLC. [(18)F]FDOPA and [(18)F]FTYR were produced in 36.3 ± 3.0% (n = 8) and 50.5 ± 2.7% (n = 10) FASTlab radiochemical yield (decay corrected). The automated radiosynthesis on the FASTlab module requires about 52 min. Total synthesis time including HPLC purification and formulation was about 62 min. Enantiomeric excesses for these two aromatic amino acids were always >95%, and the specific activity of was >740 GBq/µmol. This automated synthesis provides high amount of [(18)F]FDOPA and [(18)F]FTYR (>37 GBq end of synthesis (EOS)). The process, fully adaptable for reliable production across multiple PET sites, could be readily implemented into a clinical good manufacturing process (GMP) environment.

Mesenchymal stromal cells in the antimicrobial host response of hematopoietic stem cell recipients with graft-versus-host disease--friends or foes?

Mesenchymal stromal cells (MSCs) are multipotent cells, which exhibit broad immunosuppressive activities. Moreover, they may be administered irrespectively of human leukocyte antigen (HLA) compatibility, without inducing life-threatening immunological reactions, as they express no HLA class II and limited HLA class I antigens under resting conditions. These characteristics have made MSC an appealing candidate for cell therapy after hematopoietic stem cell transplantation (HSCT), for example, for treatment of graft-versus-host disease (GvHD) or for graft rejection prevention/treatment in allogeneic HSCT recipients. Unfortunately, information regarding the effect of MSC infusion on the host response to infectious agents is scarce, and study results on infectious complications in patients receiving MSC are conflicting. The present review focuses on the available data from in vitro studies and animal models regarding the interaction of MSC with bacterial, viral and fungal pathogens. In a clinical part, we present the current information on infectious complications in allogeneic HSCT recipients who had received MSCs as prophylaxis or treatment of GvHD disease.

Bone marrow transplantation improves the outcome of Atm-deficient mice through the migration of ATM-competent cells.

Ataxia telangiectasia (A-T) is a highly pleiotropic disorder. Patients suffer from progressive neurodegeneration, severe bronchial complications, immunodeficiency, hypersensitivity to radiotherapy and elevated risk of malignancies. Leukemia and lymphoma, along with lung failure, are the main causes of morbidity and mortality in A-T patients. At present, no effective therapy for A-T exists. One promising therapeutic approach is bone marrow transplantation (BMT) that is already used as a curative therapy for other genomic instability syndromes. We used an established clinically relevant non-myeloablative host-conditioning regimen and transplanted green fluorescent protein (GFP)-expressing ataxia telangiectasia mutated (ATM)-competent bone marrow-derived cells (BMDCs) into Atm-deficient mice. GFP expression allowed tracking of the potential migration of the cells into the tissues of recipient animals. Donor BMDCs migrated into the bone marrow, blood, thymus, spleen and lung tissue of Atm-deficient mice showing an ATM-competent phenotype. BMT inhibited thymic lymphomas, normalized T-lymphocyte populations, improved weight gain and rearing activity of Atm-deficient mice. In contrast, no GFP(+) cells were found in the cerebellum or cerebrum, and we detected decreased size index in MRI imaging of the cerebellum in 8-month-old transplanted Atm-deficient mice in comparison to wild-type mice. The repopulation with ATM-competent BMDCs is associated with a prolonged lifespan and significantly improved the phenotype of Atm-deficient mice.

Identification and selective depletion of alloreactive T-cells for adoptive immunotherapy.

BACKGROUND: T-cell-depleted allografts may exhibit delayed T-cell recovery, severe infections, and relapse after haploidentical hematopoietic stem cell transplantation (HSCT). Required donor lymphocyte infusions containing nonalloreactive cells may transfer immune function without causing graft-versus-host disease. METHODS: We developed an ex vivo approach for the immunomagnetic depletion of alloreactive CD25+, CD69+, and HLA-DR+ T-cells. To achieve highest rates of alloantigen expression, we cocultured peripheral blood mononuclear cells (PBMNCs) with PBMNCs (A/B*), dendritic cells (A/B* DCs), or cytokine-pretreated PBMNCs (A/B* cyt cells). Functional analyses were performed after depletion. RESULTS: After coculture with PBMNCs (A/B* cells), 29% of T-cells became CD25+, CD69+, and HLA-DR+. In modified mixed lymphocyte reactions (MLR) (A/B* cyt cells and A/B* DCs), 35% and 37% of T-cells became CD25+, CD69+, and HLA-DR+. Alloactivation was confirmed by interferon gamma release and proliferation. Immuno-magnetic depletion produced <1% alloactivated cells. Furthermore, this depletion strategy was allospecific and hardly impaired the immune function of the retained cells. DISCUSSION: The efficiency of immunomagnetic depletion depended on the stimulatory capacity of stimulator cells and was improved by using cytokine-pretreated PBMNCs for alloactivation. Overall, this approach might be a promising strategy for restoration of the immune system, particularly after haploidentical HSCT.

Enrichment of cell subpopulations applying automated MACS technique: purity, recovery and applicability for PCR-based chimerism analysis.

Enrichment of cell subpopulations is a prerequisite for lineage-specific chimerism analysis (LCA), a frequent approach in follow-up after allo-SCT. An efficient enrichment technique is Magnetic Cell Sorting (MACS) using the AutoMACS separator. However, evaluation of purity, recovery and applicability for PCR-based chimerism analysis of MACS-enriched subpopulations from post-transplant peripheral blood, providing reduced cell numbers and/or unbalanced proportions of subpopulations, is currently unavailable. We performed enrichment of CD3-, CD14-, CD15-, CD19- and CD56-positive subpopulations using 'Whole Blood MicroBeads' and AutoMACS separator in 137 prospectively collected peripheral blood samples from 15 paediatric patients after allo-CD3-/CD19-depleted SCT. Purity was assessed by immune phenotyping. Recovery and applicability for chimerism analysis was evaluated. Excellent purity >90% was achieved in CD14-, CD15-positive cells in 81%, 95% of the isolates and in 86% of CD3 and CD19 isolates, if ACC was >400 cells per mul. Median purity of CD56-positive isolates was 78.9%. Recovery >90% was between 93 (CD56) and 37% (CD15). Conventional and real-time PCR-based chimerism analysis was feasible in virtually all samples. Isolation of cell subpopulations by automated cell enrichment in post-transplant peripheral blood is feasible and fast providing excellent purity and recovery for routine lineage-specific chimerism analysis.

WT1 peptide-specific T cells generated from peripheral blood of healthy donors: possible implications for adoptive immunotherapy after allogeneic stem cell transplantation.

The Wilms tumor antigen, WT1, is expressed at high levels in various types of leukemia and solid tumors, including lung, breast, colon cancer and soft tissue sarcomas. The WT1 protein has been found to be highly immunogenic, and spontaneous humoral and cytotoxic T-cell responses have been detected in patients suffering from leukemia. Furthermore, major histocompatibility complexes class I- and II-restricted WT1 peptide epitopes have been shown to elicit immune responses in patients with WT1-expressing tumors. As a consequence, WT1 has become an attractive target for anticancer immunotherapy. In this study, we investigated the feasibility of generating WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation. We analyzed the incidence of T cells specific for WT1 peptide epitopes in cancer patients and healthy volunteers. It is noted that we could generate WT1-specific responses in nine of ten healthy volunteer donors and established T-cell clones specific for two WT1-derived peptide epitopes. These in vitro expanded WT1-specific T cells effectively lysed WT1-expressing tumor cell lines, indicating the potential clinical impact of ex vivo expanded donor-derived WT1-specific T cells for adoptive immunotherapy after allogeneic stem cell transplantation.

Efficient in vitro generation of adult multipotent cells from mobilized peripheral blood CD133+ cells.

OBJECTIVES: To generate non-haematopoietic tissues from mobilized haematopoietic CD133(+) stem cells. MATERIALS AND METHODS: Mobilized peripheral blood CD133(+) cells from adult healthy donors were used. In vitro ability of highly enriched CD133(+) cells from mobilized peripheral blood to generate multipotent cells, and their potential to give rise to cells with characteristics of neuroectoderm, endoderm and mesoderm layers was investigated. RESULTS: We found that a recently identified population of CD45(+) adherent cells generated in vitro after culture of highly purified CD133(+) cells for 3-5 weeks with Flt3/Flk2 ligand and interleukin-6 can, in presence of the appropriate microenvironmental cues, differentiate into neural progenitor-like cells (NPLCs), hepatocyte-like cells and skeletal muscle-like cells. We have termed them to be adult multipotent haematopoietic cells (AMHCs). AMHC-derived NPLCs expressed morphological, phenotypic and molecular markers associated with primary neural progenitor cells. They can differentiate into astrocyte-like cells, neuronal-like cells and oligodendrocyte-like cells. Moreover, AMHC-derived NPLCs produced 3,4-dihydrophenylalanine and dopamine and expressed voltage-activated ion channels, suggesting their functional maturation. In addition, AMHC-derived hepatocyte-like cells and skeletal muscle-like cells, showed typical morphological features and expressed primary tissue-associated proteins. CONCLUSION: Our data demonstrate that AMHCs may therefore serve as a novel source of adult multipotent cells for autologous replacement cell therapies.

Flow cytometry with anti HLA-antibodies: a simple but highly sensitive method for monitoring chimerism and minimal residual disease after HLA-mismatched stem cell transplantation.

Transplantation of HLA-mismatched stem cells may allow determination of chimerism status of single cells by differential expression of HLA molecules. Monoclonal antibodies against HLA antigens can be used to determine the HLA type of sub-populations by standard flow cytometry. Blood samples from 23 patients transplanted from HLA-mismatched family donors were monitored using HLA-specific antibodies. Suitable antibodies could be found for all donor recipient pairs by using differences in HLA Bw4 and Bw6 groups or other serological antigens. Pretransplant controls of donor and recipient were used to correct for variable fluorescence intensities of the antibodies and sub-populations. Owing to the high sensitivity, cell populations with a minimum frequency of 0.1% were detectable. Flow-cytometric analysis was confirmed by chimerism analysis of immunomagnetically isolated T cells by standard PCR technique. In addition to chimerism evaluation, HLA antibodies improved the detection of leukemic cells after transplantation with aberrant phenotype. In conclusion, flow cytometry using antibodies against HLA antigens is an interesting tool for determination of chimerism and minimal residual disease after HLA-mismatched transplantation. Information about the chimerism status is given on a single-cell level and allows fast and convenient analysis of sub-populations.

Determination of residual T- and B-cell content after immunomagnetic depletion: proposal for flow cytometric analysis and results from 103 separations.

BACKGROUND: T- and B-cell depletion of apheresis products is an attractive alternative to standard stem cell enrichment in haplo-identical transplantation. Thorough T- and B-cell depletion is necessary for prevention of acute GvHD and T-cell depletion-associated lymphoproliferative disorders. However, the large number of non-T and -B cells in the graft requires special protocols for the determination of extremely low frequencies of residual T cells. METHODS: Apheresis products from healthy donors were T- and B-cell depleted by the CliniMACS system using CD3 and CD19 Ab reagents and the LS tubing set. The recovery of cells and degree of depletion were determined. A four-color multigating strategy was used for enumeration of residual T and B cells. RESULTS: One-hundred and three separations were performed, with a mean cell recovery of 38+/-12%, CD34 recovery of 61+/-16% and CD56 recovery of 63+/-33%. T and B cells were depleted by log 4.15+/-0.46 and log 3.64+/-0.63, respectively. Four-color multigating flow cytometry allowed the detection of single T cells. DISCUSSION: Combined T- and B-cell depletion is a feasible method for obtaining stem cell grafts with acceptable stem cell recovery, profound T- and B-cell depletion and a very high amount of NK cells and monocytes. However, analysis of residual T cells is challenging and requires special protocols.

Clinical scale isolation of T cell-depleted CD56+ donor lymphocytes in children.

We present a clinical scale method for immunomagnetic separation of CD56+ donor natural killer cells for adoptive immunotherapy of pediatric leukemias after allogeneic transplantation. This time-saving and partially automated procedure employed CD56+ selection followed by CD3+ depletion, resulting in a median purity of 98.6% NK cells and a four-log depletion of T cells. The enriched NK cells demonstrated high cytotoxic activity against K562 target cells and fresh leukemic blasts with low HLA class I expression, which could be further enhanced by IL-2 stimulation. Lysis of NK-insensitive leukemic cells with high HLA class I expression could also be demonstrated via ADCC. Due to the high degree of T cell depletion, alloreactive proliferation in mixed lymphocyte cultures and response to T cell-specific mitogen stimulation was profoundly decreased. Our results suggest that, even in the case of mismatched donors, infusions of donor NK cells with extremely low T cell content may be a promising treatment option for leukemic minimal residual disease after allogeneic transplantation without risk of inducing severe GVHD.

Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase-containing liposomes.

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)-containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H2O2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H2O2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H2O2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H2O2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for life-threatening infections.

Immunological aspects of haploidentical stem cell transplantation in children.

Thirty-eight children with high-risk hematological malignancies underwent transplantation with megadoses of peripheral mobilized CD34+ cells from haploidentical parents (n = 24) or from matched unrelated donors (n = 14). The CD34+ cells were isolated to a purity of > 98% using magnetic-activated cell sorting. This high purity was associated with an almost complete depletion of T lymphocytes. No pharmacological prophylaxis for graft-versus-host disease (GvHD) was used, and significant primary GvHD was not seen. A final engraftment was seen in all patients. Sixteen patients are alive and disease-free with a median follow-up of 24 months. The immunological reconstitution was faster in the patients transplanted with CD34+ stem cells from the haploidentical donors compared to the matched unrelated donors, and the transplantation of large numbers of haploidentical CD34+ stem cells seems to be superior to that of the matched unrelated donors. The phenotypical and functional analysis of the immune reconstitution provided some insights into the biology of transplantation of highly purified CD34+ cells. In this article, we summarize our current results with the transplantation of highly purified stem cells and discuss possible implications for further antileukemic post-transplant therapeutic strategies.

HLA class I polymorphism in the Albanian population.

The HLA class I polymorphism was studied in a sample of the Albanian population. Ninety-three unrelated healthy Albanians were typed for HLA-A, -B and -Cw antigens by standard microlyphocytotoxicity test. The antigens with the highest frequencies were: HLA-A2 (34.4%), A3 (14.5%) and A1 (12.4%); B51 (19.3%), B35 (12.4%) and B18 (10.2%); Cw4 (16.2%), Cw7 (16.2%) and Cw6 (10.8%). The HLA haplotypes with high frequency in Albanians included A2-B51 (4.3%), A2-B18 (2.4%), A2-B35 (2.4%), Cw4-B35 (7.6%), and Cw7-B18 (6.5%), which are not significantly different from the other neighboring populations. Low frequency of HLA-A1-B8 haplotype (1.1%) is noted in the Albanian population. The frequency of HLA-B27 antigen (1.1%) is one of the lowest frequencies observed in Caucasians. Such results are important in studies of HLA-A1-B8, HLA-B27 and disease associations. These findings should be also useful in understanding the origin of Albanians, representing a base for future studies about HLA polymorphism in the Albanian population.

Clinical scale isolation of highly purified peripheral CD34+progenitors for autologous and allogeneic transplantation in children.

We present our experience with three clinical scale isolation methods for positive selection of CD34+ progenitors from peripheral blood for autologous and allogeneic transplantation in children. A combination of the CellPro device and the Magnetic Activated Cell Sorting system (MACS), as well as two different combinations of MACS systems were used (VarioMACS-SuperMACS and SuperMACS-SuperMACS). With the CellPro-MACS combination (16 separations), a median purity of 96.2% and a median recovery of 42% CD34+ cells could be achieved, whereas the two step MACS systems (55 and 29 separations) showed a median purity of 97.6% and 98.0% and a median recovery of 96.5% and 97%, respectively. Depletion of T cells was profound (4-5 log). A total of 34 patients in the autologous and 18 patients in the allogeneic setting have been transplanted with highly enriched CD34+ cells, obtained by these methods. Only one patient failed to engraft, all other patients showed a rapid and sustained hematological engraftment with the longest follow-up of 3 years. In summary, especially the two step MACS systems have proven to be appropriate tools for enrichment of CD34+ cells, yielding both high purity and good recovery, and can thus be used for tumor cell purging in the autologous setting and for effective T cell depletion in the allogeneic setting.

Isolation of highly purified autologous and allogeneic peripheral CD34+ cells using the CliniMACS device.

The CliniMACS CD34+ selection device was used for positive selection of apheresis products for autologous transplantation from 10 patients with malignant diseases and for allogeneic transplantation from 26 healthy donors. A total of 71 separations were performed. In 1 allogeneic donor, CD34+ progenitors were also isolated from bone marrow. Between 0.27 and 8.9 x 10(10) nucleated cells (median 4.9 x 10(10)) containing 0.09%-10.8% (median 0.67%) CD34+ progenitor cells were separated. After separation, a median number of 227 x 10(6) mononuclear cells (MNC) (51-524) were recovered, with a median viability of 99% (22%-100%) and a median purity of 97.0% (68.3%-99.7%) CD34+ cells. Depletion of T cells was extensive, with a median of 0.04% residual CD3+ cells (range <0.01%-0.92%). Residual CD19+ cells were between <0.01% and 17%, including CD34+CD19+ cells. Recovery of CD34+ cells was calculated according to the ISHAGE guidelines and ranged from 24% to 105% (median 71%). We conclude that with the CliniMACS device CD34+ cells with high purity and recovery can be isolated with concomitant effective T cell depletion in the allogeneic setting and with a high purging efficacy in the autologous setting.

Phenotypic and functional characterization of mobilized peripheral blood CD34+ cells coexpressing different levels of c-Kit.

In this report we evaluated the exact expression pattern of c-Kit on mobilized peripheral blood (PB) CD34+ cells. Using a monoclonal antibody against CD117 antigen (95C3), flow cytometric analysis revealed that approximately 25% of the mobilized PB CD34+ cells coexpress c-Kit. This cell fraction showed a considerable heterogeneity with respect to c-Kit expression, consisting of a small fraction with high levels of c-Kit (4.2%) (CD34+/CD117high fraction) and a larger proportion of cells expressing low levels of this antigen (21.0%) (CD34+/CD117low fraction). Clonogenic assays showed that CD34+/CD117high cell fraction consisted almost exclusively of erythroid progenitors, in contrast to CD34+/CD117low cell subset which gave rise mostly to granulocyte-monocyte colonies. The majority of CFU-GEMM and the most primitive week 6 cobblestone area forming cells (CAFCs) segregated in the CD34+/CD117low cell subset, suggesting the highest content of multipotential progenitors within this cell fraction. None of the sorted cell subsets was able to produce reactive oxygen intermediates (ROI). However, ex vivo expansion of the sorted subsets with interleukin 3, stem cell factor and FLT3 ligand for 2 weeks resulted in a significant production of O2- and H2O2/HOCl by CD34+/CD117low cell fraction, compared to the same sorted but not expanded counterparts. According to the major content of multipotential hematopoietic progenitors and highest capacity to generate sufficient amounts of ROI after ex vivo expansion, we suggest that CD34+/CD117low cell subset would be one of the most potential candidates for transplantation in patients with acute lymphoblastic leukemia, which lack c-Kit antigen expression.

Expression of CD44 isoforms by highly enriched CD34-positive cells in cord blood, bone marrow and leukaphereses.

CD34-positive cells were isolated from cord blood (n = 8), bone marrow (n = 4) and leukapheresed material (n = 7), using an immunomagnetic isolation technique, MACS (Miltenyi Biotec, Bergisch Gladbach, Germany). In flow cytometric analysis, cell populations after enrichment revealed a fraction of 96.1% (cord blood), 96.2% (bone marrow) and 98.6% (leukapheresis material) CD34-positive cells. Cells were further stained with antibodies specific for CD44 isoforms: CD44s (SFF-2), CD44v5 (VFF-8) and CD44v6 (VFF-18). CD44-positive cells were detected by directly (FITC, fluorescein isothiocyanate) or indirectly (streptavidin-PE, phycoerythrin)-conjugated fluorochromes in flow cytometric analysis. Analysis was restricted to CD34-positive cells. A high expression of CD44s was noted in all kinds of material under investigation with mean values in the range of 98.6-100%. There was little expression of CD44v6 (mean values in the range of 1.5-3.6%) and very slight expression of CD44v5 (mean values in the range of 0.6-1.4%). The finding that CD34-positive hematopoietic stem cells express CD44v5 and CD44v6 to a very small extent offers the possibility of using antibodies specific to CD44v5 and CD44v6 in immunopurging in the course of autologous stem cell transplantation.

Phagocytic activity and oxidative burst of granulocytes in persons with myeloperoxidase deficiency.

In the present study, phagocytosis and the oxidative metabolism of neutrophil granulocytes from five clinically healthy persons with different degrees of myeloperoxidase deficiency were investigated and compared to those of normal persons. The identification of individuals with myeloperoxidase deficiency was performed with the Bayer/Technicon H3 blood cell counter, which differentiates the leukocytes by measuring the peroxidase activity. Neutrophils of three out of five investigated myeloperoxidase deficient persons showed extremely low peroxidase indices (-53 and lower), but only the neutrophils of one person totally lacked myeloperoxidase. This was demonstrated by comparing myeloperoxidase mass concentration measured with an enzyme immunoassay, lack of HOCl production, and was further confirmed by measuring luminol- and lucigenin-enhanced chemiluminescence. Characteristically, myeloperoxidase deficient granulocytes showed a strikingly decreased luminol-enhanced chemiluminescence while the lucigenin-enhanced chemiluminescence was significantly increased compared to normal granulocytes. Although there is a DNA sequence homology of about 70%, the activity of peroxidase in eosinophils was not affected in any myeloperoxidase deficient person investigated. Moreover, a person with a very rare defect of eosinophil peroxidase had completely normal myeloperoxidase activity. The lack of myeloperoxidase activity is compensated for by an increased phagocytic activity, an increased production of superoxide anion (lucigenin-chemiluminescence) and probably by an alternative metabolism of H2O2; since persons lacking myeloperoxidase activity do not normally suffer from severe infections, H2O2 is obviously metabolized to other reactive oxygen substrates than HOCl, e.g. to OH-radicals.

Isolation and phenotypic characterization of CD117-positive cells.

Mononuclear cells derived from cord blood were stained using the CD1 17-specific, fluorochrome-labeled monoclonal mouse antibody 95C3. Additional staining was performed using an isotype-specific rat-anti-mouse antibody, labeled with supermagnetic microparticles. Target cells were enriched by the technique of magnetic cell separation, MACS. The resulting cell population contained 96.5% (+/-1.7% S.D.) CD1 17-expressing cells (n = 12) with different levels of CD117 antigen expression. Using flow cytometry, two cell populations differing in size were found. A majority (93%) of cells with high forward scatter revealed a phenotype positive for CD117 and CD34. Isolated cells revealed a high fraction of hematopoietic progenitors (16%). The technique presented allows for an alternative approach of stem cell enrichment and might be useful in autologous transplantation of cells with hematopoietic properties.

Interferon-gamma upregulates the susceptibility of human neuroblastoma cells to interleukin-2-activated natural killer cells.

The influence of interferon-gamma on the susceptibility of neuroblastoma cells in cell-mediated killing was investigated. Neuroblastoma cells were only weakly susceptible targets for peripheral mononuclear cells. However, enrichment of natural killer (NK) cells or activation of NK cells with interleukin-2 resulted in a considerable increase of neuroblastoma cell lysis. Pretreatment of neuroblastoma targets with interferon-gamma additionally increased the susceptibility to enriched NK cells as well as to interleukin-2-activated NK cells. The conjugate formation between enriched NK cells and the neuroblastoma targets was not affected by the pretreatment of the targets with interferon-gamma. Concomitantly, treatment of the neuroblastoma targets with interferon-gamma resulted in a strong induction of otherwise poorly expressed major histocompatibility complex (MHC) class I antigen expression. These results suggest that the increased expression of MHC class I antigens on target cells is not always correlated with decreased sensitivity for NK cells but can also be followed by an increased susceptibility for NK cells.