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.

A large-scale method for the selective depletion of alphabeta T lymphocytes from PBSC for allogeneic transplantation.

BACKGROUND: We sought to develop a method for the clinical large-scale depletion of alphabeta T lymphocytes from mobilized peripheral stem cells, which would allow the allogeneic transplantation of a graft enriched for stem cells, natural killer (NK) cells and gammadelta T lymphocytes. METHODS: Therefore, we obtained mononuclear cells from either mobilized or non-mobilized healthy adult volunteer donors and incubated the cells with a biotinylated anti-alphabeta T-cell Ab and subsequently with an anti-biotin Ab conjugated with magnetic microbeads. The depletion was then performed using a CliniMACS device. RESULTS: The median T-cell depletion was 3.9 log (range 3.5-4.1 log). The recovery of the gammadelta and NK cells was 92% and 80%, respectively. The recovery of CD34+ stem cells from the mobilized donors was 66%. DISCUSSION: This method had no negative influence on the in vitro colony formation of stem cells, and transplantation of alphabeta-depleted cells into NOD-SCID IL-2 common gamma chain knockout (NOD-scid IL2r (null)) mice resulted in a rapid engraftment of human myeloid and lymphoid cells. This method will allow large-scale depletion of alphabeta T cells from mobilized peripheral blood in clinical trials.

A one-step large-scale method for T- and B-cell depletion of mobilized PBSC for allogeneic transplantation.

BACKGROUND: The presence of T and B cells in allogeneic grafts contributes to GvHD and to EBV-associated lymphoproliferative disease (LPD). Depletion of T and B cells from the graft decreases the risk of these complications. METHODS: T and B cells were depleted from mobilized peripheral stem cells from volunteer donors (n=5) using anti-CD3 and anti-CD19 Abs conjugated to magnetic microbeads, and the CliniMACS device. The function of the stem cells after depletion was evaluated using colony assays and non-obese diabetic (NOD)/SCID repopulating experiments. RESULTS: The mean mononuclear cell (MNC) count prior to T- and B-cell depletion was 2.19x10(10) (range 1.48-3.53). After depletion, the mean percentage of contaminating T cells was 0.02% (range 0.01-0.04%) with a mean log(10) depletion of 3.4 (range 3-3.8). The mean percentage of contaminating B cells was 0.1% (range 0.01-0.4%) with a mean log(10) depletion of 2.2 (range 1.4-3). The mean recovery of CD3- and CD19-negative MNCs after depletion was 70% (range 54-88%) and the mean recovery of CD34(+) stem cells was 69% (range 52-98%). The mean number of natural killer (NK) cells after T- and B-cell depletion was 5.2x10(8) (range 2-10x10(8)). In vitro colony assays and in vivo NOD/SCID repopulation assays showed no negative impact of this method on the function of the hematopoietic stem cells. DISCUSSION: Our results show that the CliniMACS system can be used to efficiently deplete PBSC of T and B cells simultaneously, without adverse effect on the graft.

Purification of human natural killer cells using a clinical-scale immunomagnetic method.

BACKGROUND: Infection, graft failure, disease relapse, and GvHD are significant adverse events associated with allogeneic BMT. Although donor leukocyte infusion has been used to prevent or to treat infection, graft failure, and relapse, the potential clinical benefits are often outweighed by the risk of T cell-mediated GvHD. Results from animal studies suggest that donor natural killer (NK) cells may be an ideal cell type for prevention or treatment of these adverse events. We have therefore sought to develop an automated, efficient, and clinical-scale human NK cell-purification method. METHODS: Twelve leukopheresis products were purified for NK cells using a two-step immunomagnetic method. CD3(+) cells were first depleted from the apheresis products. CD56(+) cells were then enriched from the CD3(+) cell-depleted products. RESULTS: The median percentage of CD3(-)CD56(+) NK cells in the final products was 91.0%, and the median recovery was 48.7%. The median depletion for CD3(+)CD56(-) T cells was 5.3 log. Natural cytotoxicity of the purified cells was approximately five-fold higher than that of unpurified mononuclear cells, and it could be further increased by stimulation of the purified cell with IL2. DISCUSSION: We described a large-scale purification method for automated, efficient, and rapid isolation of human NK cells that yielded minimal contamination with T cells or B cells. These purified NK cells may be expedient for preclinical and clinical uses.

Large-scale isolation of CD133+ progenitor cells from G-CSF mobilized peripheral blood stem cells.

We have evaluated the feasibility of large-scale isolation of CD133+ progenitors from healthy mobilized adult donors for potential clinical use in autologous and allogeneic transplantation. A total of 11 healthy volunteer adult donors were mobilized with G-CSF. CD133+ stem cells were isolated from a single leukapheresis using the Clinimacs method. The median percentage of CD133 before positive selection was 0.75% (range 0.39-2.03%). After selection, the median purity and recovery was 94% (range 85.2-98.0%) and 69% (range 44-100%), respectively. The median log10 T-cell depletion obtained by CD133+ positive selection was 4.2 (range 3.8-4.7). The CD133+ progenitors were highly enriched in colony-forming units (CFU) and transplantation into NOD/SCID mice resulted in a high engraftment rate. Transplantation of sorted CD133+/CD34+ cells into NOD/SCID mice showed a higher engraftment compared to CD133-/CD34+ cells. Mobilized peripheral CD133+ stem cells can be purified in large scale for potential clinical use. The biological function of the cells is not impaired. The majority of the NOD/SCID repopulating cells are within the CD133+/CD34+ subpopulation. Therefore, clinical studies using purified CD133+ stem cells can be envisoned to further clarify the role of CD133+ stem cells in hematopoietic reconstitution after transplantation.

A large-scale method for T cell depletion: towards graft engineering of mobilized peripheral blood stem cells.

We have investigated the feasibility and efficacy of large-scale T cell depletion from granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood stem cells (PBSC). The method is based on the use of a CD3 antibody conjugated to magnetic microbeads and magnetic activated cell sorting (Clinimacs). A total of eight large-scale experiments were performed. In four experiments, CD3(+) T cells were depleted from PBSC obtained from volunteers mobilized with G-CSF whereas, in four experiments, T cells were depleted from PBSC from stem cell donors, in which the CD34(+) stem cells had been removed for allogeneic transplantation by positive selection prior to T cell depletion. The mean number of processed mononuclear cells (MNCs) was 3.3 x 10(10) (range 1.5 x 10(10)-5.1 x 10(10)) with a mean T cell proportion of 35.8% (range 16.7-64.0%). After T cell depletion, the percentage of contaminating T cells was 0.15% (range 0.01-1.01%) with a mean log(10) depletion of 3.4 (range 2.8-4.1). The mean recovery of CD3-negative MNCs after depletion was 76% (range 52-100%). The mean recovery of CD34(+) stem cells in the four evaluable experiments was 82% (range 75-92%). In vitro colony assays and in vivo NOD/SCID repopulation assays showed that this large-scale T cell depletion method has no negative impact on the function of the hematopoietic precursor cells. Therefore, we conclude that this T cell depletion method is a valuable tool for further graft engineering strategies involving mobilized PBSCs.

Retrovirus-mediated gene transfer of the cytokine genes interleukin-1beta and tumor necrosis factor-alpha into human neuroblastoma cells: consequences for cell line behavior and immunomodulatory properties.

We have investigated the value of a gene therapy approach for neuroblastoma (NB), based on retroviral transduction of the IL-1beta or TNF-alpha cytokine genes into human NB lines. Secretion of the corresponding cytokine, was demonstrated in all lines, although with considerable quantitative variations. Cytokine gene expression significantly reduced the proliferation index (p = 0.0001); this effect was associated with either terminal neuronal (one TNF-alpha line) or fibroblast-like differentiation (two IL-1beta lines), leading to growth arrest after a few weeks. Cell surface levels of CD54 and HLA class II remained unaffected, but HLA class I (p < 0.001) and CD58 expression (p = 0.01) increased on SKNSH after TNF-alpha gene transfer. Mononuclear cells from normal allogeneic donors cocultured with both IL-1beta (p < 0.001) and TNF-alpha lines (p < 0.01), showed a significant increase in the proportion of activated T cells (CD3+DR+); however, their cytotoxicity and proliferation rate remained unchanged. Immunotherapy of neuroblastoma will require identification of transduced lines in which cytokine secretion induces phenotypic changes in such a way as to augment their likely immunomodulatory properties without impeding cell growth. Because of the limited efficacy of IL-1beta or TNF-alpha gene transfer alone, further studies should focus on combination with other immunomodulatory agents, to improve their potential efficacy in neuroblastoma.

IL-2 adenovector-transduced autologous tumor cells induce antitumor immune responses in patients with neuroblastoma.

In many different murine models, the immunogenicity of tumor cells can be increased by transduction with a range of immunostimulatory genes, inducing an immune response that causes regression of pre-existing unmodified tumor cells. To investigate the relevance of these animal models to pediatric malignancy, we used autologous unirradiated tumor cells transduced with an adenovirus-IL-2 to immunize 10 children with advanced neuroblastoma. In a dose-escalation study, we found that this tumor immunogen induced a moderate local inflammatory response consisting predominantly of CD4(+) T lymphocytes, and a systemic response, with a rise in circulating CD25(+) and DR+ CD3(+) T cells. Patients also made a specific antitumor response, manifest by an IgG antitumor antibody and increased cytotoxic T-cell killing of autologous tumor cells. Clinically, five patients had tumor responses after the tumor immunogen alone (one complete tumor response, one partial response, and three with stable disease). Four of these five patients were shown to have coexisting antitumor cytotoxic activity, as opposed to only one of the patients with nonresponsive disease. These results show a promising correlation between preclinical observations and clinical outcome in this disease, and support further exploration of the approach for malignant diseases of children.

Interleukin-2 gene-modified allogeneic tumor cells for treatment of relapsed neuroblastoma.

Tumor cells that have been genetically modified to express immunostimulatory genes will induce effective antitumor responses in a range of syngeneic animal models. For human applications, transduced autologous tumor cell lines are often difficult or impossible to prepare, so that there are strong incentives for substituting a standardized allogeneic tumor cell line. However, such lines may be inferior immunogens if they differ from host tumors in the antigens they express. We have evaluated the safety, immunostimulatory, and antitumor activity of an interleukin-2-secreting allogeneic neuroblastoma cell line in 12 children with relapsed stage IV neuroblastoma. They received two to four subcutaneous injections of cells in a dose-escalating schedule, up to a maximum of 10(8) cells per injection. There was induration and pruritus at the injection site, and skin biopsies revealed mild panniculitis with CD3+ cells surrounding scanty residual tumor cells. There was a limited but significant peripheral monocytosis. No patient showed any increase in direct cytotoxic effector function against the immunizing cell line, but 3 patients had a rise in the frequency of neuroblastoma-reactive cytotoxic T lymphocyte precursor cells. One child had > 90% tumor response (PR), 7 had stable disease, and 4 had progressive disease in response to vaccine alone. Although these results offer some encouragement for the continued pursuit of allogeneic vaccine strategies in human cancer, the antitumor immune responses we observed are inferior to those obtained in an earlier immunization study using autologous neuroblastoma cells. Hence, we suggest that this earlier approach remains preferable, its difficulties notwithstanding.

Predominance and characteristics of Burkitt lymphoma among children with non-Hodgkin lymphoma in northeastern Brazil.

The purpose of this paper was to define the histologic distribution, clinical features, and treatment response of childhood non-Hodgkin lymphoma (NHL) in northeastern Brazil. We reviewed medical records and histopathologic studies of 98 children treated for NHL from 1980 to 1987 at a major pediatric cancer center in Recife, Brazil. Treatment outcome was evaluated in relation to tumor burden (stage and serum LDH) and type of therapy (LSA2L2 vs other multiagent chemotherapy). There was a striking predominance of the small noncleaved cell (Burkitt) subtype, which occurred in 92 of the 98 children and adolescents diagnosed with NHL. Subsequent analyses focused on these patients. The majority (n = 84) had advanced (stage III/IV) disease at diagnosis. The abdomen was the most common site of disease (84 cases); jaw involvement was rare (three cases). Five-year event-free survival (excluding treatment refusals) was significantly better for patients with limited vs advanced stage disease (75 +/- 14% vs 42 +/- 6%; P < 0.04). Elevated serum LDH (>500 U/l) was associated with a poorer outcome (P = 0.008). The type of chemotherapy did not affect EFS (P = 0.95). Only 39% of patients are long-term survivors, reflecting the high rate of septic deaths (25% of patients) and parental refusal/abandonment of therapy (10%). Epstein-Barr virus (EBV) was detected in tumor cells from eight of the 11 cases studied. In clinical presentation, these cases resemble sporadic Burkitt lymphoma, yet in their apparent responsiveness to LSA2L2 therapy and association with EBV, they do not. Childhood NHL in northeastern Brazil is predominantly of the Burkitt subtype, and is associated with clinical features that appear to distinguish it from the endemic and sporadic forms of this tumor. These cases may represent a third or intermediate subtype of Burkitt lymphoma.

Gene marking and gene therapy for transplantation medicine.

The classic application for gene therapy is in the correction of single gene defects, although this has been complicated by the low efficiency of gene transfer into hematopoietic cells. Gene therapy, however, has potential for the modulation of tumor cell growth, drug sensitivity, and antitumor immune responses. In addition, gene marking can be used, in spite of this limited transfer efficiency, to provide information on hematopoiesis, sources of cancer relapse after stem cell transplant, and the relative efficacy of graft manipulation techniques. This article reviews the applications of gene therapy and gene marking in transplantation medicine.

High-efficiency transduction of freshly isolated human tumor cells using adenoviral interleukin-2 vectors.

Tumor cells genetically modified to express immunostimulatory molecules can produce high levels of antitumor immunity in rodent models. Although a number of clinical trials are currently in progress to assess the value of the approach in human disease, almost all require ex vivo transduction of cultured tumor cells with retroviral vectors. This process is not feasible for many human malignancies, hampering clinical evaluation of the approach. We have used an E1a,1b/E3 deletion mutant of adenovirus containing either the lacZ or the human interleukin-2 (IL-2) gene to transduce human neuroblastoma cells. This vector transduces fresh neuroblastoma cells and neuroblastoma cell lines with an efficiency of 80-90%, compared to an efficiency of 0-14% obtained with retroviral vectors. Cells transduced with the IL-2 adenovector produce up to 12,000 pg of IL-2/10(6) cells/24 hr. IL-2 adenovector-transduced neuroblasts are immunostimulatory; when they are cultured with patient lymphocytes, they increase the proportion of DR+ T cells and generate major histocompatibility complex (MHC) unrestricted cytotoxic effector cells active against parental (nontransduced) tumor cells. We conclude that IL-2 adenovector can be used to transduce freshly isolated human tumor cells efficiently, which will then produce immunomodulatory quantities of the cytokine. The use of adenoviral rather than retroviral vectors facilitates preparation of human tumor "vaccines" and these vectors are now being used in our clinical study of neuroblastoma patients.

Immunomodulatory effects of human neuroblastoma cells transduced with a retroviral vector encoding interleukin-2.

We have investigated whether retroviral mediated transfer of the IL-2 gene renders human neuroblastoma cells immunogenic, justifying their use in a clinical tumor immunization study. Fourteen neuroblastoma cell lines were established from patients with disseminated neuroblastoma and transduced with the vector G1Ncvl2, which contains the neomycin phosphotransferase gene and the cDNA of the human interleukin-2 gene. Clones secreting > 150 pg/10(6) cells/24 h of IL-2 were selected for further study. Secretion of IL-2 was maintained for at least 3 weeks in nonselective media, implying that production of the cytokine would continue under in vivo conditions. Co-culture of IL-2 transduced cell lines with patient lymphocytes induced potent cytotoxic activity against both transduced and parental neuroblastoma cell lines. This activity was HLA unrestricted, and predominantly mediated by CD16+ or CD56+ and CD8- lymphocytes. These data form the preclinical justification for our current immunization protocol for patients with relapsed or resistant neuroblastoma.

Comprometimento ocular no linfoma de burkitt e na leucemia linfocítica aguda: critério de classificaçäo entre essas patologias / Ocular finding in Burkitt's lymphoma and lymphocitic leukemia: criterion of the classification of these pathologies

Os autores descrevem um caso com diagnóstico de linfoma näo-Hodgkin (Burkitt) cujas manifestaçöes oculares foram as típicas de uma leucemia linfocítica aguda e discutem clinicamente os critérios de classificaçäo destas patologias