Quantum phase transitions in the Kondo-necklace model: perturbative continuous unitary transformation approach.

The Kondo-necklace model can describe magnetic low-energy limit of strongly correlated heavy fermion materials. There exist multiple energy scales in this model corresponding to each phase of the system. Here, we study quantum phase transition between the Kondo-singlet phase and the antiferromagnetic long-range ordered phase, and show the effect of anisotropies in terms of quantum information properties and vanishing energy gap. We employ the 'perturbative continuous unitary transformations' approach to calculate the energy gap and spin-spin correlations for the model in the thermodynamic limit of one, two, and three spatial dimensions as well as for spin ladders. In particular, we show that the method, although being perturbative, can predict the expected quantum critical point, where the gap of low-energy spectrum vanishes, which is in good agreement with results of other numerical and Green's function analyses. In addition, we employ concurrence, a bipartite entanglement measure, to study the criticality of the model. Absence of singularities in the derivative of concurrence in two and three dimensions in the Kondo-necklace model shows that this model features multipartite entanglement. We also discuss crossover from the one-dimensional to the two-dimensional model via the ladder structure.

Retrovirus-mediated gene transfer into T cells: 95% transduction efficiency without further in vitro selection.

This study was designed to retrovirally transduce T cells by a protocol that would be simple, short, cost effective, applicable for clinical use, and efficient enough to avoid further selection of transduced T cells. Because retrovirally mediated infection is depending on the cell cycle, we first optimized the conditions for activating T cells in the presence of immobilized CD3 monoclonal antibodies and recombinant interleukin 2. Cell cycle analysis indicated that CD8+ and total T cells reach a maximum of cycling within 4 days whereas CD4+ T cells attain their maximum of cycling only by day 6. Taking into account these data, CD4+, CD8+, and total T cells were preactivated for 5 and 3 days, respectively, and then infected for 24 hr with supernatant containing retrovirus pseudotyped with gibbon-ape leukemia virus envelope, using a cell centrifugation protocol. Results show that approximately 95% of CD4+, CD8+, and total T cells can be transduced, this transduction efficiency being significantly higher than that obtained with amphotropic retrovirus vectors. Furthermore, under permanent growth stimulation, transduced T cells can be expanded approximately 1,000-fold in 4 weeks of culture with maintenance of transgene expression. However, Immunoscope analysis revealed alterations of T cell repertoire diversity after 2-3 weeks in culture that was not due to retroviral transduction per se. Overall, these data provide evidence that T cells can be transduced at levels that may alleviate the need for both further selection of transduced cells and in vitro expansion, thereby preserving the repertoire diversity of the transduced T cells to be reinfused.

High level of retrovirus-mediated gene transfer into dendritic cells derived from cord blood and mobilized peripheral blood CD34+ cells.

Dendritic cells (DCs), the most potent antigen-presenting cells, can be generated from CD34+ hematopoietic stem cells and used for generating therapeutic immune responses. To develop immunotherapy protocols based on genetically modified DCs, we have investigated the conditions for high-level transduction of a large amount of CD34+-derived DCs. Thus, we have used an efficient and clinically applicable protocol for the retroviral transduction of cord blood (CB) or mobilized peripheral blood (MPB) CD34+ cells based on infection with gibbon ape leukemia virus (GALV)-pseudotyped retroviral vectors carrying the nls-LacZ reporter gene. Infected cells have been subsequently cultured under conditions allowing their dendritic differentiation. The results show that using a growth factor combination including granulocyte-macrophage colony-stimulating factor plus tumor necrosis factor alpha plus interleukin 4 plus stem cell factor plus Flt3 ligand, more than 70% of DCs derived from CB or MPB CD34+ cells can be transduced. Semiquantitative PCR indicates that at least two proviral copies per cell were detected. Transduced DCs retain normal immunophenotype and potent T cell stimulatory capacity. Finally, by using a semisolid methylcellulose assay for dendritic progenitors (CFU-DCs), we show that more than 90% of CFU-DCs can be transduced. Such a highly efficient retrovirus-mediated gene transfer into CD34+-derived DCs makes it possible to envision the use of this methodology in clinical trials.

High-level gene transfer to cord blood progenitors using gibbon ape leukemia virus pseudotype retroviral vectors and an improved clinically applicable protocol.

The best methods for transducing hematopoietic progenitor cells usually involve either direct co-cultivation with virus-producing cells or human stromal supportive cells. However, these methods cannot be safely or easily applied to clinical use. Therefore, we aimed at improving retrovirus-mediated gene transfer into hematopoietic progenitors derived from cord blood CD34+ cells using viral supernatant to levels achieved at least with direct co-cultivation and under conditions that are suitable for clinical applications. In a first set of experiments, CD34+ cells were infected with supernatant containing amphotropic retroviral particles carrying the nls-lacZ reporter gene and the effects of centrifugation, cell adhesion to fibronectin, and Polybrene on the transduction of both clonogenic progenitors (CFC) and long-term culture initiating cells (LTC-IC) were studied. Transduction efficiency was evaluated on the percentage and total number of progenitors expressing the beta-galactosidase activity. Results show that a 48-hr infection of CD34+ cells with viral supernatant combining centrifugation at 1000 x g for 3 hr followed by adhesion to fibronectin allows transduction levels for both CFC and LTC-IC to be reached that are as good as using direct co-cultivation. In a second set of experiments, CD34+ cells were infected using this optimized protocol with pseudotyped retroviral particles carrying the gibbon ape leukemia virus (GALV) envelope protein. Under these conditions, between 50 and 100% of CFC and LTC-IC were transduced. Thus, we have developed a protocol capable of highly transducing cord blood progenitors under conditions suitable for a therapeutical use.

Optimization of the cycling of clonogenic and primitive cord blood progenitors by various growth factors.

The cycling status of cord blood progenitors and the culture conditions triggering their activation into S-phase have been studied using flow cytometry and a 3H-thymidine suicide assay. Mononuclear cells cultured either in Iscove's modified Dulbecco's medium (IMDM) +/- 10% fetal calf serum ([FCS]; IMDM + FCS) or in Dulbecco's modified Eagle's medium (DMEM) +/- 10% newborn bovine serum ([NBS]; DMEM + NBS) were stimulated by various growth factors (GFs). Results showed that CD34+ cells, clonogenic progenitors (colony forming cells [CFCs]) and long-term culture initiating cells (LTC-IC) present in freshly harvested cord blood were quiescent. CFC numbers were maintained without cycling after 48-h cultures in serum-containing media without GFs. Addition of interleukin 3 (IL-3) + IL-6 + stem cell factor stimulated into S-phase approximately 40% of CFCs within 24-48 h, without modifying their number except in DMEM + NBS where erythroid progenitors decreased. When cells were stimulated in IMDM + FCS by these three GFs + insulin-like growth factor I and basic fibroblast growth factor used at high concentration, more than 50% of CFCs were in S-phase and their total number was maintained. The latter culture conditions also recruited up to 66% of LTC-IC into S-phase. Our data underline the importance of the combination of GFs and culture media used for optimizing the cycling and maintenance of CFCs and LTC-IC within two days.

Direct infection of CD34+ progenitor cells by human cytomegalovirus: evidence for inhibition of hematopoiesis and viral replication.

We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.