No abstract

Background: DC derived-exosomes are nanomeric vesicles harboring functional MHC/peptide complexes capable of promoting T cell immune responses and tumor rejection. Here we report the feasability and safety of the first Phase I clinical trial using autologous exosomes pulsed with MAGE 3 peptides for the immunization of stage III/IV melanoma patients. Secondary endpoints were the monitoring of T cell responses and the clinical outcome.

ABSTRACT

Interactions between 4-1BB and its ligand, 4-1BBL, enhance CD8(+) T cell-mediated antiviral and antitumor immunity in vivo. However, mechanisms regulating the priming of CD8(+) T cell responses by 4-1BB remain unclear, particularly in humans. The 4-1BB receptor was undetectable on naive or resting human CD8(+) T cells and induced in vitro by TCR triggering. Naive cord blood cells were therefore primed in vitro against peptides or cellular antigens and then co-stimulated with 4-1BBL or agonistic antibodies. Co-stimulation enhanced effector function such as IFN-gamma production and cytotoxicity by augmenting numbers of antigen-specific and effector CD8(+) T cells. OKT3 responses also showed reduced cell death and revealed that the proliferation of CD8(+) T cells required two independently regulated events. One, the induction of IL-2 production, could be directly triggered by 4-1BB engagement on CD8(+) T cells in the absence of accessory cells. The other, expression of CD25, was induced with variable efficacy by accessory cells. Thus, suboptimal accessory cells and 4-1BB co-stimulation combined their effects to enhance IL-2 production and proliferation. Reduced apoptosis observed after co-stimulation in the presence of accessory cells correlated with increased levels of Bcl-X(L) in CD8(+) T cells, while Bcl-2 expression remained unchanged. Altogether, 4-1BB enhanced expansion, survival and effector functions of newly primed CD8(+) T cells, acting in part directly on these cells. As 4-1BB triggering could be protracted from the TCR signal, 4-1BB agonists may function through these mechanisms to enhance or rescue suboptimal immune responses.

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.

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.

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.

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.