We show that, using lentiviral vectors, efficient gene transfer in human and murine DC can be obtained and that these DC can elicit antigen-specific immune responses in vitro and in vivo. The composition of the transfer vector has a major impact on the transduction efficiency.

Summary The aim of this study was to evaluate the tissue infiltration and phenotypic adhesion profile of 5T2 multiple myeloma (MM) and 5T33 MM cells and to correlate it with that observed in human disease. For each line, 30 mice were intravenously inoculated with myeloma cells and at a clear-cut demonstrable serum paraprotein concentration; mice were sacrificed and a number of organs removed. The haematoxylin-eosin stainings on paraffin sections were complemented with immunohistochemistry using monoclonal antibodies developed against the specific MM idiotype. When analysed over time, 5T2 MM cells could be observed in bone marrow samples from week 9 after transfer of the cells. For the 5T33 MM, a simultaneous infiltration was observed in bone marrow, spleen and liver 2 weeks after inoculation. Osteolytic lesions consistently developed in the 5T2 MM, but this was not consistent for 5T33 MM. PCNA staining showed a higher proliferative index for the 5T33 MM cells. The expression of adhesion molecules was analysed by immunohistochemistry on cytosmears: both 5T2 MM and 5T33 MM cells were LFA-1, CD44, VLA-4 and VLA-5 positive. We conclude that both lines have a phenotypic adhesion profile analogous to that of human MM cells. As the 5T2 MM cells are less aggressive than the 5T33 MM cells, their organ distribution is more restricted to the bone marrow and osteolytic lesions are consistently present, the former cell line induces myeloma development similar to the human disease.Keywords: multiple myeloma; adhesion molecules; organ involvement; 5T2; 5T33 Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal expansion of malignant plasma cells secreting a monoclonal immunoglobulin (Ig). The disease is mainly localized in the bone marrow. In this microenvironment the myeloma plasma cells receive signals necessary for their proliferation, terminal differentiation and for the secretion of osteoclast-activating factors. The osteoclast-activating factors recruit osteoclasts, which induce in situ osteolytic bone lesions (Bataille et al, 1989;Alsina et al, 1996); this is one of the major characteristics of the disease. It has been suggested that both cytokines and adhesion molecules are involved in this complex network of signals (Van Riet and Van Camp, 1993).To elucidate the exact mechanisms described above, an in vivo MM model is necessary. Radl et al (1979) found that 0.5% of ageing C57BL/KaLwRij mice spontanously developed a disease reminiscent of MM. The MM cells isolated from the bone marrow of different mice (5T MM) did not grow in vitro but could be transplanted by intravenous injection into young recipients of the same strain. This transplantable model resembles the human disease in several aspects (Radl et al, 1988): myeloma occurred spontaneously, the frequency of development of the disease is age related, tumour load can be assessed by paraproteinaemia and the (Radl et al, 1988).In order to understand the homing mechanisms of the 5T MM cells to the bone marrow, it was essential to determine accurately the...

Ex vivo lentivirally transduced dendritic cells (DC) have been described to induce CD8 + and CD4 + T-cell responses against various tumor-associated antigens (TAAs) in vitro and in vivo. We report here that direct administration of ovalbumin (OVA) encoding lentiviral vectors caused in vivo transduction of cells that were found in draining lymph nodes (LNs) and induced potent anti-OVA cytotoxic T cells similar to those elicited by ex vivo transduced DC. The cytotoxic T-lymphocyte (CTL) response following direct injection of lentiviral vectors was highly effective in eliminating target cells in vivo up to 30 days after immunization and was efficiently recalled after a boost immunization. Injection of lentiviral vectors furthermore activated OVA-specific CD4 + T cells and this CD4 help was shown to be necessary for an adequate primary and memory CTL response. When tested in therapeutic tumor experiments with OVA + melanoma cells, direct administration of lentiviral vectors slowed down tumor growth to a comparable extent with the highest dose of ex vivo transduced DC. Taken together, these data indicate that direct in vivo administration of lentiviral vectors encoding TAAs has strong potential for anticancer vaccination.

Recently, human dendritic cells ( DCs ) pulsed with mRNA encoding a broad range of tumor antigens have proven to be potent activators of a primary anti -tumor -specific T -cell response in vitro. The aim of this study was to improve the mRNA pulsing of murine DC. Compared to a standard lipofection protocol and passive pulsing, electroporation was, in our hands, the most efficient method. The optimal conditions to electroporate murine bone marrow -derived DCs with mRNA were determined using enhanced green fluorescent protein and a truncated form of the nerve growth factor receptor. We could obtain high transfection efficiencies around 70 -80% with a mean fluorescence intensity of 100 -200. A maximal expression level was reached 3 hours after electroporation. A clear dose -response effect was seen depending on the amount of mRNA used. Importantly, the electroporation process did not affect the viability nor the allostimulatory capacity or phenotype of the DC. To study the capacity of mRNAelectroporated DCs to present antigen in the context of MHC classes I and II, we made use of chimeric constructs of ovalbumin. The dose -dependent response effect and the duration of presentation were also determined. Together, these results demonstrate that mRNA electroporation is a useful method to generate genetically modified murine DC, which can be used for preclinical studies testing immunotherapeutic approaches.

Until now, studies utilizing mRNA electroporation as a tool for the delivery of tumor antigens to human monocyte-derived dendritic cells (DC) have focused on DC electroporated in an immature state. Immature DC are considered to be specialized in antigen capture and processing, whereas mature DC present antigen and have an increased T-cell stimulatory capacity. Therefore, the consensus has been to electroporate DC before maturation. We show that the transfection efficiency of DC electroporated either before or after maturation was similarly high. Both immature and mature electroporated DC, matured in the presence of an inflammatory cytokine cocktail, expressed mature DC surface markers and preserved their capacity to secrete cytokines and chemokines upon CD40 ligation. In addition, both immature and mature DC can be efficiently cryopreserved before or after electroporation without deleterious effects on viability, phenotype or T-cell stimulatory capacity including in vitro antigen-specific T-cell activation. However, DC electroporated after maturation are more efficient in in vitro migration assays and at least as effective in antigen presentation as DC electroporated before maturation. These results are important for vaccination strategies where an optimal antigen presentation by DC after migration to the lymphoid organs is crucial. Gene Therapy (2005) 12, 772-782.

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

CD4(+)CD25(+) regulatory T cells (Treg) have been described as an important hurdle for immunotherapy. Engagement of glucocorticoid-induced TNF receptor-related protein (GITR) has emerged recently as an important mechanism to control the suppression of CD4(+)CD25(+) Treg. Furthermore, it has been documented extensively that GITR ligation is costimulatory for naive and activated T cells in the murine setting. However, little is known about the role of the human GITR ligand (huGITRL). We wanted to explore whether huGITRL could enhance antigen-specific T cell priming by dendritic cells (DC). First, we confirmed the endogenous expression of GITRL on HUVEC. We also detected GITRL expression on EBV-B cell lines, whereas no GITRL expression was observed on human monocyte-derived DC. Electroporation of GITRL mRNA in monocyte-derived DC resulted in a strong and long-lasting surface expression of GITRL. In contrast to data obtained in mice, no significant abrogation of Treg suppression by GITRL-expressing human DC was observed. Consistent with our mouse data, we showed that huGITRL is costimulatory for responder T cells. Furthermore, we found that GITRL-expressing DC primed increased numbers of Melan-A-specific CD8(+) T cells. We conclude that although huGITRL is not capable of alleviating Treg suppression of responder T cells, huGITRL overexpression on monocyte-derived DC enhances their capacity to induce antigen-specific T cell responses. Thus, GITRL incorporation in DC might improve the antitumor immune response after vaccination.

Dendritic cells (DCs) genetically engineered to overexpress CD95 (Fas) ligand (CD95L-DC) were proposed as tools to induce peripheral tolerance to alloantigens. Herein, we observed that CD95L-DC obtained after retroviral gene transfer in bone marrow (BM) precursors derived from CD95-deficient (lpr/lpr) mice elicit much stronger allospecific type 1 helper T-cell and cytotoxic T-cell activities than control DCs upon injection in vivo, although they induce lower T-cell responses in vitro. Indeed, a single injection of CD95L-DC prepared from C57BL/6 mice was sufficient to prime bm13 recipients for acute rejection of C57BL/6 skin allografts that were otherwise tolerated in the context of this single weak major histocompatibility complex ( IntroductionCD95 (Fas)-mediated apoptosis of activated T lymphocytes is critically involved in the homeostasis of the T-cell pool 1,2 and the maintenance of peripheral tolerance to self antigens. 3 Moreover, it has been proposed that the immune privilege status of particular anatomic sites could be related to local expression of CD95L [4][5][6] and that expression of CD95L by tumor cells might protect them from immune attack. [7][8][9] On this basis, it has been considered that expression of CD95L on allo-or xenografts might promote their acceptance by deleting host T cells specific for transplanted antigens. Indeed, CD95L expression on Sertoli cells was suggested to be directly responsible for testis allograft survival. 4,10 It was then reported that implantation of syngeneic muscle cells transfected with CD95L together with allogeneic grafted pancreatic islets allowed long-term survival of the transplanted islets. 11 More recently, CD95L overexpression on allogeneic endothelium was shown to inhibit transplant-associated intimal hyperplasia. 12 However, several of these observations have been refuted [13][14][15] so that the role of CD95L in conferring immune privilege is currently a matter of controversy. Furthermore, chemoattraction of neutrophils leading to a massive inflammatory reaction has emerged as a major consequence of CD95L overexpression. As a matter of fact, neutrophil infiltration leading to graft destruction was observed after implantation of pancreatic islets in which the CD95L gene was overexpressed. 16 Likewise, CD95L transgenic islet ␤ cells or heart allografts were shown to be more rapidly rejected than their wild-type counterparts, in association with a massive influx of neutrophils in the transplant. 17,18 In order to promote deletion of allospecific T cells without inducing inflammation at the graft level, it has been proposed to condition allograft recipients with antigen-presenting cells overexpressing CD95L prior to transplantation. Indeed, allogeneic macrophages transduced with murine CD95L induced profound alloantigen-specific T-cell unresponsiveness. 19 Dendritic cells (DCs) represent a suitable cell type for such an approach as indicated by the report of Matsue et al showing that injection of an ovalbuminpulsed DC line transfected with murine CD95L ind...