Sonja Van Meirvenne

Lentivirally transduced dendritic cells as a tool for cancer immunotherapy

Dendritic cells (DC) are the professional antigen‐presenting cells of the immune system, fully equipped to prime naive T cells and thus essential components for cancer immunotherapy.

Efficient genetic modification of murine dendritic cells by electroporation with mRNA

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 mRNA-electroporated 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.

Dendritic cells fused with mastocytoma cells elicit therapeutic antitumor immunity

Characterization of the spontaneous immune response that frequently occurs in tumor‐bearing animals, as well as immunization using dendritic cells pulsed with tumor antigens, suggests that a limiting factor of the tumor‐specific immune response may be a defect in the co‐stimulatory signal that is required for optimal activation of T cells. In this work, we describe a new approach to improve the antigen‐presenting capacity of tumor cells, which does not require a source of purified tumor‐associated antigen. We fused P815 mastocytoma cells with bone marrow‐derived dendritic cells. We obtained one hybrid that displayed the phenotypic and functional properties of dendritic cells and expressed mRNA coding for the tumor‐associated antigen P815 A/B. Injections of irradiated hybrid cells prevented the growth of pre‐implanted mastocytoma and induced long‐lasting tumor resistance. Int. J. Cancer76:250–258, 1998.© 1998 Wiley‐Liss, Inc.

Dendritic cells retrovirally overexpressing IL-12 induce strong Th1 responses to inhaled antigen in the lung but fail to revert established Th2 sensitization.

It has been postulated that low‐level interleukin (IL)‐12 production of antigen‐presenting cells is associated with the risk of developing atopic asthma. To study the relationship between IL‐12 production capacity of dendritic cells (DCs) and development of T helper type 2 (Th2) responses in the lung, we genetically engineered DCs to constutively overexpress bioactive IL‐12. Retrovirally mediated overexpression of IL‐12 in DCs strongly polarized naïve ovalbumin (OVA)‐specific CD4+ T cells toward Th1 effector cells in vitro. After intratracheal injection, OVA‐pulsed IL‐12‐overexpressing DCs failed to induce Th2 responses in vivo and no longer primed mice for Th2‐dependent eosinophilic airway inflammation upon OVA aerosol challenge, readily observed in mice immunized with sham‐transfected, OVA‐pulsed DCs. Analysis of a panel of cytokines and chemokines in the lung demonstrated that the lack of Th2 sensitization was accompanied by increased production of the Th1 cytokine interferon‐γ (IFN‐γ), chemokines induced by IFN‐γ, and the immunoregulatory cytokine IL‐10. When Th2 priming was induced using OVA/alum prior to intratracheal DC administration, DCs constitutively expressing IL‐12 were no longer capable of preventing eosinophilic airway inflammation and even enhanced it. These data show directly that high‐level expression of IL‐12 in DCs prevents the development of Th2 sensitization. Enhancing IL‐12 production in DCs should be seen as a primary prevention strategy for atopic disorders. Enhancing IL‐12 production in DCs is less likely to be of benefit in already Th2‐sensitized individuals.

Expression of human GITRL on myeloid dendritic cells enhances their immunostimulatory function but does not abrogate the suppressive effect of CD4+CD25+ regulatory T cells

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 transduced with viral interleukin 10 or Fas ligand: no evidence for induction of allotolerance in vivo.

Dendritic cells (DC) are the most potent presenters of alloantigens and therefore are responsible for the induction of allograft rejection. Genetic modifications of DC allowing the expression of a tolerogenic molecule may render them immunosuppressive. We transduced bone marrow-derived DC with recombinant MFG retrovirus encoding either viral interleukin (vIL)-10 or Fas ligand (FasL) to induce transplantation tolerance. Up to 10 ng/ml of bioactive vIL-10 was produced by DC after transfer of the corresponding gene. Although the inhibitory properties of vIL-10-transduced DC were revealed in vitro in a mixed lymphocyte culture, no clear down-regulation of the allogeneic response was observed in vivo after single or multiple injections of those DC overexpressing vIL-10. When we transduced wild-type bone marrow-derived DC with recombinant MFG retrovirus encoding murine FasL, cells quickly died, probably because of suicidal or fratricidal Fas-dependent death. Indeed, only DC from Fas-deficient lpr mice survived to FasL gene transfer. Those FasL-transduced lpr DC exhibited a strong cytotoxic activity against Fas-positive targets in vitro. DC overexpressing FasL did not behave as immunosuppressive DC in vivo. The subcutaneous injection of FasL+ lpr DC in MHC class II-disparate mice hyperactivated the allospecific proliferation of T cells in the draining lymph nodes compared with mice treated with control-transduced DC. These results argue against the development of FasL+ DC or vIL-10-secreting DC as immunosuppressive tools in vivo. The alternative pathways of T-cell activation triggered by these genetically modified DC need to be investigated.

Bispecific antibody treatment of murine B cell lymphoma.

Abstract This report summarizes our experimental data concerning the use of bispecific antibodies (bsAb) for the treatment of the murine BCL1 B cell lymphoma model. Initially we used a hybrid-hybridoma-derived bsAb with specificity for the TcR/CD3 complex on T cells and the idiotype of the membrane-bound IgM on the tumor cells. The bsAb used as a single agent could cure animals with a low tumor load (resembling minimal residual disease). However, in experiments aimed at increasing the therapeutic effect in animals with a higher tumor burden, we could demonstrate the importance of additional T-cell-costimulatory signals and the careful timing of the bsAb administration. Recently we have generated a bispecific single-chain Fv (bsscFv) fusion protein with the same dual specificity as the hybrid-hybridoma-derived bsAb. Immunotherapy with this smaller molecule also resulted in tumor elimination in BCL1-bearing mice. A second bsscFv (α-CDl9×α-CD3) with a broader applicability is now being characterized and tested in vivo.