Mesenchymal stromal cells: a key player in 'innate tolerance'?

The existence of a mesenchymal stromal cell (MSC) population with the main property of physically supporting parenchymal tissues has long been recognized in virtually all organs. However, it was only recently that MSC have been identified as playing a novel role in modulating inflammation. It has been extensively documented that, under particular circumstances, MSC potently impair virtually all cells of the immune system, including antigen-presenting-cells.

A lentiviral vector pseudotype suitable for vaccine development.

BACKGROUND: Lentiviral vectors (LV) are promising vaccines because they transduce dendritic cells (DC) in vivo. To translate LV vaccines into clinical trials, bulk production will be necessary. The present study aimed to find a suitable envelope for LV vaccine production from stable packaging cells because the commonly used vesicular stomatitis virus envelope (VSV-G) is cytotoxic. METHODS: The envelope from Ross river virus (RRV) was selected. It can infect mouse and human cells, allowing testing in animals before clinical translation. We used VSV-G for comparison. Vectors produced with each envelope were titred on human 293T cells and mouse 3T3 cells. RESULTS: RRV-pseudotyped LV (RRV-LV) infected mouse myeloid DC in culture and immunized mice. An approximately 50-fold higher dose of RRV-LV than VSV-G-LV was required to generate a similar T cell response. The RRV-LV could also be used to infect human mDC and to prime a human T cell immune response. CONCLUSIONS: RRV envelope is a suitable candidate to be used for the construction of an LV producer cell line. LV vaccines with RRV envelope can be tested in mice and in human immune cell cultures. The higher dose of RRV-LV required for vaccine efficacy compared to VSV-G-LV will partly be offset by ease of production.

Lentiviral vectors transduce proliferating dendritic cell precursors leading to persistent antigen presentation and immunization.

Lentiviral vectors (LVs) are tools for in vivo gene delivery, to correct genetic defects or to deliver antigens for vaccination. It was reported that systemic injection of LVs in mice transduced cells in liver and spleen. Here we describe the reasons for, and consequences of, persistent gene expression in spleen. After 5 days of intravenous injection, a green fluorescence protein (GFP)-expressing LV was detected in lymphocytes, macrophages and all subsets of dendritic cells (DCs) in spleen. In the case of macrophages and DCs, the percentage of transduced cells increased between 5 and 30 days after injection. We used bromodeoxyuridine (BrdU) incorporation to show that the macrophages were largely nondividing, whereas the transduced DCs arose from dividing precursor cells and could be detected in spleen 2 months after injection. Expression of ovalbumin (OVA) in the LV reduced the number of transduced DCs in spleen after 30 days. However, the remaining transduced cells stimulated proliferation and activation of OVA-specific CD8(+) T cells transferred 2 months after LV injection. The mice also maintained cytolytic activity against OVA-pulsed targets. These results show that LVs transduce DC precursors, which maintain transduced DCs in spleen for at least 2 months, leading to prolonged antigen presentation and effective T-cell memory.

Single-chain antibodies that target lentiviral vectors to MHC class II on antigen-presenting cells.

Lentiviral vectors are promising vaccines because they can transduce and express antigens in dendritic cells in vivo, leading to potent immunization. To improve the safety and efficacy of lentivector vaccination, we sought to target vector transduction to antigen-presenting cells by modifying the viral envelope. To do this we screened a nonimmunized human single-chain antibody phage display library for phage that bound mouse bone marrow-derived dendritic cells (BMDCs) and isolated three single-chain antibodies (scFvs) that bound to more than 20% of cells in the BMDC culture. The three scFvs also bound to dendritic cells, macrophages, monocytes, and B cells from mouse spleen, but not to neutrophils, eosinophils, or T cells. Immunoblotting demonstrated that two unique scFvs, C2 and C7, recognized MHC class II. We constructed chimeric envelope proteins, by fusing these two scFvs to the amino terminus of the amphotropic murine leukemia virus envelope (MLV-A). These chimeric envelopes were expressed on the surface of lentiviral vector particles and enhanced infection (5- to 10-fold) of BMDC cultures, compared with lentiviral vectors with unmodified MLV-A envelope. Similarly, the chimeric envelopes enhanced (10- to 20-fold) the infection of primary lymph node class II-positive cells. One of the envelopes, C2, gave increased interferon-gamma production from splenocytes of vaccinated mice compared with MLV-A, achieving a level similar to that obtained with vesicular stomatitis virus glycoprotein G, when used to deliver an ovalbumin model antigen gene. These results demonstrate that surface-targeting lentiviral vector transduction of antigen-presenting cells gives efficient and potentially safer immunization.

Expression of vFLIP in a lentiviral vaccine vector activates NF-{kappa}B, matures dendritic cells, and increases CD8+ T-cell responses.

Lentiviral vectors deliver antigens to dendritic cells (DCs) in vivo, but they do not trigger DC maturation. We therefore expressed a viral protein that constitutively activates NF-kappaB, vFLIP from Kaposi's sarcoma-associated herpesvirus (KSHV), in a lentivector to mature DCs. vFLIP activated NF-kappaB in mouse bone marrow-derived DCs in vitro and matured these DCs to a similar extent as lipopolysaccharide; costimulatory markers CD80, CD86, CD40, and ICAM-1 were upregulated and tumor necrosis factor alpha and interleukin-12 secreted. The vFLIP-expressing lentivector also matured DCs in vivo. When we coexpressed vFLIP in a lentivector with ovalbumin (Ova), we found an increased immune response to Ova; up to 10 times more Ova-specific CD8(+) T cells secreting gamma interferon were detected in the spleens of vFLIP_Ova-immunized mice than in the spleens of mice immunized with GFP_Ova. Furthermore, this increased CD8(+) T-cell response correlated with improved tumor-free survival in a tumor therapy model. A single immunization with vFLIP_Ova also reduced the parasite load when mice were challenged with OVA-Leishmania donovani. In conclusion, vFLIP from KSHV is a DC activator, maturing DCs in vitro and in vivo. This demonstrates that NF-kappaB activation is sufficient to induce many aspects of DC maturation and that expression of a constitutive NF-kappaB activator can improve the efficacy of a vaccine vector.

Targeting dendritic cell signaling to regulate the response to immunization.

Dendritic cells (DCs) are key regulators of the immune system; they capture antigens and then can either stimulate an immune response or induce tolerance. Our aim was to activate individual DC signaling pathways to regulate the immune response. We therefore expressed constitutive activators of mitogen-activated protein kinase (MAPK) pathways or the interferon pathway, together with tumor antigens, using lentivectors. Triggering of p38 activated DCs substantially enhanced the antitumor immune response and prolonged survival of tumor-bearing mice. Activation of extracellular signal-regulated kinase (ERK) increased TGF-beta expression while expression of a constitutively activated interferon regulatory factor-3 (IRF3) stimulated IL-10 secretion by DCs. ERK and IRF3 suppressed the immune response and stimulated expansion of regulatory T cells. These results provide a toolkit to regulate immune responses to viral vector or DC immunization; vaccine responses to foreign or tumor antigens can be enhanced and harmful responses to self-antigens or introduced transgenes can be reduced.

Immunization with a lentivector that targets tumor antigen expression to dendritic cells induces potent CD8+ and CD4+ T-cell responses.

Lentivectors stimulate potent immune responses to antigen transgenes and are being developed as novel genetic vaccines. To improve safety while retaining efficacy, we constructed a lentivector in which transgene expression was restricted to antigen-presenting cells using the mouse dectin-2 gene promoter. This lentivector expressed a green fluorescent protein (GFP) transgene in mouse bone marrow-derived dendritic cell cultures and in human skin-derived Langerhans and dermal dendritic cells. In mice GFP expression was detected in splenic dectin-2(+) cells after intravenous injection and in CD11c(+) dendritic cells in the draining lymph node after subcutaneous injection. A dectin-2 lentivector encoding the human melanoma antigen NY-ESO-1 primed an NY-ESO-1-specific CD8(+) T-cell response in HLA-A2 transgenic mice and stimulated a CD4(+) T-cell response to a newly identified NY-ESO-1 epitope presented by H2 I-A(b). As immunization with the optimal dose of the dectin-2 lentivector was similar to that stimulated by a lentivector containing a strong constitutive viral promoter, targeting antigen expression to dendritic cells can provide a safe and effective vaccine.

Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy.

Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A(27-35) epitope. The expanded cells are functional in that they release IFN-gamma upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A(27-35) peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial.

Immunization with a lentiviral vector stimulates both CD4 and CD8 T cell responses to an ovalbumin transgene.

Lentiviral vectors encoding antigens are promising vaccine candidates because they transduce dendritic cells (DC) in vivo and prime CTL responses. Here we examine their stimulation of antigen-specific CD4(+) T cells, critical for protective immunity against tumors or infectious disease. We constructed lentiviral vectors (lentivectors) expressing ovalbumin, which was secreted (OVA), cytoplasmic (OVAcyt), or fused to either invariant chain (Ii-OVA) or transferrin receptor (TfR-OVA) sequences, targeting the MHC class II presentation pathway. Murine DC infected with the various lentivectors could stimulate OT-I (CD8(+), OVA TCR transgenic) T cells and all except OVAcyt could also stimulate OT-II (CD4(+), OVA TCR transgenic) T cells in vitro. Direct injection of the OVA-, Ii-OVA-, or TfR-OVA-expressing vectors into mice resulted in a CD4(+) T cell response, as shown by expansion of adoptively transferred OT-II T cells and upregulation of CD44 on these cells. The Ii-OVA vector was the most potent inducer of IFN-gamma-secreting CD4(+) and CD8(+) T cells and was the only vector to protect mice completely from challenge with OVA-expressing tumor cells. Therefore directly injected lentivectors can stimulate CD4(+) T cells; both CD4(+) and CD8(+) responses can be enhanced by targeting the antigen to the MHC class II pathway.

Intravenous injection of a lentiviral vector encoding NY-ESO-1 induces an effective CTL response.

Lentiviral vectors can efficiently transduce a variety of nondividing cells, including APCs. We assessed the immunogenicity of a lentiviral vector encoding the melanoma Ag NY-ESO-1 in HLA-A2 transgenic mice. Direct i.v. injection of NY-ESO-1 lentivirus induced NY-ESO-1(157-165)-specific CD8(+) cells, detected ex vivo with an A2/H-2K(b) chimeric class I tetramer. These NY-ESO-1(157-165)-specific CD8(+) cells could be expanded by boosting with an NY-ESO-1 vaccinia virus and could kill NY-ESO-1(157-165) peptide-pulsed targets in vivo. Such direct lentiviral vector injection was similar in potency to the injection of in vitro-transduced dendritic cells (DC). In addition, human monocyte-derived DC transduced by the NY-ESO-1 lentivirus stimulated an NY-ESO-1(157-165)-specific specific CTL clone. These data suggest that direct lentiviral transduction of DC in vivo might provide a powerful immunotherapeutic strategy.

Modulation of dendritic cell endocytosis and antigen processing pathways by Escherichia coli heat-labile enterotoxin and mutant derivatives.

Escherichia coli heat-labile enterotoxin (LT) is known to be a potent adjuvant of both the mucosal and systemic immune systems but the mechanism of action leading to adjuvant activity remains incompletely understood. This study investigates the action of LT and LT mutants with impaired enzymatic activity, on the function of dendritic cells. Wild-type LT and LTR72, which retains some ADP ribosyltransferase activity, induced a selective increase in cell surface expression of B7.1, and a selective decrease of CD40 expression on mouse bone marrow derived dendritic cells. LTK63 and LT-B had no obvious effect on the expression of these antigens on similar dendritic cells. LT-treated dendritic cells also showed a profoundly impaired ability to present protein antigen (ovalbumin) to cognate T cells, although this effect was not observed with non-toxic LT mutants. LT and LTR72-treated cells showed a slower rate of receptor-mediated endocytosis as measured by flow cytometric analysis of uptake of fluorescently labelled dextran. Furthermore, confocal microscopy showed changes in the intracellular distribution of endocytosed molecules, and of the class II containing acidic antigen processing compartments. This response of dendritic cells to toxin is likely to play an important role in determining the adjuvant activity of these molecules.