Frédéric Paulart

Free cationic liposomes inhibit the inflammatory response to cationic lipid–DNA complex injected intravenously and enhance its transfection efficiency

In this report, we show that intravenous (i.v.) injection into mice of a complex made of the cationic lipid diC14-amidine and the luciferase reporter plasmid (pCMV-luc) results in efficient gene expression in several organs but elicits an inflammatory response characterized by a release of tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) into the serum of treated animals. A single preinjection of free diC14-amidine liposomes improves the i.v. transfection efficiency of the diC14-amidine/protamine/pCMV-luc complex as much as 40 times. This improvement is correlated with the ability of free liposomes to inhibit TNF-alpha but not IFN-gamma production resulting from complex injection. TNF-alpha-rich serum obtained from mice injected with diC14-amidine/protamine/pCMV-luc complex inhibits luciferase expression in transfected mouse lung endothelial (MLE) cells cultured in vitro, whereas IFN-gamma has no effect. This inhibitory effect can be partly abolished by treating the mouse serum with a specific anti-TNF-alpha antibody. These data point out that cationic lipids are potent inhibitors of the inflammatory response to the CpG motifs in plasmid DNA. This property is shown to enhance the in vivo transfection efficiency.

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.

An Alternative Pathway of NF-κB Activation Results in Maturation and T Cell Priming Activity of Dendritic Cells Overexpressing a Mutated IκBα

Maturation of dendritic cells (DC) is a critical step in the induction of T cell responses and depends on the activation of NF-κB transcription factors. Therefore, inhibition of NF-κB activation has been proposed as a strategy to maintain DC in an immature stage and to promote immune tolerance. Herein, we generated murine myeloid DC expressing a mutated IκBα acting as a superrepressor of the classical NF-κB pathway (s-rIκB DC) to investigate the consequences of NF-κB inhibition on the ability of DC to prime T cell responses. Upon in vitro LPS activation, maturation of s-rIκB DC was profoundly impaired as indicated by defective up-regulation of MHC class II and costimulatory molecules and reduced secretion of IL-12 p70 and TNF-α. In contrast, after injection, s-rIκB DC had the same capacity as control DC to migrate to draining lymph node and to induce Th1- and Th2-type cytokine production in a MHC class II-incompatible host mice. Likewise, s-rIκB DC pulsed with OVA were as efficient as control DC to induce Ag-specific T cell responses in vivo. Indeed, further in vitro experiments established that s-rIκB DC undergo efficient maturation upon prolonged contact with activated T cells via the alternative pathway of NF-κB activation triggered at least partly by lymphotoxin β receptor ligation and involving processing of p100/RelB complexes.

Neutrophil-dependent tumor rejection and priming of tumoricidal CD8 T cell response induced by dendritic cells overexpressing CD95L

Overexpression of CD95 (Fas/Apo‐1) ligand (CD95L) has been shown to induce T cell tolerance but also, neutrophilic inflammation and rejection of allogeneic tissue. We explored the capacity of dendritic cells (DCs) genetically engineered to overexpress CD95L to induce an antitumor response. We first found that DCs overexpressing CD95L, in addition to MHC class I‐restricted OVA peptides (CD95L‐OVA‐DCs), induced increased antigen‐specific CD8+ T cell responses as compared with DCs overexpressing OVA peptides alone. The enhanced T cell responses were associated with improved regression of a tumor expressing OVA, allowing survival of all animals. When DCs overexpressing CD95L (CD95L‐DCs) were injected with the tumor expressing OVA, in vivo tumor proliferation was strikingly inhibited. A strong cellular apoptosis and a massive neutrophilic infiltrate developed in this setting. Neutrophil depletion prevented tumor regression as well as enhanced IFN‐γ production induced by CD95L‐OVA‐DCs. Furthermore, the CD8+ T cell response induced by the coadministration of tumor cells and CD95L‐DCs led to rejection of a tumor implanted at a distance from the DC injection site. In summary, DCs expressing CD95L promote tumor rejection involving neutrophil‐mediated innate immunity and CD8+ T cell‐dependent adaptative immune responses.

CD8+ T Lymphocytes Regulating Th2 Pathology Escape Neonatal Tolerization

Transplantation tolerance induced by neonatal injection of semiallogeneic spleen cells is associated in several strain combinations with a pathological syndrome caused by Th2 differentiation of donor-specific CD4+ T lymphocytes. We investigated the role of host CD8+ T cells in the regulation of this Th2 pathology. IgE serum levels and eosinophilia significantly increased in BALB/c mice neonatally injected with (A/J × BALB/c)F1 spleen cells when CD8+ T cells were depleted by administration of anti-CD8 mAb or when β2-microglobulin-deficient mice were used as recipients. In parallel, increased serum levels of IL-5 and IL-13 were measured in blood of tolerant CD8+ T cell-deficient mice. Whereas neonatally injected mice were unable to generate anti-donor cytotoxic effectors, their CD8+ T cells were as efficient as control CD8+ T cells in reducing the severity of Th2 pathology and in restoring donor-specific cytotoxicity in vitro after in vivo transfer in β2-microglobulin-deficient mice. Likewise, CD8+ T cells from control and tolerant mice equally down-regulated the production of Th2 cytokines by donor-specific CD4+ T cells in vitro. The regulatory activity of CD8+ T cells depended on their secretion of IFN-γ for the control of IL-5 production but not for IL-4 or IL-13. Finally, we found that CD8+ T cells from 3-day-old mice were already able to down-regulate IL-4, IL-5, and IL-13 production by CD4+ T cells. We conclude that regulatory CD8+ T cells controlling Th2 responses are functional in early life and escape neonatal tolerization.

Hypereosinophilic syndrome induced by neonatal immunization against MHC class II alloantigen: critical role of IL-4

A significant proportion of patients with the hypereosinophilic syndrome suffer from oligoclonal expansion of type 2 helper T lymphocytes (Th2). Herein, we first provide evidence that mice immunized at birth against a single MHC class II alloantigen develop pathological features mimicking this variant of the hypereosinophilic syndrome. Indeed, C57BL / 6 mice injected at birth with (C57BL/ 6 × bm12)F1 spleen cells displayed T lymphocytes producing high levels of IL‐5 and IL‐13, increased blood eosinophil counts, eosinophilic infiltrates in various tissues, hyperplasia of lymphoid tissues, as well as serum hyperIgE. Moreover, eotaxin mRNA accumulated in the spleen of these animals. IL‐4‐deficient mice developed neither expansion of Th2 cells nor pathological changes except splenomegaly. Eotaxin mRNA accumulation was also prevented in these animals. We conclude that neonatal exposure to a single MHC class II alloantigen is sufficient to elicit an IL‐4‐dependent hypereosinophilic syndrome mimicking the lymphocytic variant of this disorder in humans.

Regulatory role of host CD8+ T lymphocytes in experimental graft-versus-host disease across a single major histocompatibility complex class II incompatibility.

Background. CD8+ T cells are known to regulate type 2 helper T cell (Th2) alloreactive immune responses but their mode of activation is unclear. We investigated the role of host CD8+ T cells in experimental Th2-type graft-versus-host disease (GVHD) where donor/recipient disparity is restricted to a single major histocompatibility complex (MHC) class II antigen. Methods. Immunoglobulin (Ig) E serum levels, eosinophilia and lymphoid tissue hyperplasia were compared after injection of bm12 CD4+ T cells in either wild-type or CD8+ T cell-deficient (CD8−/−) C57BL/6 mice. In vitro, we explored effects of the addition of CD8+ T cells from wild-type or IFN-γ−/− mice in mixed leukocyte cultures prepared with β2 microglobulin-deficient (β2m−/−) CD4+ T cells as responders or β2m−/− dendritic cells as stimulators. Results. HyperIgE resolved after 3 weeks in wild-type hosts whereas it persisted for 6 weeks in CD8−/− hosts. Eosinophil infiltrates in lymph nodes were significantly enhanced in CD8−/− hosts. Increased serum levels of IL-5 and IL-13 in CD8−/− hosts confirmed the enhancement of Th2-type responses in the context of recipient CD8+ T cell deficiency. Hyperplasia of lymph nodes and spleen were similar in both groups, as well as in vivo proliferation of donor CD4+ T cells. In vitro, CD8+ T cell regulation of the alloreactive Th2 response depended on their production of IFN-γ and did not require expression of β2m on CD4+ T cells or antigen-presenting cells. Conclusions. Host CD8+ T cells regulate alloreactive Th2 responses during graft-versus-host disease through an IFN-γ dependent pathway, independently of the recognition of β2m-associated MHC class I molecules.