MethodsMice. C57BL/6 and BALB/c mice were obtained from Harlan (Zeist, the Netherlands). C57BL/6.CH-2 bm12 (bm12) mice were obtained from The Jackson Laboratory (Bar Harbor, Maine, USA). C57BL/6 IL-5 knockout mice (23) were kindly provided by M. Kopf (Basel Institute for Immunology, Basel, Switzerland).Antibodies. The hamster anti-mouse CD3 mAb 145-2C11, the IgG1 rat anti-mouse IL-5 mAb TRFK-5, the IgG1 rat antimouse IL-4 mAb 11B11, and the IgG1 rat anti-mouse IL-10

Eosinophil infiltrates, typical of MHC class II disparate acute skin graft rejection, are critically dependent on the availability of IL-4. IL-4 mice reject MHC class II disparate skin grafts by a pathway of rejection where neutrophils play a direct causal role.

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.

β2-Microglobulin (β2m)-derived peptides are minor transplantation Ags in mice as β2m-positive skin grafts (β2m+/+) are rejected by genetically β2m-deficient recipient mice (β2m−/−). We studied the effector pathways responsible for the rejection induced by β2-microglobulin-derived minor transplantation Ags. The rejection of β2m+/+ skin grafts by naive β2m−/− mice was dependent on both CD4 and CD8 T cells as shown by administration of depleting mAbs. Experiments performed with β2m−/−CD8−/− double knockout mice grafted with a β2m+/+ MHC class I-deficient skin showed that sensitized CD4 T cells directed at β2m peptides-MHC class II complexes are sufficient to trigger rapid rejection. Rejection of β2m+/+ grafts was associated with the production of IL-5 in vitro, the expression of IL-4 and IL-5 mRNAs in the grafted tissue, and the presence within rejected grafts of a considerable eosinophil infiltrate. Blocking IL-4 and IL-5 in vivo and depleting eosinophils with an anti-CCR3 mAb prevented graft eosinophil infiltration and prolonged β2m+/+ skin graft survival. Lymphocytes from rejecting β2m−/− mice also displayed an increased production of IFN-γ after culture with β2m+/+ minor alloantigens. In vivo neutralization of IFN-γ inhibited skin graft rejection. Finally, β2m+/+ skin grafts harvested from B6lpr/lpr donor mice, which lack a functional Fas molecule, survived longer than wild-type β2m+/+ skin grafts, showing that Fas-Fas ligand interactions are involved in the rejection process. We conclude that IL-4- and IL-5-dependent eosinophilic rejection, IFN-γ-dependent mechanisms, and Fas-Fas ligand interactions are effector pathways in the acute rejection of minor transplantation Ags.

No abstract