title: Allograft rejection and the role of the immunoregulatory cytokine interleukin-12
Creators: name: Orr, Douglas J.
Publisher: name: University of Glasgow
Year: 1999

description: This aim of this study was to examine the effect of neutralising endogenous IL-12 on skin allograft survival in a mouse experimental model. The immune response following treatment was investigated to determine whether anti-IL-12 antibody could switch a Th1 to a Th2 response and whether this was associated with prolonged graft survival. C57B1/6 and DBA/2 mice were grafted with tail skin from semi- allogeneic (C57B1/6 X DBA/2) F1 (BDFi) donors. Skin graft survival was compared between untreated mice and those treated with a neutralising goat anti-mouse IL-12 polyclonal antibody. In addition, mice were pre-treated with donor specific transfusion in the form of BDF1 spleen cells injected intravenously prior to grafting, or with a combination of both anti-IL-12 antibody and donor cell pretreatment. Skin graft survival was not prolonged in animals which had been treated with anti-IL-12 antibody or donor cell injection alone compared to unmodified controls. Allograft survival was, however, significantly prolonged in animals pretreated with both anti-IL-12 antibody plus donor cell injection. Interestingly, when a fully allogeneic mismatch was used, this combined treatment did not prolong graft survival. The immunological processes involved with the rejection response were investigated. The spleens of treated and untreated mice were removed at various time points after grafting and single cell suspensions prepared and maintained in vitro. When re-stimulated with irradiated BDF1 cells in a MLR, cells from unmodified C57B1/6 graft recipients proliferated well. However, where animals had been pre-treated with donor cells, either with or without anti-IL-12 antibody, proliferation was markedly reduced, although such a reduction was not associated with prolonged graft survival. Pre-treatment with anti-IL-12 antibody alone did not reduce proliferation in vitro. Supernatants from cultures of spleen cells from treated animals re-stimulated with donor-specific antigen were analysed for their cytokine content by ELISA. Unmodified graft rejection was associated with increased production of the Ti1 cyokines, IL-2 and IFN-gamma. Treatment with donor cells prior to grafting abolished this Th1 response in vitro, with cells producing little Th1 or Th2 cytokines, although grafts were still rejected at the normal time. Treatment with anti-IL-12 antibody alone induced cells to produce less IFN-gamma in vitro and also slightly increased production of the Th2 cytokines, IL-5 and IL-10, although this did not alter allograft rejection. Pre-treatment with both anti-IL-12 antibody and donor cells abolished a Th1 response and significantly increased Th2 cytokine production and this was associated with prolonged allograft survival. In this model, therefore, prolonged skin graft survival appeared to be associated with an increased Th-2 response and a down-regulated Th-1 response when cells from treated animals were re-stimulated in vitro. Specific cytotoxicity, as measured by CTL activity in spleen cell preparations was not reduced by neutralising endogenous IL-12, but NK cell activity was reduced. NK cells are responsible for the elimination of injected foreign cells, and persistence of such injected cells within the host has been shown by others to prolong allograft survival. However, using FACS analysis, it was not possible to demonstrate that reduced NK cell activity directly led to increased survival of injected BDF1 cells in treated animals in this model. Thus inhibition of endogenous IL-12, together with donor cell pretreatment, prolonged skin graft survival and this was associated with an up-regulated Th2 response and a down-regulated Th1 response. This indicates that effecting a switch to a Th2 response can prove beneficial for allograft survival.