Role Of Decay Accelerating Factor In Allograft Immunity In Mice

Decay-accelerating factor (DAF) is a cell surface complement regulatory protein that accelerates the dissociation of C3convertase, which plays a pivotal role in the ampification of complement activation, and thereby prevents the complement-mediated self-damaging effect on cell.In the context of transplantation, the deficiency of DAF, which has been thought to regulate antibody-mediated rejection, was detected to aggravate ischemia-reperfusion injury. Inversely, increased expression of DAF in renal allograft was associated with better graft function and longer graft survival.In addition, DAF also play a role in regulating cellular immunity and humoral immunity. Absence of DAF in patient with paroxysmal nocturnal hemoglobinuria increase C3b on the surface of erythrocyte and hemolysis. In contrast, overexpression of DAF on tumor cell results in immune escape. In the context of transplantation, the deficiency of DAF, which has been thought to regulate antibody-mediated rejection, was detected to aggravate ischemia-reperfusion injury. Inversely, increased expression of DAF in renal allograft was associated with better graft function and longer graft survival. We construct a mouse ventral heterotopic heart transplant model and use Daf1deficiency mice to study the effect of DAF deficiency on allograft rejection and T cell immunity.Methods and Results1.Construct a mouse ventral heterotopic heart transplant model.Hearts were transplanted heterotopically by anastomosing the donor aorta and pulmonary artery to the recipient abdominal aorta and inferior vena cava. Syngeneic heart mean survival time (MST) of60days, allograft MST of9days. Microscopic examination of H&E-stained tissue sections obtained at cessation of heartbeats revealed diffuse mononuclear cell infiltration and perivascular inflammation in both groups, typical of acute cellular rejection.2.The absence of donor Dafl accelerates graft rejection through augmentation of direct pathway mediated direct anti-donor T cell responses.To delineate the role of donor DAF in T cell mediated rejection of a vascularized allograft, we examined the survival of Dafl deficient (Daf1-/-) hearts, WT hearts and C3deficient hearts from male C57BL/6(B6, H-2b) mice following transplantation into fully allogeneic recipients (male BALB/c (H-2d) mice). In H-2d recipients, B6H-2b Dafl-/-hearts were rejected on7days posttransplantation, faster than WT and C3deficient hearts which were rejected on9.5and14days posttransplantation respectively. Results were statistically significant. Similar results were observed with C3H (H-2k) male recipients.(Daf1-/-MST of8days, WT MST of17days, C3-/-MST of25days with two>60days, n=6/group, p<0.01).IFN-y ELISPOY assays of direct anti-donor T cell response performed at the time of rejection revealed that spleens of recipients of Daf1-/-allografts contained almost2-fold more donor-reactive IFN-y producers (p<0.05vs WT recipients). In contrast, the total number of anti-donor IFN-y producing T cells in recipients of C3-/-allografts was low.(Daf1-/-235.5±60.3、WT98.5±15.3、C3-/-56.3±16.7(/5×104, X±S))Direct anti-donor T cell responses in recipients of Dafl-/-B6hearts assessed by ELISPOT assay6days posttransplantation were significantly higher than those detected in recipients of WT B6hearts.(Dafl-/269.5±48.2, WT126.0±19.3(/5×104, x±S)(p<0,01)).3.Recipient DAF deficiency has no effect on graft survival nor anti-donor T cell immunity.To assess the role of recipient DAF deficiency in T cell mediated rejection of a vascularized allograft. We examined the survival of heart from BALB/c (H-2d) following transplantation into Daf1-/-(n=4) and WT (n=5) B6(H-2b) female recipients. In Dafl-/-recipients, allografts were rejected10.5days posttransplantation and the results was not significantly different from WT recipient on day10posttransplantation.(p>0.05). The frequency of recipient IFN-y-producing T cells in Daf1-/-recipients was266.8±16.9, and in WT recipients was206.0±13.5(/5×104, χ±S). The statistics was not significant.4.Dornor DAF deficiency accelerated graft rejection independent of antibody initiated, classic complement pathway.We transplanted WT or Daf1-/-B6heart into allogenic BALB/c (H-2d) recipients. On day6posttransplantation, flow cytometry was performed to detect the donor-reactive alloantibodies directed to donor thymocyte. The titer of Ab was low and was not significantly different between recipients of WT (n=3)and Daf1-/-B6(n=3) heart grafts (p>0.05).We transplanted WT or.Daf1-/-B6heart into allogeneic SCID/C3H (H-2k) recipients. Ten days later we adminstered6×106T cells from naive C3H mice thymus gland i.v. through the tail vein and followed graft survival by palpation. The adoptively transferred T cells caused acute rejection of the Dafl-/-B6hearts by day9, while WT hearts exhibited significantly prolonged survival (one survive12days, two>20days) despite transfer of the same number of WT T cells. Besides, no anti-donor antibody was detected in serum of SCID/C3H (H-2k) recipients of both groups.5.DAF enhances function of alloreactive CD8effector T cells.We administered15×106splenic cell from B6(H-2b) mice to C3H (H-2k) mice i.v. Through the tail vein. Two weeks later, CD8+effector T cell from spleen of C3H mice was isolated and stimulated with splenic cell (with APC) from Daf1-/-、WT、C3-/-B6(H-2b) mice. IFN-γ ELIDPOT assays were performed to test the T cell response when they reencounter their target Ags. IFN-γ production and CLT activity were significantly greater when the primed T cells were challenged with Daf1-/-splenic cells than those to WT and C3-/-stimulators(Daf1-/-878.5±270.9、WT389.8±53.5、C3-/-264.3±27.1(/1×105, χ±S)).(WT vs. C3-/-P<0.01;Daf1-/-vsC3-/-WT P<0.001).6. DAF affects the antigen presentation ability of APCs.Peritoneal microphages (APC) from WT or Daf1-/-B6mice was co-cultured with CD+4effector T cells and ovoalbumin (OVA) or ovoalbumin polypeptide (OVA323-339) for24Hs. After centrifugation, supernatant was assessed by CTLL assay for IL-2produced by activated T cell. The production of IL-2was significantly greater by Daf1-/-originated macrophagy than WT when co-cultered with CT4+effector T cell and OVA (P<0.0001). However, there was not discernible difference in IL-2production between Daf1-/-originated and WT originated macrophagy when co-cultered with CT4+effector T cell and OVA323-339(p>0.05). Similar results were found in CT8+effector T cells when co-cultrured with peritoneal macrophagy and OVA/OVA323-339. Conclusion1. Donor DAF deficiency augment graft rejection and anti-donor T cell response.2. Recipient DAF deficiency has no effect on graft survival nor anti-donor T cell immunity.3. DAF enhances function of alloreactive CD8effector T cells. When APC interacts with T cell, APC DAF deficiency (not T cell DAF deficiency) enhance T cell function.4. APCs DAF deficiency enhance the antigen presentation ability.5. Dornor DAF deficiency accelerated graft rejection independent of antibody initiated, classic complement pathway.