The Study Of Prolongation Of Rat Renal Allograft Survival By Cd4⁺cd25-t Cells Induced By Recipient Dendritic Cells Transfected With Ikk2dn

Objective: To establish a simple and reliable model of renal allograft transplantationin rats. Providing a more practical and high acute rejection rate model for experimentalstudies of renal transplantation and to observe whether CD4⁺CD25-T cells induced byrecipient-derived immature dendritic cells（imDC） transfected with IKK2dn gene andloaded with donor antigen can prolong the survival time of kidney transplantation in ratsand investigate its probably.Methods: DC were cultured from recipient rats （Lewis） bone marrow withgranulocyte macrophage colony stimulating factor （5ng/ml） and interleukin-4（5ng/ml）.These cells were collected on the fifth day, recombinant adenovirus expression plasmidIKK2dn was transfected into rat bone marrow-derived DC to arrest their maturation. Thenloaded with donor antigens for the last48hours. Autologous T cell proliferation stimulatedby Adv-IKK2dn-DC was assessed in primary mixed lymphocyte reaction（MLR）, and theT cells were separated by magnetic activated cell sorting after72hours. At last wecollected these cells for further experiment. Male Brown Norway rats and Lewis rats wereused as donors and recipients respectively, four groups were set up (naive T cells group,Adv0-CD4⁺T cells group, CD4⁺CD25-T cells group and rejection group), receiving1×107naive CD4⁺T-cell, Adv-0-DC-generated CD4⁺CD25-T-cell, Adv-IKK2dn-DC-generatedCD4⁺CD25-T-cell and equal volume of normal saline, respectively7days beforetransplantation. In the third party donor group, wistar rats as donors were treated the sameas CD4⁺CD25-T cells group before transplantation. After transplantation, the survival timeof recipients were observed; the T lymphocyte proliferation in recipients was measured;the levels of serum interleukin-2（IL-2）, interleukin-10（IL-10）, interferon-γ（IFN-γ）, transforming growth factor-β（TGF-β） were detected; serum creatinine levels weremonitored; pathological changes were examined to identify the grade of rejection.Results:1．To test the characteristics of DC on the fifth day by flow cytometer. TheimDC expressed lower levels of CD80、CD86and MHC-II （19.0±2.01%,17.3±1.17%and27.1±2.11%, respectively）, results suggested that DC in an immature state.2．In contrast tountransfected DC and Adv-0-DC, one-way MLR results showed that allogeneic T cellsproliferation induced by Adv-IKK2dn-DC was obviously low（P<0.05）.3.Recipient-derived imDC transfected by IKK2dn and loaded with donor antigen couldinduce lewis rat T lymphocyte into CD4⁺CD25-T cells in vitro. The expression of CD25inCD4⁺T cells induced by Adv-IKK2dn-DC was lower （17.8±0.89%） than that in the controlgroup （72.8±1.99%） and the Adv-0-DC group （63.3±1.70%）, the difference wasstatistically significant （P<0.05）. The purity of CD4⁺CD25-T cells which obtained by flowcytometry was （94.2±1.91）%, having expanded in cell number can be up to2.0².5×106/ml in vitro.4．Compared with other groups, CD4⁺CD25-T cells groupmarkedly prolonged the survival time of renal allografts （28.5±2.36d, P<0.01）, and hadhigher expression of IL-10and TGF-β （P<0.05or P<0.01）. Compared with Adv0-CD4⁺Tcells group, rejection group, the third party donor group, expression levels of IL-2andIFN-γ were lower（P<0.05）. In contrast to other groups, CD4⁺CD25-T cells group elicitedmarkedly lower proliferative responses （P<0.05or P<0.01） and lower grade as estimatedby pathological examination.Conclusions: These findings suggested that CD4⁺CD25-T cells induced byrecipient-derived immature DC transfected by IKK2dn and loaded with donor antigencould prolong renal allograft survival in a donor-specific manner, and it maybe byexpressing high levels of IL-10and TGF-β.