IMMUNE REGULATION AND TOLERANCE I

N. Satoda¹, Y. Li², H. Ohe³, S. Sakaguchi⁴, S. Uemoto⁵, H. Date¹, T. Koshiba^2
1Department Of Thoracic Surgery, Kyoto University, Gradate School of Medicine, Kyoto/JAPAN, ²Hepatopancreatobillary Surgery And Liver Transplantation, Kyoto University Hospital, Kyoto/JAPAN, ³Department Of Surgery, Kyoto University Graduate School of Medicine, Kyoto/JAPAN, ⁴, Institute for Frontier Medical Science, Kyoto University, Kyoto/JAPAN, ⁵Division Of Hepato-pancreato-biliary Surgery And Transplantation, Department Of Surgery, Kyoto University, Kyoto/JAPAN

Body: (Background) An adoptive cell transfer of regulatory T cells (Tregs) has been successful in rodent Tx models,but little is known about its potential in large animal Tx models.
(Method) Fully mismatched miniature swines (two haplotypes; C1 and C2), weighing 20-30kg, were used as recipients and donors.Orthotopic lung Tx was performed. The peripheral blood mononuclearcells were obtained from recipients by apheresis and CD4⁺CD25⁺⁺ cells were isolated by magnet beads system on pre-Tx day 2. They were stimulated by donors APC with IL-2 andrapamycin until post-operative day (POD) 10 when cultured CD4⁺CD25⁺⁺ cells (10⁶ cells/kg) were transferred into recipients in the presence of leukopenia induced bycyclophosphamide. High-dose tacrolimus was used from Tx to POD 6, which was followed by low-dose tacrolimus from POD 7 to 21(Group-Tregs+TAC,n=5). Graft survivals were compared with those in othergroups where low dose tacrolimus alone (POD7 to 21) (Group-TAC,n=4), Tregs transfer alone (POD10) (Group-Tregs,n=3), and neither low dose tacrolimus nor Tregs transfer(Group-non TAC,non Tregs, n=2)was given. High-dose tacrolimus was given to all the groups(Tx to POD6). Open biopsy of grafts was performed at the time of transfer and 4 days after transfer and FOXP3mRNA level was semiquantifiedby RT-PCR.
(Results) In Group-non TAC,non Tregs, graft was rejected on POD15.5. Adjunction of low dose tacrolimus alone or Tregs transfer alone did not prolonged survivals (Group-TAC and-Tregs; POD16.3and 15). However, in the presence of Tregs transfer and low dose tacrolimus, survival was the greatest (Group-Tregs+TAC ; POD 51.0, p<0.01 vs Group-TAC, -Tregs and -non TAC, non Tregs). In thisgroup, one recipient was rendered tolerance (>POD100). An increase of intra-graft FOXP3 expression was observed in 4 days after Tregs transfer only in Group-Tregs+TAC, but not in othergroups.
(Conclusions) Adoptive transfer of ex vivo donor alloantigen-stimulated Tregs combined with low dose tacrolimus ameliorates rejection of immunogenic lung Tx in clinically relevant miniatureswines. Early accumulation of Tregs was observed in tolerized grafts but not rejecting grafts.

Disclosure: All authors have declared no conflicts of interest.