Prevention of Donor Specific Antibody Production after Skin Allografting by Mobilization of Endogenous Stem Cells Using a Combination of AMD3100 and Low-dose FK506 in Rats

Abstract

Background: We have developed a stem cell mobilizing strategy that enables long-term liver and kidney allograft survival without sustained immunosuppression in small and large animals using a safe combination of two FDA approved drugs AMD3100 and low-dose FK506 (AF). Furthermore, no increase in serum DSA levels was detected in animals displaying long-term survival even after donor skin grafts were rejected. Therefore, the purpose of this study is to determine if AF combination treatment prevents de novo DSA production in a rat model of skin allograft rejection. Methods: Split thickness skin allografts from Dark Agouti (DA, RT1a) rats were transplanted into adult Lewis (RT11) recipients. Recipient animals were randomly divided into AF treatment group (AMD3100 1 mg/kg and FK506 0.1 mg/kg, s.c., every other day for 14 days) and control group (same volume of saline, s.c.). Serum levels of DSA including both IgG and IgM were measured by flow cytometry at 14 days, 1 and 2 months after skin transplantation. The sensitization to donor antigens in Lewis rats at 2 months after skin allografting was further confirmed by transplanting donor DA kidneys. Full dose FK506 (1 mg/kg/day, s.c) was given to prevent acute cellular rejection after kidney transplantation. Results: Skin allografts were rejected in 8–10 days after transplantation in both groups. Serum levels of DSA-IgG increased 6 to 10 folds in control animals (n = 10), but only 2 to 3 folds in animals treated with AF combination (n = 11) at 1 month and remained at similar levels at 2 months after skin transplantation. All control animals (n = 5) died within 7 days after kidney transplantation. DSA deposition and AMR in kidney allografts were confirmed with signs of IgG deposition in and around glomeruli with C4d deposition on peritubular capillaries. In contrast, about 64% (7/11) animals with AF treatment after skin allografting survived over three weeks without antibody mediated rejection (AMR) and no increase in serum levels of DSA at 14 days after kidney transplantation. Conclusion: Mobilizing endogenous stem cells with AF combination treatment does not prolong skin allograft survival but prevents de novo DSA production after allograft rejection. AF treatment may inhibit B cell sensitization, prevent de novo DSA production and improve long-term allograft function after organ transplantation.