DISTINCT TOLERANCE PATHWAYS IN LIVER TRANSPLANT RECIPIENTS:

251 We have recently demonstrated paradoxical effects of skin allograft (Tx)-induced sensitization in rat orthotopic liver transplant (OLT) model. Although LBNF1 liver Tx survive >100 d in normal LEW recipients, they are rejected in an accelerated manner in <14 d in rats presensitized with BN skin Tx. Surprisingly, however, two consecutive skin grafts one week apart, induce indefinite liver Tx survival. This study was designed to dissect putative mechanisms that govern liver Tx acceptance in untreated vs. "presensitized" OLT recipients. First, we have confirmed that normal otherwise untreated LEW rats survived >100 d following transplantation of LBNF1 liver Tx (Gr. I; n=6), as did isograft controls (n=3). Similarly, LEW recipients of LBNF1 liver Tx that were repeatedly challenged with BN skin Tx (d −14 and −7) survived >100 d (Gr. II; n=5). Serum sGOT levels (IU/L) at >100 d in Gr. I and II of OLT rats were at average of 417 and 250, resp. (p<0.01) vs. 80 in isograft controls. Long-term (>100 d) liver Tx recipients were then challenged with donor-specific or third-party cardiac Tx. Acute (7 d) rejection of DA and acceptance of LBNF1 secondary Tx suggests donor-specific tolerance in both Gr I and Gr II OLT models. Well-functioning cardiac Tx were harvested at d 35 and analyzed by immunohistochemistry. In contrast to Gr. I, cardiac Tx harvested from Gr. II animals showed good preservation of histologic detail and mild T cell (ca. 21 cells/10HPF) and macrophage (ca. 38) infiltration (vs. 55 and 126 in Gr. I, resp). We then asked whether regulatory T cells contribute to immune tolerance in our OLT models. Spleen cells (100×106) from rats bearing liver Tx for >100 d were transferred i.v. into syngeneic secondary hosts. These were then challenged 24 h later with LBNF1 cardiac Tx. Two out of 3 test cardiac Tx in Gr. I, and all three test cardiac Tx in Gr. II survived >200 d after transfer of OLT spleen cells. These Tx recipients were challenged at 210 d with secondary LBNF1 test cardiac Tx. As in the original Gr. I and Gr. II OLT models, secondary test Tx survived long-term in adoptively transferred hosts. They were harvested (d 29) along with primary test cardiac Tx (d 238) for immunohistochemical assessment. In contrast to rats given regulatory T cells from Gr. I OLT hosts, both primary and secondary test cardiac Tx in rats conditioned with cells from Gr. II OLT hosts, showed absence of IL-2R (CD25) staining and minimal ICAM-1 expression. Moreover, T cell and macrophage infiltration was sparse (14-36 cells/10HPF) as compared to corresponding Gr. I cardiac Tx (140->200 cells). Conclusion: (i) donor-specific "immune" tolerance does not correlate with a "true" tolerance in OLT models; (ii) repeated skin Tx challenge in OLT recipients exerts "protective" effects upon Tx function in an organ-nonspecific manner, and can be transferred in an "infectious" fashion by spleen cells into new cohorts of test Tx recipients; (iii) such an unexpected tolerogenic mechanism results most likely from APC modifications and/or hematopoietic microchimerism.