MECHANISMS OF TOLERANCE INDUCED BY TOLEROGENIC ALLOCHIMERIC DONOR/RECIPIENT CLASS I MHC PROTEIN

Abstract

42 Introduction: We previously reported that portal vein (p.v.) or oral administration of a donor/recipient tolerogenic protein (α1hu62-69-RT1.Aa) bearing four donor amino acids (Arg62, Glu63, Gln65, Gly69) in combination with a short course of cyclosporine (CsA) to ACI (RT1a) rats induced donor-specific transplantation tolerance to Wistar Furth (WF; RT1u) heart allografts (>100 days). The present study examines the mechanisms of tolerance using T cell adoptive transfer experiments and analysis of cytokine mRNA expression. Methods: Tolerance to WF hearts was induced by p.v. injection to ACI rats of 10 µg α1hu62-69-RT1.Aa (day 0) or oral gavage with 5 mg α1hu62-69-RT1.Aa (days 0-3) in combination with a 7-day CsA course (4 mg/kg/day; days 0-6). For adoptive transfer experiments, 40×106 T cells purified from spleen or lymph node cells of tolerant ACI recipients were injected intravenously into irradiated (500 rads) syngeneic ACI recipients of donor-type WF or third-party Brown Norway (BN; RT1n) heart allografts. Expression of cytokine mRNAs was analyzed in: WF heart allografts from tolerant ACI recipients (>100 days); WF hearts from untreated WF recipients (day 7; isograft controls); and WF heart allografts from untreated ACI recipients (day 7; allograft controls). Normal WF hearts (normal controls) were also used for analysis. Total RNA was purified by homogenizing hearts in guanidine isothiocyanate solution, followed by CsCl2 gradient with ultracentrifugation. A semi-quantitative RT-PCR method was used to measure the levels of cytokine mRNAs expression using the pairs of gene-specific primers, namely for IL-2, IFN-γ (T helper 1; Th1), IL-4, and IL-10 (Th2). Results: Irradiated ACI recipients rejected WF heart allografts at a mean survival time (MST) of 33.2±1.1 days. Adoptive transfer of 40×106 tolerant T cells, obtained from p.v.-induced (n=5) or orally induced (n=4) tolerant recipients, rendered irradiated ACI recipients tolerant to WF heart allografts (> 100 days, p<0.01). In contrast, the same tolerant 40×106 T cells did not prolong the survival of the third-party BN heart allografts; MSTs were 13.3±5.5 days in a p.v. tolerance model (n=3, NS) and 12.5±1.0 days (n=3, NS) in an oral tolerance model. Non-transferred ACI recipients rejected BN heart allografts at 11.6±0.9 days. WF heart allograft from untreated recipients had increased levels of mRNAs for IL-2 (++++), IFN-γ (++++), IL-4 (+), and IL-10 (++++) when compared with normal or isograft WF controls [IL-2 (-), IFN-γ (+), IL-4 (-), and IL-10 (+)]. In comparison to rejectors, tolerant WF heart allografts (from p.v.- or orally induced tolerant hosts) displayed reduced levels of IL-2 (+) and IFN-γ (+++) mRNAs, but not IL-4 (+) and IL-10 (++++) mRNAs. Conclusion: These results suggest that allochimeric protein-induced tolerance is mediated by regulatory T cells, possibly with selective activation of Th2-like cells.