THE ROLES OF DONOR LEUKOCYTES AND SOLUBLE MHC CLASS I IN TRANSPLANTATION TOLERANCE

Abstract

A275 Aims: Spontaneous induction of tolerance occurs in the DA (RT1a) to PVG (RT1c) rat model following orthotopic liver transplantation (OLT), while heart grafts, unless accompanied by a liver transplant, are acutely rejected in 8 days. The liver, unlike the heart allograft, is a source of both secreted soluble MHC molecules and resident leukocytes which may be responsible for spontaneous tolerance. In the present study, we measured levels of circulating soluble MHC class I molecules (sMHC) following OLT and tested whether sMHC and leukocytes could substitute for a liver transplant in protecting DA cardiac allografts from acute rejection in PVG recipients. Methods: PVG rats received either a heterotopic cardiac allograft or an OLT from DA donors. MHC class I heavy chains (RT1Aa, RT1A1c or RT1Au) and β2microglobulin were produced using a prokaryotic expression system, refolded around appropriate single peptides and purified by gel filtration using FPLC. Recombinant sMHC were continuously administered intravenously to graft recipients using osmotic mini infusion pumps. Allotype specific ELISA were developed and used to quantify levels of circulating MHC levels in the recipient sera. Results: Naïve DA rats had circulating levels of sRT1Aa of ∼350ng/ml while RT1A1c was <50ng/ml in naïve PVG rats. Following OLT, donor sMHC (RT1Aa) rise rapidly, peaking around 400ng/ml before returning to a plateau of ∼200ng/ml, 2 weeks after transplantation. Recipient RT1A1c levels also increased briefly to ∼100ng/ml following OLT. Infusion, at the time of transplant, of sRT1Aa alone failed to prolong cardiac allograft survival (median survival time, mst = 8d) despite serum levels of 2000ng /ml. However, combined administration of 10-150 million donor lymph node or spleen cells together with continuous sRT1Aa (serum levels >1000ng/ml) significantly prolonged allograft survival (mst = 12d versus 6.5d for cells alone). Similarly, pre-treatment with sRT1Aa alone also prevented rapid rejection (mst = 12d). Third party sMHC or cells were ineffective. Conclusions: These findings support the concept that soluble MHC class I may play a role in spontaneous liver tolerance. It is noteworthy that the sRT1Aa used in this study was of prokaryotic (not eukaryotic) origin and therefore lacked glycosylation and presented only a single peptide rather than a range of naturally selected peptides.