HUMAN LIVER ALLOGRAFT ACCEPTANCE AND THE ???TOLERANCE ASSAY???: IN VITRO ANTI-DONOR T CELL ASSAYS SHOW HYPOREACTIVITY TO DONOR CELLS, BUT UNLIKE DTH, FAIL TO DETECT LINKED SUPPRESSION1

Abstract

Human allograft acceptance is associated with immune regulation, characterized by donor-antigen-linked suppression of delayed-type hypersensitivity (DTH). We wished to determine if "classical" in vitro assays of alloreactivity could also detect linked suppression and thus be useful in the clinical diagnosis of active immune regulation. We analyzed peripheral blood mononuclear cells from a group of eight liver transplant recipients, one of whom had stopped all immunosuppression 4.5 years ago yet continues to have good graft function (graft acceptor). The regulator phenotype was defined as the ability to suppress a DTH response to a recall antigen in the presence of donor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro mixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specifically hyporesponsive to donor compared with third-party B-LCL stimulators. However, in contrast to the linked suppression of DTH seen when a given B-LCL expressed donor-type HLA-B antigens, there was no evidence of linked suppression in vitro, either in CTL, proliferative, or interferon-gamma cytokine release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor beta or interleukin-10, which could restore a strong DTH response to donor B-LCL. We conclude that DTH analysis can readily detect donor antigen-linked suppression in liver transplant recipients. CTL and MLC tests failed to do so. These findings may be relevant to the development of a tolerance assay suitable for use in clinical trials.