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 suppression

Human allograft acceptance is associated with immune regulation, characteri zed by donor-antigen-linked suppression of delayed-type hypersensitivity (D TH). We wished to determine if "classical" in vitro assays of alloreactivit y could also detect linked suppression and thus be useful in the clinical d iagnosis of active immune regulation. We analyzed peripheral blood mononucl ear cells from a group of eight liver transplant recipients, one of whom ha d stopped all immunosuppression 4.5 years ago yet continues to have good gr aft function (graft acceptor). The regulator phenotype was defined as the a bility to suppress a DTH response to a recall antigen in the presence of do nor antigen. Using the trans vivo DTH test, we identified four regulators, and four nonregulators. When we tested two of the regulators for in vitro m ixed lymphocyte culture (MLC) and cytotoxic T lymphocyte (CTL) responses to B-lymphoblastoid cell lines (B-LCL), we found both patients to be specific ally hyporesponsive to donor compared with third-party B-LCL stimulators. H owever, in contrast to the linked suppression of DTH seen when a given B-LC L expressed donor-type HILA-B antigens, there was no evidence of linked sup pression in vitro, either in CTL, proliferative, or interferon-gamma cytoki ne release assays. The primary CTL hyporesponsiveness to donor B-LCL could not be reversed by neutralizing antibodies to transforming growth factor be ta 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.