An In Vitro Model to Investigate the Importance for Renal Transplant Survival of Mismatches for Specific Histocompatibility Antigens

Abstract

The importance of specific donor and recipient HLA antigens for transplant survival has been difficult to estimate from clinical outcomes because of constraints on experimentation imposed by transplantation. As an alternative approach we investigated the feasibility of inducing sensitization and effector responses in vitro using assays for macrophage migration inhibition (MIF) and stimulation (MStF) factors. Supernatants were assayed for MIF and MStF activity in triplicate by the capillary tube method using the human monocytoid cell line U937 as target cells (1). The ratio of migration areas produced by supernatants of alloantigen-stimulated cells to those of unstimulated cells was multiplied by 100 to give a migration index with values of < 91, 91–109, and > 109 taken as MIF, negative, or MStF responses respectively. These responses were determined for recipient mononuclear cells stimulated by an extract of donor spleen or peripheral blood mononuclear cells at 3, 7, 10, and 14 days after renal transplantation from cadaver donors (2). The responses of cells sensitized in vitro in mixed lymphocyte reactions (MLR) were also determined using responder cells from renal transplant recipients obtained prior to transplantation and donor cells as stimulators. Lymphokine responses in MLR were also evaluated using cells from healthy individuals as stimulators and responders. Stimulator cells were pulsed with puromycin 5 µg/ml for two hours prior to culture and responder cells were stimulated with a soluble extract from the stimulator cells after 72 hours culture. Typing for HLA A,B,C, and DR antigens was performed with tissue typing trays obtained from the Canadian Red Cross.