SINGLE CELL ANALYSIS OF HUMAN ALLO AND XENORECOGNITION PATHWAYS MIRRORS THE MIXED LYMPHOCYTE RESPONSE

Abstract

51 T cell activation consequent to direct and indirect allo- and xenorecognition has usually been determined by measuring proliferation of bulk mixed lymphocyte cultures (MLC). Recently, single cell analysis of T cell activation has been reported using flow cytometric analysis of responder cells labeled with the fluorescent carboxyfluorescein diacetate succinimidyl ester (CFSE). Our study is the first to analyze single T cell proliferation in human allo and xeno responses due to direct and indirect recognition and to compare these results to the classical bulk thymidine incorporation assay. Methods: Human peripheral blood lymphocytes (PBL) were obtained by density gradient centrifugation of heparinized whole blood. One-way MLCs were set-up with 4×106 responder cells and 1×106 irradiated (25Gy) stimulator cells in 2 ml of medium. MLCs were set up to test direct recognition (APC-depleted responder PBL + stimulator adherent cells), indirect recognition (APC- depleted responder PBL + responder adherent cells + APC-depleted stimulator PBL) and both pathways (responder PBL + stimulator PBL). The stimulators were from allogeneic or xenogeneic (porcine) origin. Proliferation was assessed by 3H-thymidine incorporation (conventional MLC) and by flow cytometric analysis of CFSE labeled responder cells, counter stained with phycoerythrin labeled monoclonal antibodies for CD4 and CD8, dead cells were eliminated by gating with ViaProbe™. Results: One representative experiment of three is shown below. The conventional MLC results are given in counts per minute (cpm) and the CFSE data is expressed in number of divisions (div.) in the CD4/CD8 positive cell population at day 6. (Table)(Figure)TableFigureConclusions: We have demonstrated that single cell analysis of T-cell proliferation using CFSE can be used to study human allo and xenorecognition pathways (including preliminary studies analyzing costimulatory blockade; data not shown) and that the results mirror conventional MLC, in which indirect responses are less vigorous.