Enumeration of human alloreactive helper T lymphocyte precursor frequencies by limiting dilution analysis of
N T Young1, D L Roelen, M J Dallman
1Nuffield Department of Surgery, University of Oxford, UK. neil.young@nds.ox.ac.uk
Abstract:
We describe a limiting dilution assay for the enumeration of alloreactive interleukin-2 (IL-2) producing helper T lymphocyte precursors (HTLp). In place of the commonly used CTLL cell line, we have employed concanvalin A (ConA) stimulated rat thymocytes as IL-2 responsive indicator cells in a proliferation assay to detect IL-2 levels in limiting dilution microculture supernatants. The proliferation of ConA stimulated thymocytes induced by either recombinant IL-2 or culture supernatants could be blocked by co-incubation with a monoclonal antibody against the rat IL-2 receptor alpha chain, demonstrating the specificity of the response. Our investigations of alloantigen-induced IL-2 production show that (i) a minimum stimulator cell irradiation dose of 50-60 Gy is required to prevent backstimulation of microcultures; (ii) frequencies of alloreactive HTLp are significantly associated with HLA-DR antigen matching between responder and stimulator; (iii) HTLp frequencies detected in assays using B lymphoblastoid cell line stimulators are significantly higher than in assays employing peripheral blood lymphocyte stimulators but possibly reflect a degree of non-specific activation; and (iv) allosensitized responders exhibit altered kinetics of IL-2 production which may permit discrimination between sensitized and naive individuals. Our results both confirm and extend previous reports concerning such features of the alloresponse in humans and demonstrate that ConA stimulated thymocytes are a suitable alternative to CTLL as IL-2 responsive indicator cells in limiting dilution assays for HTLp analysis.
