ENUMERATION OF HUMAN ALLOREACTIVE HELPER T-LYMPHOCYTE PRECURSOR FREQUENCIES BY LIMITING DILUTION ANALYSIS OF INTERLEUKIN-2 PRODUCTION

We describe a limiting dilution assay for the enumeration of alloreact ive interleukin-2 (IL-2) producing helper T lymphocyte precursors (HTL p). In place of the commonly used CTLL cell line, we have employed con canavalin A (ConA) stimulated rat thymocytes as IL-2 responsive indica tor cells in a proliferation assay to detect IL-2 levels in limiting d ilution microculture supernatants. The proliferation of ConA stimulate d thymocytes induced by either recombinant IL-2 or Culture supernatant s could be blocked by co-incubation with a monoclonal antibody against the rat IL-2 receptor a chain, demonstrating the specificity of the r esponse. Our investigations of alloantigen-induced IL-2 production sho w that (i) a minimum stimulator cell irradiation dose of 50-60 Gy is r equired 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 det ected in assays using B lymphoblastoid cell line stimulators are signi ficantly higher than in assays employing peripheral blood lymphocyte s timulators but possibly reflect a degree of non-specific activation; a nd (iv) allosensitized responders exhibit altered kinetics of IL-2 pro duction which may permit discrimination between sensitized and naive i ndividuals.Our results both confirm and extend previous reports concer ning 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 analys is.