Assessment of human allospecific IL-2-secreting helper T lymphocytes in limiting dilution cultures using restimulation and split well analysis.

A limiting dilution (LD) culture was established which allows the detection of allospecific interleukin-2 (IL-2)-secreting helper T lymphocyte precursors (HTL-p) among human peripheral blood mononuclear cells (PBMNC). HTL-p stimulated with allogeneic Epstein-Barr virus-transformed B lymphoblastoid cell lines (EBV-LCL) in the presence of exogenous recombinant IL-2 (r-IL-2) clonally developed into IL-2 secreting effector cells when restimulated against the original stimulating alloantigen. Split well cultures were performed prior to restimulation to assess the antigen specificity of the response. Frequencies of alloreactive IL-2-secreting HTL-p ranged from 1/100 to 1/800. Allospecificity of effector T cells was determined by a strong decline of frequencies obtained after restimulation against third-party antigens. In (clonal) segregation analyses the vast majority of IL-2-secreting progenies (80%) were specific for the original stimulating alloantigen. Allele specificity was disclosed by using class II MHC related third-party restimulator cells. By comparison with LD short-term cultures it became evident that exogenous r-IL-2 in the initial culture period was required to reveal optimal precursor frequencies of IL-2-producing T cells. Furthermore, successful antigenic restimulation was strictly confined to these culture conditions.