Autoreactivity, backstimulation and reproducibility in a helper T lymphocyte precursor assay

Helper T lymphocyte precursor (HTLp) frequencies determined by limiting dil ution analysis (LDA) have a predictive value for alloreactivity in allogene ic bone marrow transplantation. Methodological problems in LDA include auto reactivity in the responder or stimulator cell populations and interleukin 2 (IL-2) production by the stimulator cells as a response to the responder cells (backstimulation). The extent and impact of these aspects for IL-2 pr oduction and HTLp frequency determination were studied by autologous and al logeneic mixed lymphocyte reactions with healthy volunteers and HTLp determ inations from bone marrow transplantation donor/recipient pairs. We found t hat autoreactivity occurred in the unirradiated cells with a reproducible i nter-individual variation. The immunogenicity of the stimulator cells was p reserved after gamma irradiation with 50 Gy and the risks of autoreactivity and backstimulation were limited. Higher doses of irradiation decreased th e immunogenicity. Immune reactions to antigens present in the serum supplem ent of the culture medium were seen with foetal calf serum and to a lesser extent with pooled human sera. This could be avoided by the use of autologo us serum. We were unable to ensure satisfactory culture conditions in serum -free medium. The reproducibility of the HTLp frequency determinations was tested for intra- and inter-assay variation. The coefficients of variation were estimated as 24% and 35%, respectively. This was acceptable considerin g the range of the HTLp frequencies (1:10(2) to 1:10(7)). The influence of the extent of autoreactivity of the bone marrow donors was investigated in 28 HLA-identical sibling transplantations. We found no correlation between the autoreactivity of the donors and the HTLp frequencies. The extent of au toreactivity of the donor did not correlate with the clinical outcome in te rms of acute graft-versus-host disease, treatment-related mortality, risk o f relapse and overall survival. In spite of methodological difficulties and interference from autoreactivity and backstimulation, reproducible quantif ication of clinically significant alloreactivity can be attained. (C) 2001 Elsevier Science B.V. All rights reserved.