Determination of helper T-cell precursor frequencies against non-haemopoietic cells: comparison of co-stimulation provided by anti-CD28 antibody versus the cellular ligand B7-1

Helper T-cell precursor frequency assays (HTLp-assays) are commonly used in transplantation to examine the frequency of T cells reactive against donor or host alloantigens. In these assays, peripheral blood mononuclear cells (PBMCs) are most often used as stimalator cells. However, cells targeted af ter transplantation do not always belong to the haematopoietic lineage and may express different alloantigens, especially minor histocompatibility ant igens (mHags). Non-haematopoietic cells lack expression of the B7 co-stimul atory molecules needed to activate primary T cells that can be supplied by anti-CD28 (alpha CD28) antibodies or transfection with B7-1 coding sequence s. lit present, it is not known how these two ways of supplied costimulatio n compare in HTLp assays. B7-1-transfected A431 keratinocytes (A431(B7-1)) induced higher proliferative responses in allogeneic primary T cells and mo re interleukin (IL) 2 production than that induced by A431 cells plus alpha CD28, whereas the kinetics of proliferation and IL-2 production were simil ar. Neither cross-linking of alpha CD28 bound to T cells nor prevention of IL-2 resorption by the anti-IL-2 receptor resulted in improved proliferatio n or IL-2 production. Results of HTLp assays indicated that A431(B7-1) acti vated on average 7.5 times more alloreactive IL-2-producing T cells than A4 31 cells plus alpha CD28. We conclude that primary T-cell alloresponses aga inst major histocompatibility complexes (MHCs) and mHags expressed on nonha ematopoietic cells can be measured in HTLp assays using supplied co-stimula tion, although alpha CD28 yields an intrinsic underestimation of actual fre quencies.