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 stimulator cells. However, cells targeted after transplantation do not always belong to the haematopoietic lineage and may express different alloantigens, especially minor histocompatibility antigens (mHags). Non-haematopoietic cells lack expression of the B7 co-stimulatory molecules needed to activate primary T cells that can be supplied by anti-CD28 (αCD28) antibodies or transfection with B7-1 coding sequences. At present, it is not known how these two ways of supplied costimulation compare in HTLp assays. BT-1-transfected A431 keratinocytes (A431(B7-1)) induced higher proliferative responses in allogeneic primary T cells and more interleukin (IL) 2 production than that induced by A431 cells plus αCD28, whereas the kinetics of proliferation and IL-2 production were similar. Neither cross-linking of αCD28 bound to T cells nor prevention of IL-2 resorption by the anti-IL-2 receptor resulted in improved proliferation or IL-2 production. Results of HTLp assays indicated that A431(B7-1) activated on average 7.5 times more alloreactive IL-2-producing T cells than A431 cells plus αCD28. We conclude that primary T-cell alloresponses against major histocompatibility complexes (MHCs) and mHags expressed on non-haematopoietic cells can be measured in HTLp assays using supplied co-stimulation, although αCD28 yields an intrinsic underestimation of actual frequencies.