Infrequent but efficient interferon-α-producing human mononuclear leukocytes induced by herpes simplex virus in vitro studied by immuno-plaque and limiting dilution assays

Human blood mononuclear leukocytes (PBMCs) producing interferon-alpha (IFN-alpha) after stimulation by herpes simplex virus type 1 (HSV) in vitro were identified by a filter immuno-plaque assay. Individual IFN-alpha-producing cells (IPCs) yielded between 0.5 and 2 units IFN-alpha, sufficient to protect cultures of MDBK cells against a viral challenge. Therefore, their frequency could be determined by a limiting dilution assay as well as by an immuno-plaque assay. Similar estimates of between 2 and 55 IPCs per 10(4) PBMCs at the peak of the IFN-alpha response were obtained by the two methods. IPCs were first detected 3 h after stimulation by HSV; their number peaked at 8 h and then declined. IPC frequencies were influenced by the concentrations of HSV and PBMCs during induction, but the quantity of IFN-alpha produced per IPC was relatively constant. The relation between the numbers of IPCs and PBMCs was linear at high PBMC concentrations, whereas at low PBMC concentrations fewer IPCs than expected were detected. The response could be fully restored by adding a combination of filler cells (Namalwa or U937 cells) and conditioned medium from 6-h HSV-induced PBMC cultures. Our results suggest that HSV induces an IFN-alpha response in a relatively rare population of efficient IPCs by complex mechanisms, which may involve cell cooperation and/or production of soluble factors.