Sequential Enrichment and Immunocytochemical Visualization of Human Interferon-α-Producing Cells

Abstract

Human HLA-DR⁺ peripheral blood mononuclear cells (PBMC) produce interferon-α (IFN-α) in response to herpes simplex virus type 1 (HSV-1) or HSV-1-infected fibroblasts (HSV-FS). We have developed a protocol, based partly on a technique known to enrich for dendritic cells, that allows for a > 125-fold enrichment of these IFN-α-producing cells. Nylon wool nonadherent PBMC (NWNA) were fractionated on a 48% Percoll gradient into low-density (LD) and high-density (HD) populations. The LD cells were 10- to 30-fold enriched for the production of IFN-α compared to PBMC when stimulated with HSV-FS. LD cells were treated further to deplete any contaminating monocytes, CD3⁺ T cells and CD56⁺ natural killer (NK) cells. The resulting population (CD3/CD56-depleted) produced approximately 30,000 IU/ml IFN-α compared to 3,000–10,0000 IU/ml for the corresponding LD cells and 30–300 IU/ml for PBMC. Immunocytochemistry to detect cytoplasmic IFN-α indicated that PBMC, NWNA, HD, LD, and CD3/CD56-depleted populations contained an average of <0.1%, 0.3%, <0.1%, 3%, and 12% IFN-α-producing cells, respectively. The cells responsible for IFN-α production in response to HSV-1 were of medium to large diameter and possessed eccentric nuclei that were often indented, with lightly staining perinuclear areas. The CD3/CD56-depleted populations were fivefold enriched for HLA-DR⁺ cells. This enrichment procedure partially overcomes the barrier of low frequency that has contributed to the elusive identification of these cells.