Sequential enrichment and immunocytochemical visualization of human interferon-alpha-producing cells

Abstract

Human HLA-DR+ peripheral mononuclear cells (PBMC) produce interferon-alpha (IFN-alpha) 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 greater than 125-fold enrichment of these IFN-alpha-producing cells. Nylon wood 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-alpha 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-alpha compared to 3,000-10,000 IU/ml for the corresponding LD cells and 30-300 IU/ml for PBMC. Immunocytochemistry to detect cytoplasmic IFN-alpha indicated that PBMC, NWNA, HD, LD, and CD3/CD56-depleted populations contained an average of less than 0.1%, 0.3%, less than 0.1%, 3%, and 12% IFN-alpha-producing cells, respectively. The cells responsible for IFN-alpha 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.