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Sequential enrichment and immunocytochemical visualization of human interferon-alpha-producing cells
M Feldman1, P Fitzgerald-Bocarsly
1Department of Pathology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark 07103.
Insights
Researchers enriched interferon-alpha (IFN-alpha) producing cells from human peripheral blood mononuclear cells (PBMC) responding to herpes simplex virus type 1 (HSV-1). This new method significantly increases the yield of these crucial immune cells for further study.
Area of Science:
- Immunology
- Virology
- Cell Biology
Background:
- Human peripheral mononuclear cells (PBMC) produce interferon-alpha (IFN-alpha) upon stimulation with herpes simplex virus type 1 (HSV-1).
- Identifying the specific cells responsible for IFN-alpha production has been challenging due to their low frequency in PBMC populations.
Purpose of the Study:
- To develop and optimize a protocol for enriching IFN-alpha-producing cells from human PBMC in response to HSV-1.
- To characterize the enriched cell population and overcome the limitations of low cell frequency for identification.
Main Methods:
- PBMC were fractionated using a 48% Percoll gradient to obtain low-density (LD) and high-density (HD) populations.
- LD cells underwent further depletion of monocytes, CD3+ T cells, and CD56+ natural killer (NK) cells.
- Immunocytochemistry was used to quantify IFN-alpha-producing cells and characterize their morphology.
Main Results:
- The developed protocol achieved a greater than 125-fold enrichment of IFN-alpha-producing cells.
- The CD3/CD56-depleted population showed a significant increase in IFN-alpha production (approx. 30,000 IU/ml) compared to unfractionated PBMC (30-300 IU/ml).
- The enriched cells were characterized as medium to large in diameter with specific nuclear and cytoplasmic features, and showed a fivefold enrichment for HLA-DR+ cells.
Conclusions:
- A novel enrichment protocol effectively isolates and concentrates IFN-alpha-producing cells from human PBMC stimulated by HSV-1.
- This method facilitates the identification and study of these critical immune cells, overcoming previous frequency-related barriers.
- The enriched cells possess characteristics consistent with antigen-presenting cells, highlighting their role in antiviral immune responses.
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.
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