Characterization of cell phenotype by a novel cDNA library subtraction system: expression of CD8 alpha in a mast...

We have established a unique variant cell line, MC/9.IL-4, which continuously proliferates in the presence of interleukin-4 (IL-4), from a murine interleukin-3 (IL-3)-dependent mast cell line, MC/9 (referred to as MC/9.IL-3). Compared with MC/9.IL-3 cells, MC/9.IL-4 cells are smaller, lack cytoplasmic granules and metachromasia, carry a very small amount of histamine, and express fewer high-affinity IgE receptors (IgERs) and IL-3 receptors. To further characterize MC/9.IL-4, we developed a novel method to enrich cell type-specific cDNAs by cDNA library subtraction and applied it for MC/9.IL-3 versus MC/9.IL-4. Sequence analysis of cDNA clones isolated by this technique showed that MC/9.IL-4 cells specifically express CD8 alpha and expression of mast cell-specific proteases and major histocompatibility complex class II (MHCII) is considerably decreased. It was also noted that responsiveness to the IL-3-agonistic antibody F9 and expression of the transcription factor GATA-2 is diminished in MC/9.IL-4, indicating that MC/9.IL-4 have lost major characteristics of the bone marrow-derived cultured mast cells. Because other T-cell marker antigens, CD8 beta, CD4, Thy-1, were not detected on MC/9.IL-4 cells, MC/9.IL-4 cells may represent an unknown class of hematopoietic cells that express CD8 alpha. This cell line will be useful in studies of IL-4-mediated signal transduction, as well as transcriptional regulation of mast cell characteristic genes. This study also demonstrates the effective use of the cDNA library subtraction strategy to characterize unknown types of hematopoietic cells at the molecular level.