A new approach to understanding T cell development: the isolation and characterization of immature CD4-, CD8-, CD3- T cell cDNAs by subtraction cloning

Abstract

During T cell development in the mammalian thymus, immature T cells are observed that lack the cell surface markers CD4, CD8, and CD3. A subtracted cDNA library was constructed to isolate cDNAs that are specific for these immature T cells. Tissue-specific expression of 97 individual cDNAs were examined using different cell types by Northern blot analysis, and six cDNAs were analyzed by reverse transcriptase (RT) polymerase chain reaction (PCR) detection of RNA. Approximately 50% of the clones could not be detected on Northern blots, and 40% of the clones were expressed by at least one other cell-type including monocytes, mature T cells, and B cells. Eight cDNA clones appear to be specific for the CD4-, CD8-, CD3- T cell line, used to construct the library, as determined by Northern blot analysis. In addition, 330 cDNA clones were subjected to partial automated DNA sequence determination. Database searches, with both nucleotide and protein translations, revealed cDNAs that exhibit interesting similarities to human cell-cycle gene 1, platelet-derived growth factor receptor, c-fms oncogene (CSF-1) receptor, and members of the immunoglobulin gene superfamily. This approach of employing subtraction coupled with large scale partial cDNA sequence determination can be useful to identify genes that may be involved in early T cell growth, cellular recognition or differentiation.