Gene Expression Profile and Identification of Differentially Expressed Transcripts during Human Intrathymic T-Cell Development by cDNA Sequencing Analysis

Abstract

The development of immature thymocytes to mature T-lymphocytes is a central process for establishing a functional immune system. The gene regulatory events involved in this process are of outstanding interest in understanding the generation of the T-cell repertoire as well as the differentiation of lineage-specific cells, such as CD4+ helper T-cells or CD8+ cytotoxic T-lymphocytes. While some essential genes involved in lineage decision and thymocyte differentiation have been already identified, the exact regulatory mechanisms and differential gene expressions are still unknown. The present study was performed to analyze the gene expression profile during T-cell development, in particular, during the differentiation of immature thymocytes into CD4+ mature T-cells by analyses of expressed sequence tags (ESTs), and to elucidate novel human genes involved in this process. Based on distinct developmental stages, three PCR-based cDNA libraries from immature CD3-,4-,8- triple-negative, CD4+,8+ double-positive, and mature CD4+,8- single-positive thymocytes were constructed. A total of 1477 randomly selected clones were analyzed by automated single-pass sequencing, and the assembly of ESTs resulted in 1027 different species of contig sequences. Among them, 392 contig sequences were matched to known genes, and several novel transcripts were discovered. The matched clones were classified into seven categories according to their functional aspects, and the gene expression profiles of the three thymocyte subsets were compared. The information ohtained in current study will serve as a valuable resource for elucidating the molecular mechanism of intrathymic T-cell development.