Methodology article

High-throughput gene expression profiling of memory differentiation in primary human T cells

W Nicholas Haining^1,3 , Jill Angelosanto^2,3 , Kathleen Brosnahan¹ , Kenneth Ross⁴ , Cynthia Hahn¹ , Kate Russell² , Linda Drury² , Stephanie Norton⁴ , Lee Nadler² and Kimberly Stegmaier^1,3,4

¹  Department of Pediatric Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA

²  Department of Medical Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA

³  Division of Hematology/Oncology, Children's Hospital Boston, USA

⁴  Broad Institute of Harvard and MIT, USA

author email corresponding author email

BMC Immunology 2008, 9:44doi:10.1186/1471-2172-9-44

Published:	1 August 2008

Abstract

Background

The differentiation of naive T and B cells into memory lymphocytes is essential for immunity to pathogens. Therapeutic manipulation of this cellular differentiation program could improve vaccine efficacy and the in vitro expansion of memory cells. However, chemical screens to identify compounds that induce memory differentiation have been limited by 1) the lack of reporter-gene or functional assays that can distinguish naive and memory-phenotype T cells at high throughput and 2) a suitable cell-line representative of naive T cells.

Results

Here, we describe a method for gene-expression based screening that allows primary naive and memory-phenotype lymphocytes to be discriminated based on complex genes signatures corresponding to these differentiation states. We used ligation-mediated amplification and a fluorescent, bead-based detection system to quantify simultaneously 55 transcripts representing naive and memory-phenotype signatures in purified populations of human T cells. The use of a multi-gene panel allowed better resolution than any constituent single gene. The method was precise, correlated well with Affymetrix microarray data, and could be easily scaled up for high-throughput.

Conclusion

This method provides a generic solution for high-throughput differentiation screens in primary human T cells where no single-gene or functional assay is available. This screening platform will allow the identification of small molecules, genes or soluble factors that direct memory differentiation in naive human lymphocytes.