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


CONCLUSIONS
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.
Haining
W Nicholas
WN
Department of Pediatric Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA. nicholas_haining@dfci.harvard.edu
Angelosanto
Jill
J
Brosnahan
Kathleen
K
Ross
Kenneth
K
Hahn
Cynthia
C
Russell
Kate
K
Drury
Linda
L
Norton
Stephanie
S
Nadler
Lee
L
Stegmaier
Kimberly
K
D015496:CD4-Positive T-Lymphocytes / Q000276:immunology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000276:immunology; D037502:Galectin 3 / Q000235:genetics / Q000276:immunology*; D020869:Gene Expression Profiling; D006801:Humans; D007156:Immunologic Memory / Q000235:genetics*; D018189:Immunomagnetic Separation; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics / Q000276:immunology; D054400:Receptors, CCR7 / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D019014:fas Receptor / Q000235:genetics / Q000276:immunology
C515370:CCR7 protein, human; C491901:FAS protein, human; D037502:Galectin 3; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D054400:Receptors, CCR7; D019014:fas Receptor