DNA array analysis of interleukin-2-regulated immediate/early genes

Abstract

BACKGROUND: Lymphocyte activation culminates in blastogenesis, cell cycle progression, DNA replication and mitosis. These complex cellular changes are programmed almost simultaneously by multiple ligands and receptors that trigger specific signal transduction pathways and transcription factors. Until now, the discovery of the genes regulated by each ligand/receptor pair has been hampered by the technologies available. RESULTS: To identify interleukin-2 (IL-2)-responsive genes, human peripheral blood mononuclear cells (PBMC) were pre-activated with anti-CD3, rested, and restimulated with IL-2 for 4 hr. Gene expression was analyzed using Affymetrix U95Av2 oligonucleotide arrays. To determine the most stringent parameters to score a gene as a bona fide IL-2 target, the expression of 19 known IL-2-regulated genes was examined first. All were induced at least 2-fold, with a difference in fluorescent intensity of >/= 100 at p < 0.05. An additional 53 unique genes met these criteria. To determine which of these were immediate/early IL-2 targets in T cells, purified T cells were stimulated with IL-2 for 4 hr in the presence of cycloheximide to prevent secondary gene expression. Of the 72 genes identified in PBMCs, 20 were detected as immediate/early IL-2-regulated genes in purified T cells. In addition, 27 unique genes were IL-2-regulated in T cells but not in PBMCs. CONCLUSIONS: For a successful reductionist approach to the analysis of gene expression in lymphocyte activation, it is necessary to examine purified cell populations and immediate/early gene expression regulated by each ligand/receptor pair involved. This approach should allow the discovery of genes regulated by all of the ligand/receptor pairs involved in lymphocyte activation.

Figure 1

IL-2-regulated genes in PBMCs. Pre-activated, rested human PBMCs were left untreated or restimulated for 4 hr with IL-2, and RNA probes were prepared for hybridization with Affymetrix U95Av2 arrays. A total of 84 probe sets were identified that showed fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds, right axis), and a significance value of the difference between the means of p < 0.05. The probe sets represent 72 unique genes, and can be segregated into 8 functional groups.

Figure 2

IL-2-responsive probe sets in PBMCs and purified T cells. (A). Using the criterion of p < 0.05 for the difference between the means of IL-2-treated and untreated groups, a total of 316 IL-2-sensitive probe sets were identified in PBMCs (left circle), and 439 in purified T cells (right circle). Of these, 110 were identified in both cell populations (overlap between circles). (B). When a second standard of either fold change ≥ 2 or subtracted difference ≥ 100 was included in the analysis, 251 probe sets were identified in PBMCs, and 161 in pure T cells, with 71 in common. (C). When all three criteria were used, 84 probe sets were identified in PBMCs, 52 in T cells, and 23 of these were common to both populations. Within each population, the percentages of IL-2- suppressed and induced probe sets are indicated.

Figure 3

IL-2-regulated genes in PBMCs and purified T cells. A total of 23 probe sets met all three expression criteria of fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds and circles, right axis), and p < 0.05 in both PBMCs and purified T cells. These probe sets represent 20 unique genes.

Figure 4

IL-2-responsive genes in purified T cells. A total of 29 additional probe sets met all three expression criteria of fold change ≥ 2 (columns, left axis), subtracted difference ≥ 100 (diamonds and circles, right axis), and p < 0.05 in purified T cells, but did not meet all three criteria in PBMCs. These probe sets represent 27 unique genes.