Transcriptional Regulation of the Interleukin-2 Gene in Normal Human Peripheral Blood T Cells: CONVERGENCE OF COSTIMULATORY SIGNALS AND DIFFERENCES FROM TRANSFORMED T CELLS

Christopher C.W Hughes,Jordan S Pober

doi:10.1074/jbc.271.10.5369

Abstract

To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection. Interleukin-2 (IL-2) promoter-reporter gene behavior closely parallels the endogenous gene in response to T cell receptor and costimulatory signals. As assessed with mutagenized promoters, the most important IL-2 cis-regulatory elements in normal T cells are the proximal AP-1 site and the NF- kappaB site. Both primary activation, with phytohemagglutinin or antibodies to CD3, and costimulation, provided by pairs of CD2 antibodies or B7-positive (B cells) or B7-negative (endothelial) accessory cells, are mediated through the same cis-elements. Interestingly, the nuclear factor of activated T cell sites are much less important in normal T cells than in Jurkat T cells. We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter.

Highlights

To study transcriptional regulation in normal human T cells, we have optimized conditions for transient transfection
We conclude that IL-2 transcriptional regulation differs in tumor cell lines compared with normal T cells and that different costimulatory signals converge on the same cis-elements in the IL-2 promoter
Transfection of Normal Peripheral Blood Lymphocytes—To investigate the regulation of IL-2 transcription in normal human T cells, we have developed a reproducible assay based on transient transfection by electroporation of IL-2 promoter-luciferase reporter gene constructs

Summary

CONVERGENCE OF COSTIMULATORY SIGNALS AND DIFFERENCES FROM TRANSFORMED T CELLS*

(Received for publication, September 27, 1995, and in revised form, December 20, 1995). 300 base pairs of the IL-2 promoter are sufficient to confer cellspecific, inducible expression to reporter gene constructs [21], other regulatory sequences may lie outside of this region [22] Within these 300 base pairs, several transcription factor-binding sites have been identified as positive regulatory elements in tumor T cells (see Table II), including proximal and distal specific sequences for the nuclear factor of activated T cells (NFAT) [21, 23, 24] and proximal and distal sequences for AP-1 [25, 26], for NF-␬B [27, 28], for NIL-2A [29], for CD28activated factors [30], and for octamer factors [31]. In Jurkat cells, CD28 has been found to induce a transcription factor composed of NFKB1 (p50), RelA (p65), and c-Rel [37, 38] that binds to a variant NF-␬B site called the CD28 response element (CD28RE) [39] This site has been found to be targeted by signals other than CD28 [40]. We have characterized the sites required for responsiveness to various costimulatory signals and accessory cell types

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION