Ikaros Silences T-bet Expression and Interferon-γ Production during T Helper 2 Differentiation

Rajan M Thomas,Chunxia Chen,Neelanjana Chunder,Lingzhi Ma,Justin Taylor,Edward J Pearce,Andrew D Wells

doi:10.1074/jbc.m109.038794

Abstract

CD4+ T cells can be instructed by nonantigen-specific signals to differentiate into functionally distinct lineages with mutually exclusive patterns of cytokine production. The molecular events that drive interferon-gamma (IFN gamma) production during Th1 development are well understood, but mechanisms that silence this cytokine during Th2 polarization are not clear. In this study, we find that the tbx21 gene encoding the Th1 master regulator T-bet is a direct target of the transcriptional repressor Ikaros. In Th2 cells, which do not express T-bet, strong Ikaros binding could be detected at the endogenous tbx21 promoter, whereas this gene was not occupied by Ikaros in T-bet-expressing Th1 cells. Inhibition of Ikaros DNA binding activity during Th2 polarization resulted in loss of Ikaros promoter occupancy, increased T-bet expression, and inappropriate T-bet-dependent production of IFN gamma. Ikaros was also required for epigenetic imprinting of the ifn gamma locus during Th2 polarization, and loss of Ikaros function in vivo led to an inappropriate Th1 response to the parasite Shistosoma mansoni. These studies demonstrate that Ikaros, a factor with an established role in lymphocyte development, also regulates the development of peripheral T helper responses.

Highlights

Using short hairpin RNA-mediated knockdown of Ikaros or a dominant-negative allele of Ikaros that allows lymphoid development but results in peripheral T cells with 90% reduced Ikaros DNA binding activity [7, 9], we show that Ikaros activity is not necessary for induction of IL-4 during T helper 2 (Th2) differentiation, but that it is required to silence ifn␥ gene expression in CD4ϩ T cells responding to Th2-promoting signals in vitro and in vivo
We have found that Ikaros actively represses the production of the T helper 1 (Th1) cytokine IFN␥ in anergic T cells,3 leading us to ask whether Ikaros plays a broader role in controlling “inappropriate” cytokine gene expression under other circumstances, such as during polarized T helper responses
We transduced wild-type CD4ϩ T cells with retroviral vector encoding a short hairpin RNA targeting the Ik1 transcript or a scrambled control shRNA and cultured these cells under Th1or Th2-polarizing conditions. This approach resulted in a Ͼ80% decrease in Ikaros protein expression by both Th1 and Th2 cells compared with mock-transduced or control shRNA-transduced cells and likewise led to a marked increase in IL-2 production by transduced Th2 cells (Fig. 1B, top panels)

Summary

Introduction

Ikaros DNA Binding Activity Is Required for Polarized Patterns of Cytokine Production by Helper T cells in Vitro—Recent studies have shown that Ikaros fulfills a previously unappreciated role in mature CD4ϩ T cells as a repressor of il2 gene expression during the induction of anergy [7, 8]. Inhibition of Ikaros DNA binding activity dur- GFPϩ IkDN/ϩ cells were strongly affected by dnT-bet ing Th2 differentiation resulted in the failure to down-regulate expression, exhibiting a 5-fold reduction in the frequency

Results

Conclusion