Shoichiro Miyatake

Cutting Edge: Chromatin Remodeling at the IL-4/IL-13 Intergenic Regulatory Region for Th2-Specific Cytokine Gene Cluster

During the differentiation of naive Th cells into Th2 effector cells, the entire IL-4/IL-13 locus is remodeled into an accessible chromatin conformation. Here we show that ectopic expression and activation of Stat6 or GATA-3 in Th cells developing under Th1-polarizing conditions lead to the induction of chromatin remodeling not only at the flanking regions of the IL-4 and IL-13 genes but also at the IL-4/IL-13 intergenic regulatory region for the IL-4/IL-13/IL-5 gene cluster. Furthermore, we demonstrate that GATA-3 and another Th2-specific, inducible protein complex interact with the IL-4/IL-13 intergenic DNase I hypersensitive region specifically in Th2 cells.

Role of NFATx (NFAT4/NFATc3) in Expression of Immunoregulatory Genes in Murine Peripheral CD4+ T Cells

Ca2+-regulated NFAT family members are transcription factors crucial for the expression of various cytokine genes and other immunoregulatory genes. Analyses of mice defective in one or two NFAT family members have revealed functions specific to each NFAT gene. However, the redundant functions of several family members limit the usefulness of gene disruption analysis. For example, CD4+ T cells isolated from NFATx-disrupted mice do not show any modulation in cytokine gene expression, perhaps because other family members compensate for its absence. To analyze the role of NFATx in the regulation of immunoregulatory genes in T cells, we made a gain-of-function mutant by creating transgenic mice expressing a constitutively nuclear form of NFATx in T cell lineages. In naive CD4+ T cells, NFATx up-regulated the expression of several cytokine genes and activation markers and suppressed the expression of CD154. In Th1 cells, NFATx enhanced the expression of the Th1 cytokine genes, IFN-γ and TNF-α. In contrast, NFATx suppressed Th2 cytokine genes such as IL-4 and IL-5 in Th2 cells. It has been reported that both NFAT1 and NFATx are required to maintain the homeostasis of the immune system. Our results suggest that NFATx exerts this function by inhibiting the expression of some critical immunoregulatory genes.

Interleukin-7 inhibits pre-T-cell differentiation induced by the pre-T-cell receptor signal and the effect is mimicked by hGM-CSF in hGM-CSF receptor transgenic mice.

We have previously reported that human granulocyte–macrophage colony‐stimulating factor (hGM‐CSF) causes a stage‐specific inhibition of T‐cell receptor (TCR) αβ cell development in the thymus of transgenic mice constitutively expressing the hGM‐CSF receptor. Since it has been reported that the addition of interleukin‐7 (IL‐7) to fetal thymic organ culture (FTOC) has similar effects, we compared the effects of IL‐7 and hGM‐CSF on TCRαβ cell development in hGM‐CSF receptor transgenic mice. We reconstituted fetal lobes with sorted pre‐T, or post pre‐T CD4−CD8− precursor cells. The addition of either IL‐7 or hGM‐CSF to these cultures suppressed further differentiation of pre‐T cells but not post pre‐T cells. At the same time, the cell number was increased, suggesting that pre‐T‐cell proliferation is stimulated by these cytokines. Furthermore, the differentiation of recombination‐activating gene‐1 (RAG‐1)‐deficient pre‐T cells in response to anti‐CD3 antibody stimulation was suppressed by either IL‐7 or hGM‐CSF, suggesting that these cytokines inhibit the pre‐T‐cell receptor (pre‐TCR) signal. This inhibition is unexpected because the pre‐TCR signal and the IL‐7 signal have previously been considered to be co‐operative. Recent analysis of the downstream events of IL‐7 receptor and GM‐CSF receptor revealed that they share common signal transduction molecules. Our results show that IL‐7 is able to promote pre‐T cell proliferation and to suppress differentiation induced by the pre‐TCR signal. GM‐CSF can mimic these biological activities of IL‐7 when the pre‐T cells express GM‐CSF receptors. Our data suggest that both timing and level of activation of the IL‐7 signalling pathway must be precisely regulated to facilitate the differentiation of thymocytes.

Chromatin remodeling and T helper subset differentiation.

The T helper subsets Th1 and Th2 regulate specific types of immune responses by producing distinct sets of cytokines. Differentiation of the T helper subsets from their common precursors, naive CD4+ T cells, is induced by antigen stimulation and controlled by various other conditions. Of these conditions, the contributions of the cytokine environment have been the best characterized. The presence of interleukin‐4 (IL‐4) directs the differentiation towards Th2 cells, whereas IL‐12 induces Th1 differentiation. The Th2 signature cytokine genes encoding IL‐4, IL‐13, and IL‐5 are clustered, and noncoding regions such as the intergenic region of the IL‐4 and IL‐13 genes are highly conserved from mice to humans. Alteration of the chromatin structure of this Th2 cytokine cluster region is detected as nuclease‐accessible regions specific to Th2 cells. Activation of STAT6 promotes Th2 differentiation by inducing Th2‐specific transcription factors, including GATA3. Expression of GATA3 induces Th2 differentiation and enhances the Th2 cell?specific chromatin accessibility, indicating that GATA3 is a key protein that regulates differentiation through chromatin remodeling. T helper subset differentiation provides a good system to study gene expression regulation at the chromatin level.

Cytokines, Extracellular Transport and Processing

The expression of various cytokines is regulated either transcriptionally or posttranscriptionally. In addition, for some cytokines, processing of cytokine proteins and releasing from cells are important steps for the regulation of their biological activities.

T Cell Activation Signals and Regulation of Lymphokine Gene by Viral and Cellular Transactivators

T cells activated by antigen stimulation produce a set of lymphokines. By employing protein kinase C (PKC) which is active without stimulation or viral transactivator HTLV-I p40tax or BPV E2 protein, we characterized the T cell antigen receptor signal transduction pathway downstream of PKC. Consistent with the earlier observations that activation of PKC and Ca2+ influx are necessary for T cell activation, the IL-2 promoter is activated by actions of constitutively active PKC and Ca2+ ionophore in the human T cell leukemia line Jurkat. We found that p40tax or E2 protein activate transfected GM-CSF gene as well as SV40 and HIV promoters without external stimuli. The sequence of GM-CSF promoter required for stimulation by PMA/A23187 is localized between positions -95 and -73 (CLE2). The same region responds to p40tax or E2 protein. Another sequence, located between -113 and -96 (CLE1), mediates inducible response to p40tax but not to E2 protein or PMA/A23187 stimulation. Activation of the SV40 promoter by p40tax or E2 protein is dependent on SV40 enhancer sequences. Only one copy of the segment carrying the NF-κ B binding site is sufficient to mediate the induction by E2 protein, p40tax or PMA/A23187 stimulation. HIV LTR promoter also responds to E2 protein or p40tax through the same DNA element. These results indicate that p40tax or E2 protein activate GM-CSF and viral promoters by interacting with cellular component(s) in the T cell activation signal transduction pathway.