HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McKarns, S. C. Articles by Schwartz, R. H. Search for Related Content PubMed PubMed Citation Articles by McKarns, S. C. Articles by Schwartz, R. H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH

Biphasic Regulation of Il2 Transcription in CD4⁺ T Cells: Roles for TNF- Receptor Signaling and Chromatin Structure¹

Susan C. McKarns^2,*, and Ronald H. Schwartz^*

^* Laboratory of Cellular and Molecular Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Center for Cellular & Molecular Immunology, Hugh E. Stephenson, Jr. Department of Surgery and Department of Molecular Microbiology and Immunology, University of Missouri School of Medicine, Columbia, MO 65212

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
We describe a novel biphasic regulation of Il2 transcription in naive CD4⁺ T cells. Few (5%) CD4⁺ T cells transcribe Il2 within 6 h of anti-TCR-β plus anti-CD28 stimulation (early phase). Most naive CD4⁺ T cells do not initiate Il2 transcription until after an additional 12 h of T cell stimulation (late phase). In comparison, essentially all previously activated (Pre-Ac) CD4⁺ T cells that transcribe Il2 do so with an early-phase response. Late-phase Il2 expression mostly requires c-Rel, CD28, and TNFR signaling. In contrast, early-phase transcription is only partly c-Rel and CD28 dependent and TNFR independent. There was also increased stable DNA accessibility at the Il2 locus and elevated c-Rel expression in resting Pre-Ac CD4⁺ cells. Upon T cell activation, a faster and greater increase in DNA accessibility as well as c-Rel nuclear expression were observed in Pre-Ac CD4⁺ cells relative to naive CD4⁺ T cells. In addition, both acetylated histone H3 and total H3 decreased at the Il2 locus upon rechallenge of Pre-Ac CD4⁺ T cells, whereas increased acetylated histone H3 with no change in total H3 was observed following activation of naive CD4⁺ T cells. We propose a model in which nucleosome disassembly facilitates rapid initiation of Il2 transcription in CD4⁺ T cells, and suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4⁺ T cells. This is provided, at least partially, by TNFR signaling during priming, but not during recall.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Interleukin-2 plays a critical role in maintaining T cell homeostasis. Signaling through the high-affinity IL-2R regulates a plethora of immunological parameters, including the proliferation, differentiation, and survival of effector T cells and the production and function of regulatory T cells (1). Dysregulation of IL-2 bioavailability has profound consequences, including lethal autoimmunity (2, 3, 4, 5, 6, 7). IL-2 is mainly produced by activated peripheral CD4⁺ T cells, but regulation of its production is complex. For example, memory CD4⁺ T cells make IL-2 faster and more abundantly than do naive CD4⁺ T cells (8); CD4⁺CD25⁺ T regulatory CD4⁺ T cells do not make IL-2 at all (9); and naive CD4⁺ T cells either made anergic through deprivation of costimulation or converted to regulatory T cells in the periphery lose their ability to make IL-2 upon restimulation (10, 11).

In quiescent naive CD4⁺ T cells, the Il2 locus is maintained in a transcriptionally silent state and is transiently activated following Ag presentation to the TCR in the context of an appropriate MHC molecule and costimulation (6, 12, 13, 14, 15). Induction of Il2 expression is controlled at the level of transcription through the assembly of transcription factor complexes, coregulators, chromatin-remodeling complexes, and complexes responsible for signal-specific histone modifications at the promoter and enhancer regions (16). A minimal essential regulatory region is located in the first 300-bp region upstream of the transcription start site of the Il2 gene. Numerous well-characterized cis-acting elements for multiple inducible and constitutive transcription factors, including members of the AP-1, NF-B, NFAT, and Oct families, are assembled at this site (17, 18), as well as multiple binding sites for the architectural protein HMGI(Y) (19). Maximal induction of Il2 gene expression requires all of these elements (20). The CD28 response element (RE)³ located between –164 and –152 bp upstream of the transcription start site is particularly critical for Il2 gene transcription (18, 21, 22, 23). A number of studies demonstrate that changes in chromatin structure accompany induction of Il2 transcription (20, 24, 25, 26, 27). Acetylation, methylation, and phosphorylation of histones as well as demethylation of individual CpGs occupying the –300 to +1 proximal promoter accompany transcriptional activation (20, 28, 29, 30, 31), and it is likely that a functional cooperativity among transcription factors and epigenetic mechanisms governs the transient nature of Il2 expression.

The objective of this study was to investigate whether rapid IL-2 production was associated with changes at the level of chromatin structure. At the cellular level, rapid IL-2 production in CD4⁺ T cells during rechallenge has been attributed to a greater frequency of Ag-specific memory cells, a lower threshold for activation, less dependence on accessory costimulation, and an increased ability to respond to a wider range of APC. However, the underlying molecular mechanisms remain elusive. To determine whether chromatin structure contributes to the kinetics of Il2 transcription, we studied micrococcal nuclease (MNase) hypersensitivity and histone modifications during Ag priming and rechallenge of a population of mono-TCR-specific CD4⁺ T cells. We identify a previously unrecognized biphasic regulation for the induction of Il2 transcription. The late phase is mostly cRel, CD28, and TNFR dependent, whereas the early phase is much less so. The Shannon laboratory has previously associated nucleosome occupancy within the regulatory region (from –60 to –210 bp) of the human Il2 gene with impaired transcription factor binding and promoter silencing (32). Our data now suggest that Il2 promoter nucleosome loss only occurs during secondary reactivation, not during primary Ag stimulation. This loss contributes to a more rapid onset of Il2 production but does not act as the ON/OFF switch for transcriptional initiation. We propose a model in which stable chromatin modifications in the proximal promoter of the Il2 gene during priming of CD4⁺ T cells allow for a T cell-intrinsic mechanism of rapid Il2 transcription in a secondary immune rechallenge.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Mice

All of the mice used in these experiments were obtained from the National Institute of Allergy and Infectious Diseases contract facility at Taconic Farms, an American Association for the Accreditation of Laboratory Animal Care-accredited specific pathogen-free barrier, and housed in sterile caging at the National Institutes of Health. B10.A (H-2^a) TCR-Cyt 5C.C7, Rag2^/ mice (33) carry the V11/Vβ3 CD4⁺ T cell transgenic receptor specific for the moth cytochrome c 88–103 and pigeon cytochrome c 81–104 peptides, and are referred to as wild type (WT). The IL-2-GFP knock-in mouse (34) was received on a C57BL/6 (N11) Rag1^/ (N2) background and crossed twice onto the B10.A 5C.C7 Rag2^/ background before selecting for homozygosity of the 5C.C7 TCR transgene, the GFP allele, and the B10.A MHC. B10.A TCR-Cyt 5C.C7 Rag2^/ IL-2-GFP heterozygous (IL-2-GFP^WT/KI) mice carrying one WT IL-2 allele and one knocked-in IL-2 GFP allele and B10.A TCR-Cyt 5C.C7 Rag2^/ IL-2-GFP homozygous (IL-2-GFP^KI/KI) mice carrying two knocked-in GFP alleles were used in these experiments. IL-2-GFP^WT/KI mice were obtained as F₁ offspring from B10.A TCR-Cyt 5C.C7 Rag2^/ IL-2-GFP^KI/KI x B10.A TCR-Cyt 5C.C7 Rag2^/ WT matings. The TNFR P55^/ TNFR P75^/ mouse generated by Peschon et al. (35) was received on a C57BL/6 (N4) background. These mice were crossed five times onto the B10.PL background and then crossed onto the B10.A TCR-Cyt 5C.C7 Rag2^/ background for four to six times before intracrossing and selecting for the 5C.C7 TCR transgene; the p55^/, p75^/, and Rag2^/ alleles; and the B10.A MHC.

Cell preparations

Naive CD4⁺ T cells were obtained from cervical, axillary, inguinal, and popliteal lymph nodes. To generate previously activated (Pre-Ac) CD4⁺ T cells, single-cell lymph node preparations were stimulated with moth cytochrome c peptide in the presence of irradiated (3000 rad) B10.A splenocytes for 72 h, then expanded 20-fold in 10 U/ml rIL-2 (Biosource International) for 14 days before isolation on a Ficoll gradient and use as Pre-Ac CD4⁺ T cells. The NIH3T3 fibroblast-transformed cell line (NIH/Swiss strain, CRL-1658; American Type Culture Collection) was used as a non-T cell control. FACS was generally used to purify populations of Vβ3 TCR⁺ CD4⁺ T cells. Briefly, cells from B10.A TCR-Cyt 5C.C7 Rag2^/ mice were labeled with Abs to B220, CD11b, CD11c, CD16/32, and NK1.1 before sorting. At times, Miltenyi Biotec magnetic beads rather than flow cytometry were used for negative selection. Purity was typically >95% Vβ3⁺CD4⁺ T cells in either case. At other times, FACS was used to positively purify either V11⁺TCR⁺CD4⁺ or Vβ3⁺CD4⁺ T cells; purity achieved was typically >99%. CD4⁺ T cells were stimulated with plate-bound anti-TCR-β (clone H57; 1–10 µg/ml). Some cultures were supplemented with ascites fluid containing the 37.51 mAb to CD28 (a gift from J. Allison, Memorial Sloan-Kettering Cancer Center, New York, NY) at a final dilution of 1/1000. Pentoxifylline (PF) and cyclosporin A (CsA) were purchased from Sigma-Aldrich; treatments (300 µg/ml and 200 ng/ml, respectively) were selected on titrations for maximal suppression of GFP expression with minimal cell death as measured by annexin V and 7-aminoactinomycin D (7-AAD) staining at 48 h (data not shown) (36, 37). PE-conjugated CD120a and CD120b, purchased from BD Pharmingen, were used for detecting surface TNFRI and TNFRII expression.

RNA purification and real-time quantitative PCR

Total RNA was extracted using the Qiagen RNeasy kit and treated with RNase-free DNase I. Taqman reverse-transcription reagents, probes, and primers for murine Il2 and c-rel were purchased from Applied Biosystems. For analyses, Il2 and c-rel samples were normalized to GAPDH; the normalized threshold cycle (Ct) values were subtracted from the target Ct values of each sample (Ct). Relative levels of target mRNA were calculated as 2^–Ct.

IL-2 secretion

The cytokine secretion assay-detection kit (Miltenyi Biotec) was used to quantify IL-2 secretion, as previously described (38).

MNase sensitivity

MNase digestion was performed essentially as described previously (39). Briefly, 5 million purified CD4⁺ T cells/digestion were lysed in a 5% sucrose homogenation buffer, nuclei were separated using sucrose gradient centrifugation, and aliquots of 1 million nuclei were digested with 25, 50, or 100 U MNase (Roche) in 1 mM CaCl₂, with the middle concentration optimized to generate predominantly tri-, di-, and mononucleosomes. Proteins were extracted via overnight proteinase K digestion. DNA was extracted using phenol:chloroform, precipitated with ethanol and 3 M sodium acetate, and then dissolved in TE buffer to 0.4 ng/ml. Genomic DNA (5 ng) was used to perform SYBR Green quantitative PCR on an ABI PRISM 7700 or 7900 sequence detector (Applied Biosystems) using SYBR Green PCR master mix (Applied Biosystems) and primers spanning the proximal promoter of the murine Il2 gene sequence (GenBank Accession M39728) and as described previously (26, 27); the primer set and the corresponding amplified region are as follows: G = –1982 to –1890; F = –652 to –460; E = –459 to –342; D = –309 to –225; C = –201 to –111; B = –110 to –16; A = +38 to +107. Data are expressed as described previously (40).

Chromatin immunoprecipitation (ChIP)

The ChIP protocol described in this study is a variation of a previously published protocol (40). Briefly, nuclei were prepared from either 5 x 10⁷ naive or Pre-Ac CD4⁺ T cells cross-linked with 1% formaldehyde, and DNA was sheared by sonication to lengths of 200-1000 bp using a Bioruptor (Diagenode). Alternatively, because the c-Rel RE overlaps a nucleosome binding site, 5 x 10⁷ naive or Pre-Ac CD4⁺ T cells were digested with 200 U MNase at room temperature for 15 min to obtain a primarily mononucleosome preparation. One-tenth of the sonicated or digested material was kept as an input sample; the remainder was divided into five aliquots and precleared with salmon sperm DNA/protein A agarose. Rabbit polyclonals to histone H3 (H3; Abcam), diacetylated (K9, K14) histone H3 (AcH3; Abcam), c-Rel (Santa Cruz Biotechnology), and control rabbit IgG (Santa Cruz Biotechnology) were added and incubated overnight. Protein-DNA cross-linking was reversed by incubation at 65°C for 4 h with 0.2 M NaCl. The protein was digested at 45°C for 1 h with proteinase K. DNA was purified by phenol-chloroform extraction, followed by ethanol precipitation. Each immunoprecipitation was done at least three times. Chromatin immune precipitates were quantified using SYBR Green quantitative PCR and primers amplifying –201 to –111 bp (region containing c-Rel B site) or –1982 to –1890 bp (non-c-Rel RE control) of the Il2 promoter. Unbound chromatin in the no-Ab-added sample was used as an input control. Data are reported as fold induction of each cross-linked sample, as determined by comparing the Ct value of the target to the Ct value of the unstimulated naive T cell immune precipitates.

Immunofluorescent detection and analysis of c-Rel nuclear translocation

Freshly isolated lymphocytes or rested Pre-Ac CD4⁺ T cells from 5C.C7 transgenic mice were stimulated with 10 µg/ml anti-TCR-β plus soluble anti-CD28 (as described above) for the times indicated. Cells were stained with anti-c-Rel (Santa Cruz Biotechnology), Alexa Fluor 555 (Invitrogen), PE Texas Red-conjugated anti-CD4 (Caltag Laboratories), and DRAQ5 (BioStatus), and then analyzed for fluorescence image-based intracellular and nuclear c-Rel translocation using the ImageStream 100 multispectral imaging flow cytometer (Amnis), as described previously (41). Briefly, a minimum of 7400 CD4⁺ T cells was collected and analyzed. Cells were excited with a 488 nm laser light and imaged on a time delay integration charge-coupled device camera. Light was then passed through a spectral decomposition element to direct different spectral bands to spatially distinct channels on the time delay integration charge-coupled device detector. The images of individual cells were then optically decomposed into a series of subimages, with each corresponding to a different color component and each having an identical spatial registry of pixels from channel to channel. c-Rel translocation was assessed by the similarity of pixel intensities between the c-Rel image in channel 4 (Alexa Fluor 555) and the nuclear image in channel 6, based on the image mask of DRAQ5 on a pixel-by-pixel basis for each cell. The similarity score for each cell was calculated using a log transformation of Pearson’s correlation coefficient. Positive translocation events were assessed by comparison with the similarity score for the negative control (correlation of darkfield scatter image with the nuclear image), followed by visual inspection of progressive bins on the similarity score histogram using the composite image of c-Rel stain and nuclear stain. Data were analyzed using the IDEAS software package (Amnis).

	Results

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Onset of transcription of the Il2 gene in the majority of naive CD4⁺ T cells is delayed until 12–18 h after TCR ligation

Vβ3⁺V11⁺CD4⁺ T cells from B10.A Rag2^/ 5C.C7 TCR mice were used to compare rates of induction and duration of steady-state Il2 mRNA expression during priming relative to rechallenge of CD4⁺ cells. Following restimulation of resting effector Pre-Ac CD4⁺ T cells with anti-TCR-β plus anti-CD28, steady-state Il2 mRNA expression was rapid and transient, with peak production by 6 h (Fig. 1A). Induction of Il2 mRNA in naive CD4⁺ T cells was minimal (although detectable) during this same period but was followed by a prolonged burst of expression beginning 10–12 h after TCR ligation, peaking at 18 h, and then persisting for up to 42 h (Fig. 1A). The small amount of Il2 mRNA produced by naive T cells during the early phase correlated with 5% of cells actively secreting IL-2 protein (Fig. 1B); peak IL-2 protein production was at 48 h. In contrast, >60% of Pre-Ac CD4⁺ T cells actively secrete IL-2 at 6 h (Fig. 1B). Surface expression of CD44 and CD62L on naive CD4⁺/Vβ3/V11 cells did not differ between early and late IL-2 producers (data not shown). In addition, there was no demonstrable difference in constitutive CD4, Vβ3 or V11 TCR, CD2, CD27, CD28, CD103, CD127, CD122, CD132, CD134, CD137, CD154, CD223, CD278, CTLA-4, or PD-1 surface expression or the induction (rate and magnitude) of CD69 or CD25 surface expression between these two populations (data not shown), thus arguing against the possibility that early IL-2 producers represent a subpopulation of cells with effector or memory status. Collectively, these results establish that the limited Il2 mRNA produced by naive CD4⁺ T cells early after T cell stimulation reflects a very low frequency of IL-2 producers at that time. Pre-Ac CD4⁺ T cells were also heterogenous for IL-2 production, but no differences in Vβ3, CD4, CD69, or CD25 expression were noted between IL-2 producers and nonproducers.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Rapid Il2 transcription in Pre-Ac CD4+ T cells. Freshly isolated naive () or resting Pre-Ac (•) WT CD4+ T cells were stimulated with anti-TCR-β plus anti-CD28 for the times indicated. A, Cells were lysed, and steady-state Il2 mRNA was quantified using quantitative PCR (qPCR); data are representative of one of two experiments; values are expressed relative to GAPDH mRNA. B, The frequency of IL-2 producers was determined by in vitro cytokine capture assay; 7-AAD– Vβ3+ cells were gated for analyses; one of four experiments is shown. C and D, Freshly isolated naive () or resting Pre-Ac (•) CD4+ T cells from IL-2-GFPKI//KI mice were stimulated with anti-TCR-β plus anti-CD28 for the times indicated. The frequency (C) and MFI (D) of 7-AAD– Vβ3+ IL-2 GFP+ CD4+ T cells were analyzed by flow cytometry.

Accessibility of the Il2 proximal promoter differs between naive and Pre-Ac CD4⁺ T cells

Given that the competence of a cell to transcribe the Il2 gene is associated with chromatin remodeling at the Il2 promoter, we investigated whether accessibility changes might contribute to the rate of onset for Il2 transcription. CD4⁺ T cells, MNase digestion, RT-PCR, and a series of seven primers spanning the Il2 promoter (–1980 to +107 bp) were used to compare chromatin accessibility, as previously described (27). This region of the Il2 locus contains cis acting elements for transcription factors, including NF-B, AP-1, NFAT, and Oct family members; a CD28RE located at –164 to –152 bp; at least one nucleosome positioned at –200 to –60 bp; and at least one and perhaps two regions that have been shown to be DNase I hypersensitive before activation, located from –313 to –361 and at the TATA cis element (Fig. 2A) (16, 25, 27, 42). In our studies, we found naive CD4⁺ T cells to be slightly but reproducibly more sensitive to MNase digestion relative to fibroblasts, a population of cells that never transcribes the Il2 gene. More importantly, they were significantly less sensitive to digestion than resting Pre-Ac T cells (Fig. 2B). We further show a different time-dependent increase in MNase sensitivity for naive vs Pre-Ac T cells (Fig. 2, C and D) that closely parallels the temporal increase in the frequency of IL-2 producers (Fig. 1C). This suggests that the progressively increasing hypersensitivity of nucleosomal DNA might be a consequence of an increased frequency of responding cells rather than a time-dependent change in cell-intrinsic chromatin modification. Maximal MNase sensitivity in naive T cells (12-fold) occurred at 40 h and was considerably smaller (50-fold) than the rapid 600-fold increase observed in Pre-Ac cells at 6 h (Fig. 2, compare C and D).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. MNase sensitivity at the –300-bp Il2 promoter correlates with the kinetics of Il2 gene transcription. A, The proximal promoter of the mouse Il2 promoter. The minimal essential regulatory region (–300 to +1) (blue), nucleosome (–200 to –60) (gray), hypersensitivity sites in resting cells (–361 to –313 and TATA box) (red), c-Rel B site (–164 to –152) (green), and the primer sets in the corresponding amplified regions (G = –1982 to –1890; F = –652 to –460; E = –459 to –342; D = –309 to –225; C = –201 to –111; B = –110 to –16; A = +38 to +107) are shown. B, Nuclei were prepared from unstimulated naive () or Pre-Ac (•) WT CD4+ T cells or NIH3T3 fibroblasts (), digested with MNase and analyzed using qPCR with seven amplicons across the Il2 gene promoter. The generated Ct values were converted to fold increase relative to undigested genomic DNA using the 2ct method. C, Nuclei were isolated from naive CD4+ T cells that were stimulated with anti-TCR-β plus anti-CD28 for 0, 6, 18, or 40 h and analyzed for MNase sensitivity. D, Nuclei were isolated from Pre-Ac CD4+ T cells that were stimulated with anti-TCR-β plus anti-CD28 for 0, 1, 3, or 6 h and analyzed for MNase sensitivity. In both C and D, the generated Ct values for stimulated cells were converted to fold increase relative to respective digested unstimulated controls using the 2Ct method. Means ± SEM are shown. E, ChIP. SYBR Green qPCR and primer set C (–201 to –111) were used to determine total and acetylated histone H3 from sonicated nuclei obtained from naive and Pre-Ac WT CD4+ T cells following anti-TCR-β plus anti-CD28 for the times indicated. Data are expressed as fold induction relative to unstimulated naive cells.

Enhanced basal expression of c-Rel in resting, Pre-Ac CD4⁺ T cells

c-Rel is essential for IL-2 production in naive CD4⁺ T cells presumably—at least in part—because of its requirement for induction of localized chromatin accessibility at the proximal Il2 promoter following T cell activation (27). A similar role for c-Rel in effector cells is unclear (43). To determine whether c-Rel expression correlates with the rate of induction of Il2 transcription and/or magnitude of MNase accessibility, we used multispectral imaging flow cytometry (41) to quantify the frequency of CD4⁺ T cells expressing c-Rel. Before rechallenge, the frequency of Pre-Ac CD4⁺ T cells producing detectable amounts of c-Rel was 26 (trial 1) to 41% (trial 2) (Fig. 3A). After activation, essentially all (98%) of the Pre-Ac cells rapidly up-regulated c-Rel with nuclear accumulation at any given time in 50–83% of these. The majority of naive T cells (up to 89%) also received signaling sufficient to produce detectable c-Rel, albeit quite slowly (Fig. 3A). This slow increase in c-Rel expression paralleled the delayed onset of Il2 transcription described in Fig. 1A. Finally, using ChIP (Fig. 3B), we found that c-rel promoter occupancy was rapidly up-regulated in Pre-Ac cells, i.e., within 6 h of T cell stimulation, whereas c-Rel binding in naive cells was delayed, i.e., 18–40 h following activation.

View larger version (20K): [in this window] [in a new window]   FIGURE 3. Enhanced basal c-Rel expression in Pre-Ac CD4+ T cells and inhibition by PF. A, Pre-Ac (solid) or naive (dashed) WT CD4+ T cells were stimulated with anti-TCR-β plus anti-CD28 for the times indicated, paraformaldehyde fixed, stained with anti-c-Rel, anti-CD4, and DRAQ5; and then analyzed for intracellular c-Rel expression and c-Rel nuclear translocation using the ImageStream 100 multispectral imaging flow cytometer. B, ChIP, SYBR Green qPCR and primer set C (–201 to –111) were used to determine c-Rel promoter occupancy in naive and Pre-Ac WT CD4+ T cells following anti-TCR-β plus anti-CD28 for the times indicated; data are expressed as fold induction relative to unstimulated naive cells. C, Pre-Ac (solid) or naive (dashed) IL-2-GFPKI/WT CD4+ T cells were stimulated with anti-TCR-β plus anti-CD28; PF or CsA was added at various times after T cell activation, as indicated. Cells were harvested at 54 h (naive) or 24 h (Pre-Ac) and gated on 7-AAD– CD4+ T cells for GFP analyses. D, PF or CsA was added to IL-2-GFPKI/WT CD4+at the time of anti-TCR-β plus anti-CD28 stimulation. Cells were harvested for GFP analyses at the times indicated.

TNF- regulates late-phase c-Rel transcription in naive CD4⁺ T cells

TNF- has been shown to regulate c-Rel activity in CD4⁺ T cells and IL-2 production in CD8⁺ T cells (44). We hypothesized that TNFR signaling might provide the costimulation necessary for the late-phase Il2 transcription in CD4⁺ T cells. To test this, CD4⁺ T cells from B10.A TCR-Cyt 5C.C7 Rag2^/ TNFR P55^/ TNFR P75^/ mice were stimulated with anti-TCR-β plus anti-CD28 in vitro. Supernatants were analyzed for IL-2 by ELISA (Fig. 4A). Relative to WT, IL-2 secretion from the double-deficient TNFR CD4⁺ T cells was greatly impaired at times after 18 h. This reduction corresponded to a decreased frequency of IL-2 producers (Fig. 4B) but not MFI (Fig. 4C). This suggests that TNFR signaling is required for late-phase IL-2 production. We then stimulated FACS-sorted, purified naive CD4⁺ T cells with anti-TCR-β plus anti-CD28 to show that this TNFR requirement was T cell autonomous (Fig. 4D). Unaltered CD69 surface expression (Fig. 4D) indicated unperturbed proximal TCR signaling in TNFR^/ CD4⁺ T cells (45). Overall, we interpret these results to mean that T cell autonomous TNFR P55 and/or TNFR P75 costimulation overcomes a threshold for activation of the Il2 promoter during late- but not early-phase IL-2 production.

View larger version (38K): [in this window] [in a new window]   FIGURE 4. Defective IL-2 production in TNFR/ CD4+ T cells. IL-2 production in naive WT (•) or TNFR/ () CD4+ T cells was measured by ELISA (A) or cytokine capture (B and C) at different times after anti-TCR-β plus anti-CD28 stimulation. Data shown are from three separate experiments in triplicate; bar indicates geometric mean. D, CD4+ FACS-sorted WT or TNFR/ T cells were stimulated with anti-TCR-β plus anti-CD28 for 0, 6, 18, or 48 h; IL-2 production was quantified using cytokine capture. Data are representative of three separate experiments. Naive (E) or Pre-Ac (F) WT or TNFR/ CD4+ T cells were FACS purified, stimulated with anti-TCR-β plus anti-CD28 for the times indicated, lysed, and analyzed for steady-state c-rel mRNA using qPCR. The data are normalized to GAPDH and expressed as fold induction (mean ± SEM) relative to unstimulated naive WT and are from three separate experiments. G, ChIP analyses to determine c-Rel association with the Il2 promoter. MNase-digested nuclei were obtained from WT or TNFR/ CD4+ T cells that had been stimulated with anti-TCR-β plus anti-CD28 for the times indicated. Data are expressed as fold induction relative to unstimulated naive cells.

/

/

/

/

CD28, but not TNFR costimulation is required for optimal induction of Il2 transcription in resting, Pre-Ac CD4⁺ T cells

We next compared the relative roles of TNFR and CD28 costimulation in IL-2 production in naive and Pre-Ac CD4⁺ T cells. CD28/B7 costimulation is critical for IL-2 production in naive CD4⁺ T cells (10); however, CD28-independent IL-2 production has been suggested for effector CD4⁺ T cells (46). Moreover, augmentation of IL-2 production by CD28 has been suggested to be entirely c-Rel dependent (43). This is consistent with CD28-mediated degradation of the inhibitory IB (43) and a c-Rel-binding B motif in the Il2 promoter (22). Using FACS-purified IL-2-GFP^KI/KI CD4⁺ T cells, we observed that CD28 costimulation is required for Il2 transcription in both naive and Pre-Ac CD4⁺ T cells, although the latter are only partially dependent (Fig. 5A). Early (24-h) as well as late (48-h) Il2 transcription in naive IL-2-GFP^KI/KI CD4⁺ T cells stimulated with anti-TCR alone was barely detectable (Fig. 5A). In contrast, by 24 h, 22% of the total Pre-Ac CD4⁺ T cells responded to anti-TCR in the absence of CD28 (Fig. 5A). Nonetheless, costimulation provided by anti-CD28 increased the IL-2-GFP⁺ frequency at 24 h to 58% and the production of GFP by individual cells (MFI) by 50-fold. This partial requirement for CD28 costimulation in Pre-Ac cells is consistent with the partial c-Rel-dependent IL-2 production in most Pre-Ac T cells (see Fig. 3C).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. CD28, but not TNFR costimulation is required for optimal induction of Il2 transcription in resting, Pre-Ac CD4+ T cells. A, IL-2-GFP expression in naive or Pre-Ac IL-2-GFPKI/WT CD4+ T cells following stimulation with anti-TCR-β alone (–-CD28) or anti-TCR-β plus anti-CD28 for the times indicated; cells are gated on 7-AAD– Vβ3+ for analyses. B, Pre-Ac WT (•) or TNFR/ () CD4+ T cells were rechallenged with anti-TCR-β or anti-TCR-β plus anti-CD28; IL-2 production is reported as the frequency of CD4+ T cells producing IL-2 at 6 h. Data from A and B are representative of two separate experiments. C, Naive or Pre-Ac WT CD4+ T cells were stimulated with anti-TCR-β plus anti-CD28 for 24 or 6 h, respectively, and gated on CD4 for analyses. Unstimulated lymph node CD4+ T cells from TNFR/ mice are also gated for negative controls. The MFI ± SEM for three separate experiments of TNFRI and TNFRII are shown. Data shown are from one of three representative experiments.

Unlike CD28 costimulation, TNFR signaling was not required for IL-2 production during rechallenge (Fig. 5B), in contrast to its major role in the late phase of IL-2 production by naive T cells (Fig. 4A). To look more closely at this pathway, we examined TNFR expression on naive and Pre-Ac CD4⁺ T cells. TNFRII expression is low, but detectable, in resting naive cells and was up-regulated 10-fold upon activation (Fig. 5C). TNFRII expression remained elevated in resting Pre-Ac cells and further increased during rechallenge. In contrast, the small amount of TNFRI in resting naive cells was down-regulated upon activation. TNFRI expression was highest in resting Pre-Ac cells, and this was also rapidly down-regulated upon activation. Thus, the lack of an apparent role for TNFR in IL-2 production in Pre-Ac cells does not stem from an absence of TNFRs. Instead, CD28 costimulation alone may be adequate for c-Rel induction in Pre-Ac cells. Alternatively, other inducible TNFR/TNF ligand superfamily members, including CD134 (OX40)/OX40L, CD137 (4-1BB)/4-1BBL, herpesvirus entry mediator (HVEM)/lymphotoxin-like inducible protein that competes with glycoprotein D for binding herpesvirus (LIGHT), CD30/CD30L, glucocorticoid-induced TNF receptor family related protein (GITR)/GITR ligand (GITRL), or CD27/CD70, may compensate for the loss of TNFR/TNF signaling. Finally, the up-regulation of TNFRII following activation of naive T cells may be the rate-limiting step required for late IL-2 production.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The kinetics of IL-2 production differs between naive and memory CD4⁺ T cells. This change in IL-2 bioavailability can significantly alter effector function and the outcome of an adaptive immune response. Rapid IL-2 production in memory CD4⁺ T cells is attributed to a host of factors, including recognition of a wider range of APCs, a lower threshold for activation, and less stringency for costimulation (8); yet the molecular mechanism(s) underlying the kinetics of Il2 transcription remains enigmatic. In this study, we contribute to the unraveling of this important question by presenting new mechanistic information regarding the regulation of Il2 promoter activity. First, we identify a novel biphasic regulation of Il2 transcription in naive CD4⁺ T cells. Second, we establish that chromatin structure and histone modifications at the proximal Il2 promoter differ markedly between naive and Pre-Ac CD4⁺ T cells. Third, we report enhanced c-Rel expression in resting Pre-Ac CD4⁺ T cells and suggest that this transcription factor may contribute to more extensive chromatin remodeling. Fourth, we suggest that the presence of a nucleosome in the proximal Il2 promoter may play a role in delaying the time of onset for Il2 transcription, but it does not serve as an all-or-nothing ON/OFF switch for Il2 transcription initiation. Lastly, we identify a role for TNFR signaling as a critical mediator of c-Rel activity and presumably chromatin remodeling and IL-2 production in naive, but not Pre-Ac CD4⁺ T cells.

Our present results expand upon earlier experiments suggesting that naive CD4⁺ T cells transcribe only a small amount of Il2 mRNA in comparison with Pre-Ac T cells. Using single-cell analysis of Il2 promoter activity, we show that most naive CD4⁺ T cells, in fact, do transcribe Il2, but with delayed kinetics (Fig. 1). The time-dependent increase in the MFI pattern of GFP expression in naive T cells (Fig. 1D) is consistent with independent transcriptional competence at each of the two Il2 alleles. These new findings further support our previously described two-step quantitative biallelic model for expression of the Il2 locus (47, 48). In contrast to naive T cells, GFP expression in Pre-Ac cells was rapid and MFI was maximal from the onset. This is consistent with rapid promoter activity at both alleles. This kinetic contrast in Il2 transcription between naive and Pre-Ac T cells led us to speculate that there was differential regulation at the level of chromatin structure. In agreement with this hypothesis, our MNase hypersensitivity assays showed a striking difference in accessibility at the regulatory region of the Il2 promoter between naive and Pre-Ac CD4⁺ T cells (Fig. 2B). DNA from resting Pre-Ac cells was more readily digested in comparison with unstimulated naive cells. This observation establishes, for the first time, a stable increase in DNA accessibility at the Il2 locus following Ag priming. We speculate that a poised chromatin structure in resting Pre-Ac cells permits rapid binding of readily available c-Rel (Fig. 3A), NFAT (49), high mobility group proteins (32), and perhaps other trans-acting transcription factors (50, 51) to their respective cognate REs within the Il2-proximal promoter to potentiate rapid chromatin remodeling and biallelic transcription (48). Following activation, both naive and Pre-Ac T cells showed an increase in MNase hypersensitivity; however, the effect in Pre-Ac cells was 50-fold greater (Fig. 2, C and D). We attribute this difference to nucleosome loss seen only in Pre-Ac T cells (Fig. 2E). The ChIP assays indicate differences at the level of histone modification between naive and Pre-Ac cells. Specifically, we observed a decrease in both Ac-H3 and total H3 following rechallenge of Pre-Ac cells; presumably, the observed H3 hypoacetylation reflects nucleosome eviction. As of yet, it is unclear whether the nucleosome is lost from the DNA in trans or rather removed by sliding along the DNA in cis (52, 53). Nonetheless, we think that this nucleosome is removed from both of the Il2 alleles upon rechallenge, and that this removal accounts for the rapid and maximal GFP expression from the onset of transcription in Pre-Ac cells. In contrast, there was no change in the total amount of H3 associated with the Il2 promoter in naive T cells. Instead, the amount of Ac-H3 increased. This suggests that the nucleosome remains at each of the two Il2 alleles during Ag priming of naive cells, although transcription is eventually initiated despite its presence. These findings are supported by previous observations from other laboratories reporting a loss of total H3 and Ac-H3 following activation of EL-4 cells, a Pre-Ac tumor population (30, 54). In contrast, Wells et al. (55) recently showed an increased Ac-H3 with no change in total H3 following activation of a population of 85% naive CD4⁺ cells. Our naive vs Pre-Ac results now provide a plausible biological explanation for the accuracy of both of these sets of experiments.

Collectively, our results show that removal of a pre-existing nucleosome in the proximal Il2 promoter is not essential for transcription. RNA polymerase II can in fact elongate through DNA containing a nucleosome, but the process is cumbersome and slow (56). We think sustained transcription through a nucleosome accounts for the late phase in our biphasic model for Il2 transcription and for the eventual ability to produce as much IL-2 as seen with Pre-Ac cells. After initial activation, however, we propose that a stable chromatin remodeling takes place that facilitates nucleosome loss during rechallenge and removal of this rate-limiting step for a rapid onset of Il2 transcription initiation during restimulation. Despite this more rapid onset, however, the final total IL-2 mRNA and protein production by Pre-Ac T cells is not much greater than that of naive T cells (Fig. 1A and our unpublished data). This is because IL-2 mRNA levels also decrease quickly in Pre-Ac T cells following peak activation (Fig. 1A). The mechanism for this might be a decreased mRNA stability in Pre-Ac T cells (57, 58) and/or a greater autocrine negative feedback loop by the burst of IL-2 protein on Il2 transcription mediated by STAT5 signaling through the IL-2R and induction of the transcriptional repressor Blimp-1 (59, 60, 61, 62).

What about the small percentage of naive T cells that respond early? These cells are from Rag2^/ TCR transgenic mice and show no surface marker or size evidence for preactivation. Nonetheless, their early IL-2 production following anti-TCR stimulation behaves in several ways, like that of Pre-Ac T cells. Their Il2 transcription (GFP⁺) is resistant to PF (Fig. 3D) and does not require TNFR signaling (Fig. 4A). It is, however, totally dependent on CD28 costimulation (Fig. 5A), and the MFI of its response (Fig. 1D) suggests that only one allele is being actively transcribed. It is tempting to speculate that this minor population, where there is evidence of rapid, monoallelic, and perhaps stochastic Il2 expression, arises from polymerase II elongation through a nucleosome-freed promoter.

The question of what determines nucleosome loss at the proximal Il2 promoter is now raised. As alluded to earlier, cell signaling, chromatin remodeling, and histone modifications have all been proposed to destabilize nucleosome interactions with the DNA. Although the underlying mechanism(s) remains unknown, c-Rel convincingly plays a role in chromatin remodeling of several proinflammatory genes, including Il2 (27, 63). Naive CD4⁺ T cells from c-Rel-deficient mice do not transcribe Il2, and chromatin at the proximal Il2 promoter does not remodel upon T cell activation (27, 43). We now show that c-Rel is at least partially required for Il2 transcription during rechallenge. In addition, c-Rel is overexpressed and more rapidly up-regulated in Pre-Ac CD4⁺ T cells (Fig. 3A). It is also more readily chromatin bound (Fig. 3B). We correlate c-Rel abundance with a poised Il2 locus in resting Pre-Ac CD4⁺ T cells and propose that overexpression of c-Rel may facilitate nucleosome displacement by rapidly recruiting other transcription factors (64, 65) and/or transcription factor IID-containing transcription complexes (66). Those cells that do not require c-Rel for Il2 transcription (Fig. 3C) may in fact already have their nucleosomes removed (Fig. 2E). These cells may represent a residual cohort of non-quiescent effector cells that harbor an open chromatin configuration reflective of S-phase DNA generated through TCR tickling or peripheral Ag cross-reactivity (67). This hypothesis is consistent with earlier studies showing unperturbed production of IL-2 in Ag-induced effector CD4⁺ T cells from c-Rel-deficient mice (43). Alternatively, the c-Rel-independent cells reflect those cells that have undergone stochastic epigenetic remodelling at the transcriptionally permissive Il2 locus (68).

Finally, TNFR signaling appears to cooperate with TCR signaling and CD28 costimulation to provide a previously undescribed regulation of late-, but not early-phase Il2 transcription during Ag priming of naive CD4⁺ T cells. Specifically, in naive T cells, c-Rel is sequestered in the cytoplasm, predominantly as a complex with the inhibitory IBβ; in contrast, p65/RelA is complexed with both IB and IBβ (37). TCR and CD28 signaling selectively degrade IB (69). As a consequence, the majority of NF-B proteins translocated into the nucleus early after TCR ligation are p65/RelA. These B proteins are insufficient in most of the T cells to induce the necessary chromatin remodeling of the Il2 locus essential for promoter activity (27). A third signal is required to activate c-Rel/IBβ. Inducible TNFR can help provide this signal (Fig. 5C). TNFR signaling not only leads to the ubiquitination of IBβ, but also induces synthesis of c-Rel and IB. This ultimately shifts c-Rel mostly into an IB complex (43), a form degradable by TCR signaling. It is likely that different concentrations of c-Rel inhibitory partners, including IB, IBβ, and IB, which control nuclear to cytoplasmic oscillation of NF-B function over time, contribute to the difference in c-Rel nuclear expression between naive and Pre-Ac cells (Fig. 3A). Alternatively, new c-Rel synthesis in Pre-Ac cells, exceeding the amount of newly synthesized IB, will result in enhanced nuclear localization. In our studies and others, abundant constitutive c-Rel and NFAT (70) in resting, Pre-Ac cells is insufficient for IL-2 production in the absence of TCR signaling (compare Figs. 3A and 1B) and reinforces the concept that cooperative interaction of multiple transcription factors is required to drive transcription at this promoter (20). Using other genetic mutants of Rag2^/ TCR transgenic mouse models, we have determined that neither IL-6 nor IL-1R deficiency impairs IL-2 production in naive CD4⁺ T cells (data not shown). This potentially makes TNFR the critical signaling component necessary to achieve the threshold level of c-Rel activity required for Il2 promoter activity. We propose that the small amount of TCR/CD28-inducible, preformed c-Rel/IB in naive and a relatively greater amount in Pre-Ac T cells accounts for the early-phase chromatin remodeling of the Il2 locus, whereas late-phase IL-2 production in the majority of the naive T cells is delayed until induction of TNFRII and sufficient concentrations of TNF-. Addition of soluble TNF- (up to 10 ng/ml) to the culture medium shortly after T cell activation did not shorten the time to onset of Il2 transcription in naive T cells (data not shown). Thus, the increase in TNFRII would appear to be the rate-limiting step. We are also exploring the possibility of a role for membrane-bound TNF- signaling (71). Overall, we propose a model in which nucleosome loss during rechallenge, but not during Ag priming, facilitates rapid Il2 induction in CD4⁺ T cells. We suggest that a threshold level of c-Rel must be reached for Il2 promoter activity in both naive and Pre-Ac CD4⁺ T cells, and that this is provided, at least partially, by TNFR signaling during priming, but not during recall.

	Acknowledgments

 
We thank Dr. Phil Morrissey for acquiring and analyzing all of the c-Rel multispectral images; Drs. Ann Dean, Gordon Hager, Christine Keifer, Sam John, and Ainhoa Perez-Diez for valuable discussions and technical assistance; Betsy Majane and Dr. Charles Mainhart for maintenance of mouse models; Dr. Kevin Holmes, Carol Henry, Calvin Eigsti, and Tom Moyer for FACS sorting; Scott Greathouse for technical assistance with the graphics; and Drs. Mike Pazin and Howard Young for valuable comments on the manuscript.

	Disclosures

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The authors have no financial conflict of interest.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.

² Address correspondence and reprint requests to Dr. Susan C. McKarns, Center for Cellular & Molecular Immunology, Departments of Surgery and Molecular Microbiology and Immunology, University of Missouri-Columbia School of Medicine, M616 Medical Sciences Building, One Hospital Drive, Columbia, MO 65212. E-mail address: mckarnss{at}health.missouri.edu

³ Abbreviations used in this paper: RE, response element; 7-AAD, 7-aminoactinomycin D; ChIP, chromatin immunoprecipitation; CsA, cyclosporin A; Ct, threshold cycle; MFI, mean fluorescence intensity; MNase, micrococcal nuclease; PF, pentoxifylline; Pre-Ac, previously activated; qPCR, quantitative PCR; WT, wild type; KI, Knock in.

Received for publication January 16, 2008. Accepted for publication May 13, 2008.

	References

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 

1. Bachmann, M. F., A. Oxenius. 2007. Interleukin 2: from immunostimulation to immunoregulation and back again. EMBO Rep. 8: 1142-1148. [Medline]
2. Sadlack, B., H. Merz, H. Schorle, A. Schimpl, A. C. Feller, I. Horak. 1993. Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75: 253-261. [Medline]
3. Schorle, H., T. Holtschke, T. Hunig, A. Schimpl, I. Horak. 1991. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature 352: 621-624. [Medline]
4. Suzuki, H., T. M. Kundig, C. Furlonger, A. Wakeham, E. Timms, T. Matsuyama, R. Schmits, J. J. Simard, P. S. Ohashi, H. Griesser, et al 1995. Deregulated T cell activation and autoimmunity in mice lacking interleukin-2 receptor β. Science 268: 1472-1476. [Abstract/Free Full Text]
5. Willerford, D. M., J. Chen, J. A. Ferry, L. Davidson, A. Ma, F. W. Alt. 1995. Interleukin-2 receptor chain regulates the size and content of the peripheral lymphoid compartment. Immunity 3: 521-530. [Medline]
6. Schimpl, A., I. Berberich, B. Kneitz, S. Kramer, B. Santner-Nanan, S. Wagner, M. Wolf, T. Hunig. 2002. IL-2 and autoimmune disease. Cytokine Growth Factor Rev. 13: 369-378. [Medline]
7. Horak, I.. 1995. Immunodeficiency in IL-2-knockout mice. Clin. Immunol. Immunopathol. 76: S172-S173. [Medline]
8. Dutton, R. W., L. M. Bradley, S. L. Swain. 1998. T cell memory. Annu. Rev. Immunol. 16: 201-223. [Medline]
9. Takahashi, T., Y. Kuniyasu, M. Toda, N. Sakaguchi, M. Itoh, M. Iwata, J. Shimizu, S. Sakaguchi. 1998. Immunologic self-tolerance maintained by CD25⁺CD4⁺ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int. Immunol. 10: 1969-1980. [Abstract/Free Full Text]
10. Schwartz, R. H.. 2003. T cell anergy. Annu. Rev. Immunol. 21: 305-334. [Medline]
11. Chen, W., W. Jin, N. Hardegen, K. J. Lei, L. Li, N. Marinos, G. McGrady, S. M. Wahl. 2003. Conversion of peripheral CD4⁺CD25^– naive T cells to CD4⁺CD25⁺ regulatory T cells by TGF-β induction of transcription factor Foxp3. J. Exp. Med. 198: 1875-1886. [Abstract/Free Full Text]
12. Fehervari, Z., T. Yamaguchi, S. Sakaguchi. 2006. The dichotomous role of IL-2: tolerance versus immunity. Trends Immunol. 27: 109-111. [Medline]
13. Malek, T. R., A. L. Bayer. 2004. Tolerance, not immunity, crucially depends on IL-2. Nat. Rev. Immunol. 4: 665-674. [Medline]
14. Nelson, B. H.. 2004. IL-2, regulatory T cells, and tolerance. J. Immunol. 172: 3983-3988. [Abstract/Free Full Text]
15. Acuto, O., S. Mise-Omata, G. Mangino, F. Michel. 2003. Molecular modifiers of T cell antigen receptor triggering threshold: the mechanism of CD28 costimulatory receptor. Immunol. Rev. 192: 21-31. [Medline]
16. Bunting, K., J. Wang, M. F. Shannon. 2006. Control of interleukin-2 gene transcription: a paradigm for inducible, tissue-specific gene expression. Vitam. Horm. 74: 105-145. [Medline]
17. Jain, J., C. Loh, A. Rao. 1995. Transcriptional regulation of the IL-2 gene. Curr. Opin. Immunol. 7: 333-342. [Medline]
18. Serfling, E., A. Avots, M. Neumann. 1995. The architecture of the interleukin-2 promoter: a reflection of T lymphocyte activation. Biochim. Biophys. Acta 1263: 181-200. [Medline]
19. Himes, S. R., R. Reeves, J. Attema, M. Nissen, Y. Li, M. F. Shannon. 2000. The role of high-mobility group I(Y) proteins in expression of IL-2 and T cell proliferation. J. Immunol. 164: 3157-3168. [Abstract/Free Full Text]
20. Rothenberg, E. V., S. B. Ward. 1996. A dynamic assembly of diverse transcription factors integrates activation and cell-type information for interleukin 2 gene regulation. Proc. Natl. Acad. Sci. USA 93: 9358-9365. [Abstract/Free Full Text]
21. Crabtree, G. R., N. A. Clipstone. 1994. Signal transmission between the plasma membrane and nucleus of T lymphocytes. Annu. Rev. Biochem. 63: 1045-1083. [Medline]
22. Shapiro, V. S., K. E. Truitt, J. B. Imboden, A. Weiss. 1997. CD28 mediates transcriptional up-regulation of the interleukin-2 (IL-2) promoter through a composite element containing the CD28RE and NF-IL-2B AP-1 sites. Mol. Cell. Biol. 17: 4051-4058. [Abstract]
23. Verweij, C. L., M. Geerts, L. A. Aarden. 1991. Activation of interleukin-2 gene transcription via the T-cell surface molecule CD28 is mediated through an NF-B-like response element. J. Biol. Chem. 266: 14179-14182. [Abstract/Free Full Text]
24. Siebenlist, U., D. B. Durand, P. Bressler, N. J. Holbrook, C. A. Norris, M. Kamoun, J. A. Kant, G. R. Crabtree. 1986. Promoter region of interleukin-2 gene undergoes chromatin structure changes and confers inducibility on chloramphenicol acetyltransferase gene during activation of T cells. Mol. Cell. Biol. 6: 3042-3049. [Abstract/Free Full Text]
25. Ward, S. B., G. Hernandez-Hoyos, F. Chen, M. Waterman, R. Reeves, E. V. Rothenberg. 1998. Chromatin remodeling of the interleukin-2 gene: distinct alterations in the proximal versus distal enhancer regions. Nucleic Acids Res. 26: 2923-2934. [Abstract/Free Full Text]
26. Rao, S., E. Procko, M. F. Shannon. 2001. Chromatin remodeling, measured by a novel real-time polymerase chain reaction assay, across the proximal promoter region of the IL-2 gene. J. Immunol. 167: 4494-4503. [Abstract/Free Full Text]
27. Rao, S., S. Gerondakis, D. Woltring, M. F. Shannon. 2003. c-Rel is required for chromatin remodeling across the IL-2 gene promoter. J. Immunol. 170: 3724-3731. [Abstract/Free Full Text]
28. Murayama, A., K. Sakura, M. Nakama, K. Yasuzawa-Tanaka, E. Fujita, Y. Tateishi, Y. Wang, T. Ushijima, T. Baba, K. Shibuya, et al 2006. A specific CpG site demethylation in the human interleukin 2 gene promoter is an epigenetic memory. EMBO J. 25: 1081-1092. [Medline]
29. Thomas, R. M., L. Gao, A. D. Wells. 2005. Signals from CD28 induce stable epigenetic modification of the IL-2 promoter. J. Immunol. 174: 4639-4646. [Abstract/Free Full Text]
30. Chen, X., J. Wang, D. Woltring, S. Gerondakis, M. F. Shannon. 2005. Histone dynamics on the interleukin-2 gene in response to T-cell activation. Mol. Cell. Biol. 25: 3209-3219. [Abstract/Free Full Text]
31. Bruniquel, D., R. H. Schwartz. 2003. Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat. Immunol. 4: 235-240. [Medline]
32. Attema, J. L., R. Reeves, V. Murray, I. Levichkin, M. D. Temple, D. J. Tremethick, M. F. Shannon. 2002. The human IL-2 gene promoter can assemble a positioned nucleosome that becomes remodeled upon T cell activation. J. Immunol. 169: 2466-2476. [Abstract/Free Full Text]
33. Tanchot, C., D. L. Barber, L. Chiodetti, R. H. Schwartz. 2001. Adaptive tolerance of CD4⁺ T cells in vivo: multiple thresholds in response to a constant level of antigen presentation. J. Immunol. 167: 2030-2039. [Abstract/Free Full Text]
34. Naramura, M., R. J. Hu, H. Gu. 1998. Mice with a fluorescent marker for interleukin 2 gene activation. Immunity 9: 209-216. [Medline]
35. Peschon, J. J., D. S. Torrance, K. L. Stocking, M. B. Glaccum, C. Otten, C. R. Willis, K. Charrier, P. J. Morrissey, C. B. Ware, K. M. Mohler. 1998. TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation. J. Immunol. 160: 943-952. [Abstract/Free Full Text]
36. Wang, W., W. F. Tam, C. C. Hughes, S. Rath, R. Sen. 1997. c-Rel is a target of pentoxifylline-mediated inhibition of T lymphocyte activation. Immunity 6: 165-174. [Medline]
37. Randak, C., T. Brabletz, M. Hergenrother, I. Sobotta, E. Serfling. 1990. Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. EMBO J. 9: 2529-2536. [Medline]
38. McKarns, S. C., R. H. Schwartz. 2005. Distinct effects of TGF-β1 on CD4⁺ and CD8⁺ T cell survival, division, and IL-2 production: a role for T cell intrinsic Smad3. J. Immunol. 174: 2071-2083. [Abstract/Free Full Text]
39. Gong, Q. H., J. C. McDowell, A. Dean. 1996. Essential role of NF-E2 in remodeling of chromatin structure and transcriptional activation of the -globin gene in vivo by 5' hypersensitive site 2 of the β-globin locus control region. Mol. Cell. Biol. 16: 6055-6064. [Abstract]
40. Litt, M. D., M. Simpson, F. Recillas-Targa, M. N. Prioleau, G. Felsenfeld. 2001. Transitions in histone acetylation reveal boundaries of three separately regulated neighboring loci. EMBO J. 20: 2224-2235. [Medline]
41. George, T. C., S. L. Fanning, P. Fitzgeral-Bocarsly, R. B. Medeiros, S. Highfill, Y. Shimizu, B. E. Hall, K. Frost, D. Basiji, W. E. Ortyn, et al 2006. Quantitative measurement of nuclear translocation events using similarity analysis of multispectral cellular images obtained in flow. J. Immunol. Methods 311: 117-129. [Medline]
42. Brunvand, M. W., A. Krumm, M. Groudine. 1993. In vivo footprinting of the human IL-2 gene reveals a nuclear factor bound to the transcription start site in T cells. Nucleic Acids Res. 21: 4824-4829. [Abstract/Free Full Text]
43. Banerjee, D., H. C. Liou, R. Sen. 2005. c-Rel-dependent priming of naive T cells by inflammatory cytokines. Immunity 23: 445-458. [Medline]
44. Pimentel-Muinos, F. X., J. Mazana, M. Fresno. 1995. Biphasic control of nuclear factor-B activation by the T cell receptor complex: role of tumor necrosis factor . Eur. J. Immunol. 25: 179-186. [Medline]
45. Kim, E. Y., H. S. Teh. 2004. Critical role of TNF receptor type-2 (p75) as a costimulator for IL-2 induction and T cell survival: a functional link to CD28. J. Immunol. 173: 4500-4509. [Abstract/Free Full Text]
46. Dubey, C., M. Croft. 1996. Accessory molecule regulation of naive CD4 T cell activation. Immunol. Res. 15: 114-125. [Medline]
47. Umlauf, S. W., B. Beverly, S. M. Kang, K. Brorson, A. C. Tran, R. H. Schwartz. 1993. Molecular regulation of the IL-2 gene: rheostatic control of the immune system. Immunol. Rev. 133: 177-197. [Medline]
48. Chiodetti, L., D. L. Barber, R. H. Schwartz. 2000. Biallelic expression of the IL-2 locus under optimal stimulation conditions. Eur. J. Immunol. 30: 2157-2163. [Medline]
49. Dienz, O., S. M. Eaton, T. J. Krahl, S. Diehl, C. Charland, J. Dodge, S. L. Swain, R. C. Budd, L. Haynes, M. Rincon. 2007. Accumulation of NFAT mediates IL-2 expression in memory, but not naive, CD4⁺ T cells. Proc. Natl. Acad. Sci. USA 104: 7175-7180. [Abstract/Free Full Text]
50. Shi, L., W. R. Godfrey, J. Lin, G. Zhao, P. N. Kao. 2007. NF90 regulates inducible IL-2 gene expression in T cells. J. Exp. Med. 204: 971-977. [Abstract/Free Full Text]
51. Shi, L., D. Qiu, G. Zhao, B. Corthesy, S. Lees-Miller, W. H. Reeves, P. N. Kao. 2007. Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in vivo in activated T-cells. Nucleic Acids Res. 35: 2302-2310. [Abstract/Free Full Text]
52. Boeger, H., J. Griesenbeck, J. S. Strattan, R. D. Kornberg. 2004. Removal of promoter nucleosomes by disassembly rather than sliding in vivo. Mol. Cell 14: 667-673. [Medline]
53. Korber, P., T. Luckenbach, D. Blaschke, W. Horz. 2004. Evidence for histone eviction in trans upon induction of the yeast PHO5 promoter. Mol. Cell. Biol. 24: 10965-10974. [Abstract/Free Full Text]
54. Adachi, S., E. V. Rothenberg. 2005. Cell-type-specific epigenetic marking of the IL2 gene at a distal cis-regulatory region in competent, nontranscribing T-cells. Nucleic Acids Res. 33: 3200-3210. [Abstract/Free Full Text]
55. Thomas, R. M., N. Chunder, C. Chen, S. E. Umetsu, S. Winandy, A. D. Wells. 2007. Ikaros enforces the costimulatory requirement for IL2 gene expression and is required for anergy induction in CD4⁺ T lymphocytes. J. Immunol. 179: 7305-7315. [Abstract/Free Full Text]
56. Ercan, S., M. J. Carrozza, J. L. Workman. 2004. Global nucleosome distribution and the regulation of transcription in yeast. Genome Biol. 5: 243[Medline]
57. Lindstein, T., C. H. June, J. A. Ledbetter, G. Stella, C. B. Thompson. 1989. Regulation of lymphokine messenger RNA stability by a surface-mediated T cell activation pathway. Science 244: 339-343. [Abstract/Free Full Text]
58. Umlauf, S. W., B. Beverly, O. Lantz, R. H. Schwartz. 1995. Regulation of interleukin 2 gene expression by CD28 costimulation in mouse T-cell clones: both nuclear and cytoplasmic RNAs are regulated with complex kinetics. Mol. Cell. Biol. 15: 3197-3205. [Abstract]
59. Gong, D., T. R. Malek. 2007. Cytokine-dependent Blimp-1 expression in activated T cells inhibits IL-2 production. J. Immunol. 178: 242-252. [Abstract/Free Full Text]
60. Martins, G. A., L. Cimmino, M. Shapiro-Shelef, M. Szabolcs, A. Herron, E. Magnusdottir, K. Calame. 2006. Transcriptional repressor Blimp-1 regulates T cell homeostasis and function. Nat. Immunol. 7: 457-465. [Medline]
61. Villarino, A. V., C. M. Tato, J. S. Stumhofer, Z. Yao, Y. K. Cui, L. Hennighausen, J. J. O'Shea, C. A. Hunter. 2007. Helper T cell IL-2 production is limited by negative feedback and STAT-dependent cytokine signals. J. Exp. Med. 204: 65-71. [Abstract/Free Full Text]
62. Jameson, S. C.. 2006. T cells climb on board Blimp-1. Trends Immunol. 27: 349-351. [Medline]
63. Brettingham-Moore, K. H., S. Rao, T. Juelich, M. F. Shannon, A. F. Holloway. 2005. GM-CSF promoter chromatin remodelling and gene transcription display distinct signal and transcription factor requirements. Nucleic Acids Res. 33: 225-234. [Abstract/Free Full Text]
64. Xu, X., C. Prorock, H. Ishikawa, E. Maldonado, Y. Ito, C. Gelinas. 1993. Functional interaction of the v-Rel and c-Rel oncoproteins with the TATA-binding protein and association with transcription factor IIB. Mol. Cell. Biol. 13: 6733-6741. [Abstract/Free Full Text]
65. Cha-Molstad, H., D. P. Young, I. Kushner, D. Samols. 2007. The interaction of C-Rel with C/EBPβ enhances C/EBPβ binding to the C-reactive protein gene promoter. Mol. Immunol. 44: 2933-2942. [Medline]
66. Kerr, L. D., L. J. Ransone, P. Wamsley, M. J. Schmitt, T. G. Boyer, Q. Zhou, A. J. Berk, I. M. Verma. 1993. Association between proto-oncoprotein Rel and TATA-binding protein mediates transcriptional activation by NF-B. Nature 365: 412-419. [Medline]
67. Geginat, J., S. Campagnaro, F. Sallusto, A. Lanzavecchia. 2002. TCR-independent proliferation and differentiation of human CD4⁺ T cell subsets induced by cytokines. Adv. Exp. Med. Biol. 512: 107-112. [Medline]
68. Dodd, I. B., M. A. Micheelsen, K. Sneppen, G. Thon. 2007. Theoretical analysis of epigenetic cell memory by nucleosome modification. Cell 129: 813-822. [Medline]
69. Zhou, X. Y., Y. Yashiro-Ohtani, M. Nakahira, W. R. Park, R. Abe, T. Hamaoka, M. Naramura, H. Gu, H. Fujiwara. 2002. Molecular mechanisms underlying differential contribution of CD28 versus non-CD28 costimulatory molecules to IL-2 promoter activation. J. Immunol. 168: 3847-3854. [Abstract/Free Full Text]
70. Serfling, E., F. Berberich-Siebelt, A. Avots. 2007. NFAT in lymphocytes: a factor for all events?. Sci STKE 2007: pe42[Abstract/Free Full Text]
71. Gerspach, J., A. Gotz, G. Zimmermann, C. Kolle, H. Bottinger, M. Grell. 2000. Detection of membrane-bound tumor necrosis factor (TNF): an analysis of TNF-specific reagents. Microsc. Res. Tech. 50: 243-250. [Medline]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via Google Scholar Google Scholar Articles by McKarns, S. C. Articles by Schwartz, R. H. Search for Related Content PubMed PubMed Citation Articles by McKarns, S. C. Articles by Schwartz, R. H. Pubmed/NCBI databases Gene GEO Profiles HomoloGene UniGene Compound via MeSH Substance via MeSH