Abstract

T helper type 2 (T_H2) bias, which is the propensity of naive CD4⁺ T cells to differentiate into interleukin 4 (IL-4)-secreting T_H2 cells, is a genetic trait that affects susceptibility to infectious, autoimmune and allergic diseases. T_H2 bias correlates with the amount of IL-4 initially secreted by newly activated helper T cells that feeds back positively through the pathway of the IL-4 receptor and the transcription factors STAT6 and GATA-3 to drive T_H2 development. Here we identify Mina, a member of the jumonji C (JmjC) protein family, as a genetic determinant of T_H2 bias. Mina specifically bound to and repressed the Il4 promoter. Mina overexpression in transgenic mice impaired Il4 expression, whereas its knockdown in primary CD4⁺ T cells led to Il4 derepression. Our findings collectively provide mechanistic insight into an Il4-regulatory pathway that controls helper T cell differentiation and genetic variation in T_H2 bias.

Introduction

Naive CD4⁺ T cells are multipotent sentinels of the immune system, poised to respond to instructive signals from antigen-presenting cells by differentiating into distinct effector cell lineages. These include T helper type 1 (T_H1) and T_H2 cells, which are adapted differently for the control of intracellular and extracellular pathogens, respectively, in part through developmentally acquired potential for high expression of distinct cytokine genes¹. Dysregulated CD4⁺ T cell development can promote susceptibility to infectious, autoimmune and allergic diseases^{2, 3, 4, 5, 6, 7, 8, 9}.

Interleukin 4 (IL-4; A001262), the canonical T_H2 effector cytokine, is also a critical developmental determinant that promotes T_H2 differentiation and inhibits T_H1 differentiation¹⁰. Recently activated helper T cells make small but functional amounts of IL-4 that induce positive feedback through the IL-4 receptor (A001263) and the transcription factors STAT6 (A002236) and GATA-3 (refs. 11,12) to promote the differentiation of T_H2 cells able to secrete copious amounts of IL-4 (refs. 10,13,14,15,16,17,18). Thus, regulation of autocrine IL-4 expression by activated helper T cells is a key control point in T helper cell lineage commitment. Nonetheless, the molecular mechanism underlying this regulation is incompletely understood.

T_H2 bias is a complex genetic trait characterized by variation in the propensity of naive helper T cells to differentiate into T_H2 (rather than T_H1) cells. T_H2 bias, measured experimentally as the amount of IL-4 produced by effector CD4⁺ T cells differentiated in vitro from naive helper T cells activated in 'neutral' conditions (that is, activated without the addition of exogenous cytokines, except IL-2, and cultured without cytokine-specific antibodies), varies over 50-fold from the high-producer phenotype of BALB/c mice to the low-producer phenotype of B10.D2 mice and correlates with susceptibility to T_H2-dependent diseases such as bronchial asthma and leishmaniasis^{14, 15, 16, 19, 20, 21, 22}. Various cellular mechanisms have been suggested as the basis for T_H2 bias, including variation in the sensitivity to prostaglandin E₂–dependent inhibition of interferon- production²³, the timing of IL-12 receptor-2 downregulation^{24, 25} and the capacity of activated helper T cells to produce autocrine IL-4 (refs. 14,15,16,20). Genetic approaches to delineate T_H2 bias have yielded many quantitative trait loci spread across mouse chromosomes 5, 12, 14 15, 16, 17, 18 and 19 (refs. 14,26,27,28,29) and a single discrete genetic locus on chromosome 11 (refs. 24,25). Several quantitative trait loci have been confirmed and isolated as discrete genetic loci by interval-specific congenic mapping^{14, 26, 28}. However, so far none of the underlying genetic determinants have been identified.

Here we combine classical genetic and transcriptional profiling analyses to identify Mina, a member of the jumonji C (JmjC) protein family, as a determinant of the T_H2 bias–regulatory locus Dice1b (called 'Dice1.2' here), which was identified by its activity in the autocrine IL-4 pathway of activated helper T cells²⁸. We found that T_H2 bias and autocrine Il4 expression correlated inversely with the Mina transcriptional rate, which in turn correlated with a Mina locus haplotype. Consistent with those findings, Mina bound to the Il4 promoter, where it repressed Il4 expression. We propose that a regulatory polymorphism that controls Mina expression in activated helper T cells determines the strength of Mina-dependent Il4 repression and hence the degree of autocrine IL-4 production and, ultimately, the extent of T_H2 differentiation, thereby accounting at least in part for strain-specific differences in T_H2 bias.

Top of page

---

Results

Mina is a Dice1.2 candidate gene

Through the use of quantitative trait loci and interval-specific congenic mapping, an Il4-regulatory locus controlling T_H2 bias, Dice1.2, has been mapped to a 26.8-megabase (Mb) genomic interval in chromosome 16 (refs. 14,28; Fig. 1a). To resolve that interval further, we generated the subcongenic strain C16D2/8D, in which the Dice1.2-spanning genomic segment derived from B10.D2 mice (low T_H2 bias) that is present in the congenic strain C16D2/8 on the BALB/c background (high T_H2 bias)²⁸ is bisected to 14.1 Mb (D16MIT138–MB04; Fig. 1a). We primed splenic CD4⁺ T cells from BALB/c, B10.D2 and C16D2/8D mice for 16 h before analyzing Il4 expression by quantitative RT-PCR. As expected, control BALB/c and B10.D2 cells showed phenotypes of high and low T_H2 bias, respectively (Fig. 1b). The production of Il4 transcripts by C16D2/8D cells was similar to that of B10.D2 cells and was significantly less than that of BALB/c cells (P = 0.0317). This result indicates that Dice1.2 is in the 14.1-Mb C16D2/8D congenic interval that spans from D16MIT138 to MB04 and contains 121 predicted or known genes³⁰. As 29 of these encode olfactory receptors, 92 remained as Dice1.2 candidates.

Figure 1: Mina is a Dice1.2 candidate gene.

(a) Location of congenic intervals in chromosome 16 of the C16D2/8 (–) and C16D2/8D (D) mouse strains, showing the chromosomal regions inherited from the B10.D2 strain (gray), the BALB/c strain (white) or ambiguous (striped); cyan dots indicate genotyped locations. Right margin, genotyping markers (red, Mina). Left margin, original (26.8 Mb) and newly identified (14.1 Mb) Dice1.2 genetic intervals, respectively. (b) Il4 expression in TCR crosslinking–activated CD4⁺ T cells from BALB/c, B10.D2 and C16D2/8D mice (n = 2–5 per strain; results are presented in arbitrary units relative to the expression of Hprt1 (encoding hypoxanthine guanine phosphoribosyl transferase). Each symbol represents an individual mouse; small horizontal lines indicate the mean. ^*P = 0.0317 (t-test). Data are representative of two experiments with similar results.

Full size image (44 KB)

The polymorphisms in Dice1.2 that underlie the genetic variability in T_H2 bias may reside in coding or regulatory sequence. To test for the latter possibility, we investigated genes in the Dice1.2 interval by transcriptional profiling with mRNA isolated from BALB/c and C16D2/8D CD4⁺ T cells stimulated by T cell antigen receptor (TCR) crosslinking for 16 h, a time at which differences in Il4 expression were nearly maximal. The Affymetrix gene chip we used for initial screening contained 30 Dice1.2 candidate genes. Of these, half were undetectable, and of the remainder, all but one had similar expression in BALB/c and C16D2/8D cells. The one exception was Mina, whose expression was two- to threefold higher in C16D2/8D cells than in BALB/c cells (Supplementary Fig. 1). Although expression profile analysis of the remaining genes in the Dice1.2 interval might identify additional Dice1.2 candidates, we explored here the possibility that Mina acts as a negative regulator of Il4 expression and T_H2 bias.

Inverse correlation of IL-4 and Mina

First, to investigate whether the inverse correlation in the expression of Mina and Il4 in BALB/c and B10.D2 cells could be generalized, we used quantitative RT-PCR to explore their expression in recently activated CD4⁺ T cells isolated from a panel of independent inbred mouse strains representing phenotypes of high and low T_H2 bias. Whereas the kinetics of the transcriptional induction of Mina and Il4 was similar across the strains tested, peak magnitudes were very dissimilar and segregated the strains into two discrete groups (Fig. 2a). In the C57BL/6, B10.D2 and C3H/HeN strains, which have low T_H2 bias, high Mina expression correlated with low Il4 expression, whereas in the BALB/c, DBA/1J, DBA/2J and DBA/2N strains, which have high T_H2 bias, the converse was true (Fig. 2a). Thus, the inverse correlation in Il4 and Mina expression could be generalized across strains with varying phenotypes of T_H2 bias.

Figure 2: Inverse correlation of Mina and Il4 expression in naive helper T cells.

(a) Time course of the expression of Mina mRNA (left) and Il4 mRNA (right) in TCR crosslinking–activated CD4⁺ T cells isolated from strains with low T_H2 bias (B10.D2, C57BL/6 and C3H/HeN; filled symbols) and high T_H2 bias (BALB/c, DBA/1J, DBA/2J and DBA/2N; open symbols); results are presented in arbitrary units relative to the expression of Actb (encoding -actin). Data are from three independent experiments (mean and s.e.m.). (b) Time course of the expression of Mina, Il4 and Ifng mRNA in BALB/c or C57BL/6 naive helper T cells activated by TCR crosslinking (top row) or from T_H1- or T_H2-primed C57BL/6 naive helper T cells (bottom); results are presented in arbitrary units relative to Hprt1 expression. Data are representative of two independent experiments (error bars, s.e.m. of triplicate PCR). (c) Mina expression in CD4⁺ T cells stimulated for 24 h with TCR crosslinking in the presence of 'graded' doses of IL-4 (B10.D2) or anti-IL-4 (BALB/c); results are presented in arbitrary units relative to Actb expression. Data are from three independent experiments (mean and s.e.m.). (d) Mina expression in B10.D2 CD4⁺ T cells stimulated for 24 h in the presence of anti-IL-12; results are presented (in arbitrary units) relative to Actb expression. Data are from three independent experiments (mean and s.e.m.).

Full size image (63 KB)

From the analysis reported above of splenic T cell samples enriched for CD4⁺ cells, it was not clear whether the inverse correlation in the expression of Il4 and Mina occurred in naive progenitor helper T cells or effector helper T cells. To investigate this question, we compared the expression of genes encoding cytokines in highly purified naive helper T cells from BALB/c and C57BL/6 mice. Quantitative RT-PCR analysis of naive helper T cells (99% CD4⁺CD62L^hiCD44^lo; less than 0.2% contamination by natural killer T cells) stimulated for 24 h with plate-bound antibody to TCR (anti-TCR) and anti-CD28 demonstrated an inverse correlation in the expression of Mina and Il4, similar to that in the cell samples enriched for CD4⁺ cells (Fig. 2b, top). Next we compared restimulated memory effector cells generated from C57BL/6 helper T cells primed in T_H1 and T_H2 conditions. As expected, Il4 expression was low and high in T_H1- and T_H2-primed cells, respectively (Fig. 2b, bottom). However, in contrast to naive helper T cells from C57BL/6 and BALB/c mice, T_H1 and T_H2 cells had similar kinetics and magnitude of Mina expression. Thus, the disparate Il4 expression potential of T_H1 and T_H2 cells is not inversely correlated with Mina expression. In contrast, natural variation in the capacity of naive helper T cells to express Il4 is inversely correlated with Mina expression.

Mina acts 'upstream' of IL-4 and IL-12

Two possibilities could account for the inverse correlation in the expression of Il4 and Mina: negative regulation of Il4 by Mina, or negative regulation of Mina by IL-4 signaling. To distinguish between these possibilities, we assessed the abundance of Mina mRNA in activated B10.D2 CD4⁺ T cells in the presence and absence of exogenously added IL-4. The addition of IL-4 to B10.D2 CD4⁺ T cells did not diminish its 'high-Mina' phenotype. Conversely, IL-4 neutralization with an IL-4-specific antibody did not enhance the 'low-Mina' phenotype of BALB/c CD4⁺ T cells (Fig. 2c). We obtained similar results with highly purified naive helper T cells (Supplementary Fig. 2a). Given the ability of IL-12 to promote and inhibit T_H1 development and T_H2 development, respectively, it was also possible that Mina expression was regulated by IL-12. However, neutralization of IL-12 with an IL-12-specific antibody did not diminish the high Mina expression of B10.D2 cells (Fig. 2d). Furthermore, the addition of IL-12 did not enhance the low Mina expression of BALB/c naive helper T cells (Supplementary Fig. 2b). Together these results indicate that neither the IL-4 signaling pathway nor the IL-12 signaling pathway regulates Mina expression, which provides support for the idea that Mina acts as a negative regulator of Il4 expression in naive helper T cells.

Mina is transcriptionally regulated

We next sought to determine whether differences in Mina transcript abundance led to differences in Mina protein abundance. We activated purified BALB/c and C57BL/6 naive helper T cells over a 72-hour time course with plate-bound anti-TCR and anti-CD28 and analyzed the resulting nuclear and cytoplasmic extracts by immunoblot with a Mina-specific antibody (Fig. 3a). Whereas cytosolic Mina increased with time, nuclear Mina appeared transiently, peaking around 24 h in both BALB/c cells and C57BL/6 cells. In both subcellular compartments, Mina protein amounts were consistently higher (about two- to fivefold) in C57BL/6 cells than in BALB/c cells, which demonstrated tight linkage between the abundance of Mina protein and that of Mina mRNA. To determine whether Mina was regulated transcriptionally, we exploited the fact that the abundance of pre-mRNA correlates with transcriptional rate³¹. Measurement of Mina pre-mRNA abundance showed higher expression in newly activated C57BL/6 helper T cells than in newly activated BALB/c helper T cells, which correlated with the differences in Mina mRNA abundance (Fig. 3b). These results collectively indicate that Mina protein abundance is transcriptionally regulated.

Figure 3: Mina is transcriptionally regulated.

(a) Immunoblot analysis of Mina and actin in cytosolic and nuclear extracts of BALB/c and C57BL/6 naive helper T cells activated for various times (above lanes) with phorbol 12-myristate 13-acetate (PMA) and ionomycin. The top band in the nuclear Mina immunoblot is nonspecific. Right, quantification of Mina expression, presented relative to actin expression. Data are representative of two independent experiments. (b) Time course of the expression of Mina pre-mRNA in naive helper T cells isolated from pools of BALB/c mice (n = 5) and C57BL/6 mice (n = 20) and activated by TCR crosslinking, measured by quantitative PCR with primers targeting sequences from Mina intron 1 and presented (in arbitrary units) relative to Hprt1 expression. Data are representative of two independent experiments (error bars, s.e.m. of triplicate PCR).

Full size image (45 KB)

To explore whether regulatory polymorphisms could account for the differences in Mina transcription in strains with high and low T_H2 bias, we sequenced the 5' end of the Mina genomic locus (including intron 1 and 1,304 base pairs (bp) of the promoter) from strains with high T_H2 bias (BALB/c and DBA/2J) and those with low T_H2 bias (C57BL/6, B10.D2/OsnJ and C3H/HeN). We identified 21 single-nucleotide polymorphisms (SNPs) that precisely defined two haplotypes (Fig. 4) that correlated with the phenotypes of T_H2 bias and Mina expression (Fig. 2). Thus, Mina haplotypes are a useful genetic marker for T_H2 bias and may contain regulatory polymorphisms responsible for differences in Mina expression across strains with distinct phenotypes of T_H2 bias.

Figure 4: Mina haplotype can be used to predict T_H2-bias phenotype.

(a) Promoter-proximal end of the Mina genomic locus, showing the 21 SNPs identified across this interval, along with exons (red), the coding region (yellow) and conserved noncoding sequences (CNS1–CNS6; cyan). (b) Position (relative to the translational start site in exon 2) and allelic identity of each SNP for three strains with low T_H2 bias (red) and two strains with high T_H2 bias (green; - indicates a gap in the sequence).

Full size image (74 KB)

Mina specifically binds and represses the Il4 promoter

To investigate whether Mina can repress transcription from the Il4 promoter, we did transient reporter assays, cotransfecting 68-41 mouse T cell hybridoma cells with a Mina or empty expression vector and a luciferase reporter driven by either an Il4 promoter or an Il2 promoter³². Mina inhibited luciferase activity driven by the Il4 promoter but not activity driven by the Il2 promoter (Fig. 5a). To define the Mina-responsive region in the 766-bp Il4 promoter, we used the transient reporter assay to study a series of Il4 promoter deletion mutants. Neither truncation of the Il4 promoter to 300 bp nor truncation to 140 bp was sufficient to eliminate sensitivity to Mina-dependent repression (Fig. 5a). Thus, a target site for the repressive activity of Mina is in the first 140 bp of the Il4 promoter.

Figure 5: Mina can bind to and repress transcription from the Il4 promoter.

(a) Transcriptional response of the Il4 promoter (left) and Il2 promoter (right) to Mina, assessed in cells transduced with a Mina or empty (-) expression vector and a luciferase reporter driven by the Il4 promoter (promoter deletion mutant length below (in bp): 766, positions -830 to -64; 300, positions -364 to -64; 140, positions -204 to -64 (all positions relative to the translational start site)) or Il2 promoter and then left unstimulated (TCR–) or stimulated for 12 h with anti-TCR (TCR+). ^*P < 0.05 (t-test). Data are from three independent experiments (average and s.e.m.). (b) Nucleotide sequence of the proximal Il4 promoter, showing regions used as oligonucleotide probes (A, B, C and D; underlined) in EMSA. (c) EMSA of nuclear extracts of B10.D2 CD4⁺ T cells stimulated for 24 h by TCR crosslinking and incubated with end-labeled probes A–D in the presence (right; probes A and B only) or absence (left) of 2 g anti-Mina. Arrows indicate the location of the nucleoprotein complex containing Mina. Data are representative of three independent experiments. (d) ChIP analysis of chromatin-bound Mina at five sites along the Il4 promoter (horizontal axis; positions relative to translational start site) and at the locus encoding CD3 (negative control) in BALB/c and B10.D2 CD4⁺ T cells stimulated for 24 h by TCR crosslinking; results are presented as immunoprecipitated relative to input. ^*P < 0.05 (t-test). Data are from five independent experiments (mean and s.e.m.).

Full size image (73 KB)

To further refine the Mina responsive region, we used electrophoretic mobility-shift assay (EMSA). We tested nuclear extracts isolated from TCR-stimulated B10.D2 CD4⁺ T cells against a series of end-labeled DNA probes spanning the first 140 bp of the Il4 promoter (Fig. 5b). Nucleoprotein complexes were formed with probe A (positions -188 to -158) and probe B (positions -157 to -128) but not probe C (positions -127 to -98) or probe D (positions -97 to -69; Fig. 5c); however, only the probe B complex was sensitive to disruption by a Mina-specific antibody. Thus, Mina can associate with the Il4 promoter over the 30-bp region of probe B that extends from position -157 to position -128.

To determine whether Mina binds to the Il4 promoter in vivo, we used anti-Mina for chromatin immunoprecipitation (ChIP) analysis of BALB/c and B10.D2 CD4⁺ T cells that had been activated for 24 h. Consistent with the binding activity detected by EMSA in the region from position -157 to position -128, we found Mina enrichment in the Il4 promoter but not in the promoter of the gene encoding CD3 (Fig. 5d). Furthermore, the magnitude of binding activity at the Il4 promoter was consistently higher in B10.D2 cells than in BALB/c cells, which correlated with the relative expression of Mina in these two strains (Fig. 2e). A broad ChIP survey of the entire T_H2 locus in B10.D2 CD4⁺ T cells activated for 24 h also detected a strong peak of Mina binding at the Il4 promoter (Supplementary Fig. 3). These data collectively support a model in which Mina is recruited to the proximal Il4 promoter, where it exerts a concentration-dependent transcription-repressive effect.

Recruitment of Mina to the Il4 promoter requires NFAT

As Mina lacks a predicted DNA-binding domain, it is likely that its recruitment to the Il4 promoter is indirect. Probe B contains published binding sites for the transcription factors NFAT (NFATc1 (A001640) and NFATc2 (A000024)) and Oct^{33, 34}, which raises the possibility of their involvement in recruiting Mina to the Il4 promoter (Fig. 6a). To test this possibility, we used wild-type probe B and a mutant probe B (B_NM) unable to bind NFAT³⁴ for EMSA with nuclear extracts from the T cell thymoma line EL4, which expresses Mina³⁵. Nuclear extracts from activated but not resting EL4 cells (and C57BL/6 CD4⁺ T cells) formed a nucleoprotein complex with wild-type probe B (Supplementary Fig. 4). This complex was sensitive to disruption by a Mina-specific antibody (Fig. 6b), which indicated that the probe B complex formed with EL4 extracts, like that formed with B10.D2 CD4⁺ T cell extracts, contained Mina. Although a wild-type NFAT consensus sequence was able to compete with probe B for complex formation, a wild-type Oct consensus sequence did not (data not shown), which suggests that the Mina complex contains NFAT but not Oct. Furthermore, a probe B mutant unable to bind Oct was still able to form the Mina complex (data not shown). Thus, Oct is neither a constituent of the Mina–probe B complex nor required for recruitment of Mina to the Il4 promoter.

Figure 6: Recruitment of Mina to the Il4 promoter requires NFAT.

(a) Oligonucleotide probes from the Il4 promoter (B and B_NM) and Il2 promoter (IL-2) used in the EMSA and DNA-precipitation experiments in b,c; above, NFAT- and Oct-binding sites. Probe B_NM is identical to probe B except for a dinucleotide mutation in the NFAT-binding site (bold italics). (b) EMSA of nuclear extracts of EL4 cells induced for 4 h with PMA-ionomycin and incubated with end-labeled probe B (^*B) or probe B_NM (^*B_NM) in the presence or absence of 100, 33 or 11 ng (wedges) of unlabeled probe B or probe B_NM (left) or with end-labeled probe B and 3 g anti-Mina, 2 g anti-NFATc1, 2 g anti-NFATc2 or rabbit serum (right). NE–, absence of nuclear extract; NE+, presence of nuclear extract. Thick and thin arrows indicate locations of the Mina complex and the anti-NFAT 'supershifted' complex, respectively. Data are representative of three independent experiments. (c) Blots of precipitation products formed by the combination of biotin-labeled probes (IL-2, B and B_NM) with nuclear extracts from EL4 cells induced for 4 h with PMA and ionomycin (Input), followed by isolation of complexes of DNA and protein (NFATc2 and Mina) with streptavidin-agarose beads. Data are representative of two independent experiments.

Full size image (61 KB)

The formation of higher-order complexes with anti-NFATc1 and anti-NFATc2 confirmed the presence of both NFAT proteins in the probe B–Mina complex (Fig. 6b and data not shown). The mutant probe B_NM that is unable to bind NFAT³⁴ did not form the Mina complex and failed to compete with probe B for complex formation (Fig. 6b), which suggests a requirement for NFAT in Mina recruitment. To confirm that idea, we did DNA precipitation assays with biotinylated probes B and B_NM. Whereas both Mina and NFAT (NFATc1 and NFATc2) could be precipitated together with the biotinylated wild-type probe B, probe B_NM failed to precipitate together with either NFAT protein or Mina (Fig. 6c and data not shown). Notably, a biotinylated NFAT-binding sequence from the Il2 promoter failed to precipitate Mina despite binding both NFATc1 and NFATc2 (Fig. 6c and data not shown), which indicated that NFATc1 and NFATc2 were insufficient for Mina recruitment. Finally, nuclear extracts of cells treated with the calcineurin inhibitor cyclosporin A, to prevent NFAT dephosphorylation and nuclear translocation, failed to form the Mina nucleoprotein complex (Supplementary Fig. 4). These data collectively suggest that NFAT is necessary but not sufficient for recruitment of Mina to the Il4 promoter–specific nucleoprotein complex.

Mina is necessary and sufficient to constrain IL-4 expression

To directly test whether Mina can repress Il4 transcription in CD4⁺ T cells, we generated three independent BDF1 C57BL/6 mouse lines expressing a Mina transgene driven by a lymphocyte-specific proximal promoter–E enhancer of the gene encoding the kinase Lck³⁶. We analyzed mice after three backcrosses to BALB/c mice, at which point they showed normal T cell development and expression of activation markers (data not shown). Using quantitative RT-PCR to compare Mina expression in CD4⁺ T cells, we found that all three transgenic lines had high basal Mina expression that diminished over a 48-h activation time course to lower expression similar to or higher than the peak of expression by their respective wild-type littermate controls (Fig. 7). To determine the effect of a transient enforced increase in Mina abundance on cytokine gene expression, we examined the transcriptional induction of Il2 and Il4 and of the gene encoding interferon- (Ifng). Expression of Il4 was considerably impaired in all three Mina-transgenic lines relative to its expression in wild-type littermate controls, but expression of Il2 and Ifng was not (Fig. 7). Thus, a transient enforced increase in Mina in CD4⁺ T cells specifically impairs expression of Il4 but not of Il2 or Ifng.

Figure 7: A transient enforced increase in Mina impairs Il4 expression in CD4⁺ T cells.

Quantitative real-time RT-PCR analysis of the expression of Mina, Il4, Il2 and Ifng in CD4⁺ T cells isolated from three independent lines from Mina-transgenic mice (lines 6, 38 and 21; filled symbols; n = 2 mice each) and their corresponding littermate controls (open symbols; n = 2 mice each), then stimulated for various times (horizontal axis) with anti-TCR and anti-CD28. Results are presented in arbitrary units relative to Actb expression; each line represents an individual mouse. Data are from two independent experiments (Exp 1 and 2).

Full size image (75 KB)

To test whether Mina-dependent repression is required to maintain normal expression of Il4, we used antisense morpholino technology to 'knock down' Mina protein in primary CD4⁺ T cells. At 24 h after activation, C57BL/6 CD4⁺ T cells pretreated for 16 h with Mina-morpholino expressed half the amount of Mina protein expressed by cells given mock pretreatment or pretreatment with control-morpholino (Fig. 8). In contrast, cells pretreated with Mina-morpholino expressed about twofold more Il4 mRNA (but not more Ifng mRNA) than did cells given mock pretreatment or pretreatment with control-morpholino (Fig. 8). Thus, the quantitative potential of CD4⁺ T cells to express Il4 is normally constrained by tight Mina-dependent negative regulation.

Figure 8: Il4 expression in CD4⁺ T cells is constrained by Mina-dependent repression.

Left, immunoblot analysis of the expression of Mina and actin in total cellular extracts from CD4⁺ T cells (4 10⁶ to 6 10⁶) given 16 h of mock pretreatment or pretreatment with control morpholino (CoMO) or Mina-morpholino (MinaMO) and then activated for 24 h with plate-bound anti-TCR and anti-CD28. Middle, quantification of Mina expression in the blot at left, presented relative to actin expression. Right, expression of Il4 and Ifng in the cells at left; results are presented (in arbitrary units) relative to Hprt1 expression. Data are representative of two independent experiments (mean of duplicate PCR).

Full size image (30 KB)

Acknowledgments

We thank R. Abe (Science University of Tokyo) for hybridoma PV-1; J. Allison (Memorial Sloan Kettering Cancer) for hybridoma 37N51.1; G. Trinchieri (US National Insitutes of Health) for hybridoma C17.8; A. Matsuno, M. Nakamura, M. Natsume, J. Epler, Y. Zhang, N. Li, S. Brown and R. Cross for technical help; J. Partridge for discussions; and D. Green, H. Beere and J. Kang (U. Mass. Medical School) for comments on the manuscript. Supported by the Cancer Research Institute (M.B.), the Burroughs Wellcome Fund (M.B.), American Lebanese Syrian Associated Charities (M.B.), the RIKEN Research Center for Allergy and Immunology International Collaboration Award Program (M.B. and M.Ku.), a Grant-in-Aid for Scientific Research (B) (M.Ku.), a Grant-in-Aid for Scientific Research on Priority Areas of the Ministry of Education, Culture, Sports, Science, and Technology (Japan) (M.Ku.), the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation (M.Ku.) and the US National Institutes of Health (AI048636 to M.B.).

Author Contributions

M.O. and M.V.S. did the experiments; L.C. did the Mina transcriptional analysis and Figures 2b and 3b and Supplementary Figure 2; M.Ka. did the Mina immunoblots; X.S. analyzed the C16D2/8D mice; Y.S. did the transgenic experiments; Y.S. and L.C. maintained the mouse colonies; O.O., H.K. and A.H. did the expression profiling; M.O., M.V.S., M.Ku and M.B. designed and conceptualized the research and analyzed the data; and M.B. prepared the manuscript.

Received 2 September 2008; Accepted 6 May 2009; Published online 28 June 2009; Corrected 5 July 2009.

References

1. Mosmann, T.R. & Coffman, R.L. TH1 and TH2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu. Rev. Immunol. 7, 145–173 (1989). | Article | PubMed | ISI | ChemPort |
2. Rook, G.A. Th2 cytokines in susceptibility to tuberculosis. Curr. Mol. Med. 7, 327–337 (2007). | Article | PubMed | ChemPort |
3. Wilson, K.T. & Fantry, G.T. Pathogenesis of Helicobacter pylori infection. Curr. Opin. Gastroenterol. 15, 66–71 (1999). | Article | PubMed | ChemPort |
4. Umetsu, D.T. & Dekruyff, R.H. Immune dysregulation in asthma. Curr. Opin. Immunol. 18, 727–732 (2006). | Article | PubMed | ChemPort |
5. Crane, I.J. & Forrester, J.V. Th1 and Th2 lymphocytes in autoimmune disease. Crit. Rev. Immunol. 25, 75–102 (2005). | Article | PubMed | ChemPort |
6. Ngoc, P.L., Gold, D.R., Tzianabos, A.O., Weiss, S.T. & Celedon, J.C. Cytokines, allergy, and asthma. Curr. Opin. Allergy Clin. Immunol. 5, 161–166 (2005). | PubMed |
7. Pearce, E.J. et al. Th2 response polarization during infection with the helminth parasite Schistosoma mansoni. Immunol. Rev. 201, 117–126 (2004). | Article | PubMed | ChemPort |
8. Maizels, R.M. et al. Helminth parasites–masters of regulation. Immunol. Rev. 201, 89–116 (2004). | Article | PubMed | ChemPort |
9. Wynn, T.A. Fibrotic disease and the T_H1/T_H2 paradigm. Nat. Rev. Immunol. 4, 583–594 (2004). | Article | PubMed | ISI | ChemPort |
10. Swain, S.L., Weinberg, A.D., English, M. & Huston, G. IL-4 directs the development of Th2-like helper effectors. J. Immunol. 145, 3796–3806 (1990). | PubMed | ISI | ChemPort |
11. Mowen, K.A. & Glimcher, L.H. Signaling pathways in Th2 development. Immunol. Rev. 202, 203–222 (2004). | Article | PubMed | ISI | ChemPort |
12. Zhu, J., Yamane, H., Cote-Sierra, J., Guo, L. & Paul, W.E. GATA-3 promotes Th2 responses through three different mechanisms: induction of Th2 cytokine production, selective growth of Th2 cells and inhibition of Th1 cell-specific factors. Cell Res. 16, 3–10 (2006). | Article | PubMed | ISI | ChemPort |
13. Le Gros, G., Ben-Sasson, S.Z., Seder, R., Finkelman, F.D. & Paul, W.E. Generation of interleukin 4 (IL-4)-producing cells in vivo and in vitro: IL-2 and IL-4 are required for in vitro generation of IL-4- producing cells. J. Exp. Med. 172, 921–929 (1990). | Article | PubMed | ISI | ChemPort |
14. Bix, M., Wang, Z.E., Thiel, B., Schork, N.J. & Locksley, R.M. Genetic regulation of commitment to interleukin 4 production by a CD4⁺ T cell-intrinsic mechanism. J. Exp. Med. 188, 2289–2299 (1998). | Article | PubMed | ISI | ChemPort |
15. Noben-Trauth, N., Hu-Li, J. & Paul, W.E. IL-4 secreted from individual naive CD4⁺ T cells acts in an autocrine manner to induce Th2 differentiation. Eur. J. Immunol. 32, 1428–1433 (2002). | Article | PubMed | ISI | ChemPort |
16. Yagi, R. et al. The IL-4 production capability of different strains of naive CD4⁺ T cells controls the direction of the T_H cell response. Int. Immunol. 14, 1–11 (2002). | Article | PubMed | ChemPort |
17. Murphy, K.M. & Reiner, S.L. The lineage decisions of helper T cells. Nat. Rev. Immunol. 2, 933–944 (2002). | Article | PubMed | ISI | ChemPort |
18. Liu, Z. et al. Requirements for the development of IL-4-producing T cells during intestinal nematode infections: what it takes to make a Th2 cell in vivo. Immunol. Rev. 201, 57–74 (2004). | Article | PubMed | ChemPort |
19. Hsieh, C.S., Macatonia, S.E., O'Garra, A. & Murphy, K.M. T cell genetic background determines default T helper phenotype development in vitro. J. Exp. Med. 181, 713–721 (1995). | Article | PubMed | ISI | ChemPort |
20. Yagi, J. et al. Genetic background influences Th cell differentiation by controlling the capacity for IL-2-induced IL-4 production by naive CD4⁺ T cells. Int. Immunol. 18, 1681–1690 (2006). | Article | PubMed | ChemPort |
21. Reiner, S.L. & Locksley, R.M. The regulation of immunity to Leishmania major. Annu. Rev. Immunol. 13, 151–177 (1995). | Article | PubMed | ISI | ChemPort |
22. Yamashita, M., Onodera, A. & Nakayama, T. Immune mechanisms of allergic airway disease: regulation by transcription factors. Crit. Rev. Immunol. 27, 539–546 (2007). | PubMed | ChemPort |
23. Kuroda, E., Sugiura, T., Zeki, K., Yoshida, Y. & Yamashita, U. Sensitivity difference to the suppressive effect of prostaglandin E2 among mouse strains: a possible mechanism to polarize Th2 type response in BALB/c mice. J. Immunol. 164, 2386–2395 (2000). | PubMed | ChemPort |
24. Gorham, J.D. et al. Genetic mapping of a murine locus controlling development of T helper 1/T helper 2 type responses. Proc. Natl. Acad. Sci. USA 93, 12467–12472 (1996). | Article | PubMed | ADS | ChemPort |
25. Guler, M.L. et al. Tpm1, a locus controlling IL-12 responsiveness, acts by a cell-autonomous mechanism. J. Immunol. 162, 1339–1347 (1999). | PubMed | ISI | ChemPort |
26. Kosarova, M., Havelkova, H., Krulova, M., Demant, P. & Lipoldova, M. The production of two Th2 cytokines, interleukin-4 and interleukin-10, is controlled independently by locus Cypr1 and by loci Cypr2 and Cypr3, respectively. Immunogenetics 49, 134–141 (1999). | Article | PubMed | ChemPort |
27. Zhang, F. et al. A murine locus on chromosome 18 controls NKT cell homeostasis and Th cell differentiation. J. Immunol. 171, 4613–4620 (2003). | PubMed | ChemPort |
28. Baguet, A., Epler, J., Wen, K.W. & Bix, M. A Leishmania major response locus identified by interval-specific congenic mapping of a T helper type 2 cell bias-controlling quantitative trait locus. J. Exp. Med. 200, 1605–1612 (2004). | Article | PubMed | ChemPort |
29. Choi, P. et al. Linkage analysis of the genetic determinants of T-cell IL-4 secretion, and identification of Flj20274 as a putative candidate gene. Genes Immun. 6, 290–297 (2005). | Article | PubMed | ChemPort |
30. Eppig, J.T., Blake, J.A., Bult, C.J., Kadin, J.A. & Richardson, J.E. The mouse genome database (MGD): new features facilitating a model system. Nucleic Acids Res. 35, D630–D637 (2007). | Article | PubMed | ChemPort |
31. Lipson, K.E. & Baserga, R. Transcriptional activity of the human thymidine kinase gene determined by a method using the polymerase chain reaction and an intron-specific probe. Proc. Natl. Acad. Sci. USA 86, 9774–9777 (1989). | Article | PubMed | ADS | ChemPort |
32. Tokoyoda, K., Takemoto, Y., Nakayama, T., Arai, T. & Kubo, M. Synergism between the calmodulin-binding and autoinhibitory domains on calcineurin is essential for the induction of their phosphatase activity. J. Biol. Chem. 275, 11728–11734 (2000). | Article | PubMed | ChemPort |
33. Bruhn, K.W., Nelms, K., Boulay, J.L., Paul, W.E. & Lenardo, M.J. Molecular dissection of the mouse interleukin-4 promoter. Proc. Natl. Acad. Sci. USA (1993).
34. Chuvpilo, S. et al. Multiple closely-linked NFAT/octamer and HMG I(Y) binding sites are part of the interleukin-4 promoter. Nucleic Acids Res. 21, 5694–5704 (1993). | Article | PubMed | ISI | ChemPort |
35. Farrar, J.J. et al. Thymoma production of T cell growth factor (Interleukin 2). J. Immunol. 125, 2555–2558 (1980). | PubMed | ISI | ChemPort |
36. Iritani, B.M., Forbush, K.A., Farrar, M.A. & Perlmutter, R.M. Control of B cell development by Ras-mediated activation of Raf. EMBO J. 16, 7019–7031 (1997). | Article | PubMed | ISI | ChemPort |
37. Klose, R.J., Kallin, E.M. & Zhang, Y. JmjC-domain-containing proteins and histone demethylation. Nat. Rev. Genet. 7, 715–727 (2006). | Article | PubMed | ISI | ChemPort |
38. Chang, B., Chen, Y., Zhao, Y. & Bruick, R.K. JMJD6 is a histone arginine demethylase. Science 318, 444–447 (2007). | Article | PubMed | ADS | ChemPort |
39. Dann, C.E., Bruick, R.K. & Deisenhofer, J. Structure of factor-inhibiting hypoxia-inducible factor 1: an asparaginyl hydroxylase involved in the hypoxic response pathway. Proc. Natl. Acad. Sci. USA 99, 15351–15356 (2002). | Article | PubMed | ADS | ChemPort |
40. Elkins, J.M. et al. Structure of factor-inhibiting hypoxia-inducible factor (HIF) reveals mechanism of oxidative modification of HIF-1. J. Biol. Chem. 278, 1802–1806 (2003). | Article | PubMed | ISI | ChemPort |
41. Baguet, A. & Bix, M. Chromatin landscape dynamics of the Il4-Il13 locus during T helper 1 and 2 development. Proc. Natl. Acad. Sci. USA 101, 11410–11415 (2004). | Article | PubMed | ADS | ChemPort |
42. Tanaka, S. et al. The interleukin-4 enhancer CNS-2 is regulated by Notch signals and controls initial expression in NKT cells and memory-type CD4 T cells. Immunity 24, 689–701 (2006). | Article | PubMed | ISI | ChemPort |
43. Amsen, D. et al. Instruction of distinct CD4 T helper cell fates by different notch ligands on antigen-presenting cells. Cell 117, 515–526 (2004). | Article | PubMed | ISI | ChemPort |
44. Mowen, K.A., Schurter, B.T., Fathman, J.W., David, M. & Glimcher, L.H. Arginine methylation of NIP45 modulates cytokine gene expression in effector T lymphocytes. Mol. Cell 15, 559–571 (2004). | Article | PubMed | ChemPort |
45. Nishimune, Y., Ohta, K., Tanaka, H., Iida, H. & Inokuchi, T. Expression of Mina53, a product of a Myc target gene in mouse testis. Int. J. Androl. 29, 323–330 (2006). | Article | PubMed | ChemPort |
46. Tsuneoka, M. et al. Mina53 as a potential prognostic factor for esophageal squamous cell carcinoma. Clin. Cancer Res. 10, 7347–7356 (2004). | Article | PubMed | ISI | ChemPort |
47. Teye, K. et al. Increased expression of a Myc target gene Mina53 in human colon cancer. Am. J. Pathol. 164, 205–216 (2004). | PubMed | ISI | ChemPort |
48. Tsuneoka, M., Koda, Y., Soejima, M., Teye, K. & Kimura, H. A novel myc target gene, mina53, that is involved in cell proliferation. J. Biol. Chem. 277, 35450–35459 (2002). | Article | PubMed | ISI | ChemPort |
49. Dubchak, I. et al. Active conservation of noncoding sequences revealed by three-way species comparisons. Genome Res. 10, 1304–1306 (2000). | Article | PubMed | ISI | ChemPort |
50. Hijikata, A. et al. Construction of an open-access database that integrates cross-reference information from the transcriptome and proteome of immune cells. Bioinformatics 23, 2934–2941 (2007). | Article | PubMed | ChemPort |
51. Seki, N. et al. IL-4-induced GATA-3 expression is a time-restricted instruction switch for Th2 cell differentiation. J. Immunol. 172, 6158–6166 (2004). | PubMed | ChemPort |
52. Rooney, J.W., Sun, Y.L., Glimcher, L.H. & Hoey, T. Novel NFAT sites that mediate activation of the interleukin-2 promoter in response to T-cell receptor stimulation. Mol. Cell. Biol. 15, 6299–6310 (1995). | PubMed | ISI | ChemPort |
53. Ragione, F.D. et al. p21Cip1 gene expression is modulated by Egr1: a novel regulatory mechanism involved in the resveratrol antiproliferative effect. J. Biol. Chem. 278, 23360–23368 (2003). | Article | PubMed | ChemPort |
54. Koyanagi, M. et al. EZH2 and histone 3 trimethyl lysine 27 associated with Il4 and Il13 gene silencing in T_H1 cells. J. Biol. Chem. 280, 31470–31477 (2005). | Article | PubMed | ChemPort |
55. Weinmann, A.S., Bartley, S.M., Zhang, T., Zhang, M.Q. & Farnham, P.J. Use of chromatin immunoprecipitation to clone novel E2F target promoters. Mol. Cell. Biol. 21, 6820–6832 (2001). | Article | PubMed | ISI | ChemPort |
56. Attar, R.M., Macdonald-Bravo, H., Raventos-Suarez, C., Durham, S.K. & Bravo, R. Expression of constitutively active IB in T cells of transgenic mice: persistent NF-B activity is required for T-cell immune responses. Mol. Cell. Biol. 18, 477–487 (1998). | PubMed | ISI | ChemPort |
57. Summerton, J.E. Endo-Porter: a novel reagent for safe, effective delivery of substances into cells. Ann. NY Acad. Sci. 1058, 62–75 (2005). | Article | PubMed | ChemPort |

Online methods

MORE ARTICLES LIKE THIS

These links to content published by NPG are automatically generated.

NPG Journals

by Subject Area

• Chemistry

  • Chemistry
  • Drug discovery
  • Biotechnology
  • Materials
  • Methods & Protocols

• Clinical Practice & Research

  • Cancer
  • Cardiovascular medicine
  • Dentistry
  • Endocrinology
  • Gastroenterology & Hepatology
  • Methods & Protocols
  • Pathology & Pathobiology
  • Urology

• Earth & Environment

  • Earth sciences
  • Evolution & Ecology

• Life sciences

  • Biotechnology
  • Cancer
  • Development
  • Drug discovery
  • Evolution & Ecology
  • Genetics
  • Immunology
  • Medical research
  • Methods & Protocols
  • Microbiology
  • Molecular cell biology
  • Neuroscience
  • Pharmacology
  • Systems biology

• Physical sciences

  • Physics
  • Materials

by A - Z Index