Effect of JAK Inhibition on the Induction of Proinflammatory HLA-DR+CD90+ Rheumatoid Arthritis Synovial Fibroblasts by Interferon-γ.

OBJECTIVE: Findings from recent transcriptome analyses of the synovium of patients with rheumatoid arthritis (RA) have revealed that 15-fold expanded HLA-DR+CD90+ synovial fibroblasts potentially act as key mediators of inflammation. The reasons for the expansion of HLA-DR+CD90+ synovial fibroblasts are unclear, but genetic signatures indicate that interferon-γ (IFNγ) plays a central role in the generation of this fibroblast subset. The present study was undertaken to investigate the generation, function and therapeutically intended blockage of HLA-DR+CD90+ synovial fibroblasts. METHODS: We combined functional assays using primary human materials and focused bioinformatic analyses of mass cytometry and transcriptomics patient data sets. RESULTS: We detected enriched and activated Fcγ receptor type IIIa-positive (CD16+) NK cells in the synovial tissue from patients with active RA. Soluble immune complexes were recognized by CD16 in a newly described reporter cell model, a mechanism that could be contributing to the activation of natural killer (NK) cells in RA. In vitro, NK cell-derived IFNγ induced HLA-DR on CD90+ synovial fibroblasts, leading to an inflammatory, cytokine-secreting HLA-DR+CD90+ phenotype. HLA-DR+CD90+ synovial fibroblasts consecutively activated CD4+ T cells upon receptor crosslinking via superantigens. HLA-DR+CD90+ synovial fibroblasts also activated CD4+ T cells in the absence of superantigens, an effect that was initiated by NK cell-derived IFNγ and that was 4 times stronger in patients with RA compared to patients with osteoarthritis. Finally, JAK inhibition in synovial fibroblasts prevented HLA-DR induction and blocked proinflammatory signals to T cells. CONCLUSION: The HLA-DR+CD90+ phenotype represents an activation state of synovial fibroblasts during the process of inflammation in RA that can be induced by IFNγ, likely generated from infiltrating leukocytes such as activated NK cells. The induction of these proinflammatory, interleukin-6-producing, and likely antigen-presenting synovial fibroblasts can be targeted by JAK inhibition.

Targeting of Janus Kinases Limits Pro-Inflammatory but Also Immunosuppressive Circuits in the Crosstalk between Synovial Fibroblasts and Lymphocytes.

Crosstalk between synovial fibroblasts (SF) and immune cells plays a central role in the development of rheumatoid arthritis (RA). Janus kinase inhibitors (JAKi) have proven efficacy in the treatment of RA, although clinical responses are heterogeneous. Currently, little is known regarding how JAKi affect pro- and anti-inflammatory circuits in the bidirectional interplay between SF and immune cells. Here, we examined the effects of tofacitinib, baricitinib and upadacitinib on crosstalk between SF and T or B lymphocytes in vitro and compared them with those of biologic disease modifying anti-rheumatic drugs (bDMARDs). JAKi dose-dependently suppressed cytokine secretion of T helper (Th) cells and decreased interleukin (IL)-6 and matrix metalloproteinase (MMP)3 secretion of SF stimulated by Th cells. Importantly, JAK inhibition attenuated the enhanced memory response of chronically stimulated SF. Vice versa, JAKi reduced the indoleamine-2,3-dioxygenase (IDO)1-mediated suppression of T cell-proliferation by SF. Remarkably, certain bDMARDs were as efficient as JAKi in suppressing the IL-6 and MMP3 secretion of SF stimulated by Th (adalimumab, secukinumab) or B cells (canakinumab) and combining bDMARDs with JAKi had synergistic effects. In conclusion, JAKi limit pro-inflammatory circuits in the crosstalk between SF and lymphocytes; however, they also weaken the immunosuppressive functions of SF. Both effects were dose-dependent and may contribute to heterogeneity in clinical response to treatment.

Increase of aerobic glycolysis mediated by activated T helper cells drives synovial fibroblasts towards an inflammatory phenotype: new targets for therapy?

BACKGROUND: A dysregulated glucose metabolism in synovial fibroblasts (SF) has been associated with their aggressive phenotype in rheumatoid arthritis (RA). Even though T helper (Th) cells are key effector cells in the propagation and exacerbation of synovitis in RA, little is known about their influence on the metabolism of SF. Thus, this study investigates the effect of Th cells on the glucose metabolism and phenotype of SF and how this is influenced by the blockade of cytokines, janus kinases (JAKs) and glycolysis. METHODS: SF from patients with RA or osteoarthritis (OA) were cultured in the presence of a stable glucose isotopomer ([U-13C]-glucose) and stimulated with the conditioned media of activated Th cells (ThCM). Glucose consumption and lactate production were measured by proton nuclear magnetic resonance (1H NMR) spectroscopy. Cytokine secretion was quantified by ELISA. The expression of glycolytic enzymes was analysed by PCR, western blot and immunofluorescence. JAKs were blocked using either baricitinib or tofacitinib and glycolysis by using either 3-bromopyruvate or FX11. RESULTS: Quiescent RASF produced significantly higher levels of lactate, interleukin (IL)-6 and matrix metalloproteinase (MMP) 3 than OASF. Stimulation by ThCM clearly changed the metabolic profile of both RASF and OASF by inducing a shift towards aerobic glycolysis with strongly increased lactate production together with a rise in IL-6 and MMP3 secretion. Interestingly, chronic stimulation of OASF by ThCM triggered an inflammatory phenotype with significantly increased glycolytic activity compared to unstimulated, singly stimulated or re-stimulated OASF. Finally, in contrast to cytokine-neutralizing biologics, inhibition of JAKs or glycolytic enzymes both significantly reduced lactate production and cytokine secretion by Th cell-stimulated SF. CONCLUSIONS: Soluble mediators released by Th cells drive SF towards a glycolytic and pro-inflammatory phenotype. Targeting of JAKs or glycolytic enzymes both potently modulate SF's glucose metabolism and decrease the release of IL-6 and MMP3. Thus, manipulation of glycolytic pathways could represent a new therapeutic strategy to decrease the pro-inflammatory phenotype of SF.

Hypoxia decreases the T helper cell-suppressive capacity of synovial fibroblasts by downregulating IDO1-mediated tryptophan metabolism.

OBJECTIVE: The development of RA is linked to local infiltration of immune cells and to changes in the phenotype of synovial fibroblasts. Synovial fibroblasts possess the capacity to suppress T cell responses through indoleamine 2, 3-dioxygenase 1 (IDO1)-mediated tryptophan metabolism. However, synovial fibroblasts from RA patients are restricted in this immune-modulatory function. Moreover, hypoxic conditions are detected within synovial tissues of RA patients, with oxygen tensions of only 3.2% O2. This study aims at investigating the effects of hypoxia on the interaction between T cells and synovial fibroblasts, particularly on the T cell-suppressive capacities of synovial fibroblasts. METHODS: Synovial fibroblasts were cultured with Th cells under normoxic and hypoxic conditions (3% O2). Th cell proliferation was detected by flow cytometry. Tryptophan and kynurenine amounts were measured by HPLC. IDO1 expression and signal transducer and activator of transcription 1 (STAT1) phosphorylation were quantified by real-time PCR or western blot, and cytokine secretion by ELISA. RESULTS: Hypoxic conditions strongly diminished the Th cell-suppressive capacities of both OA synovial fibroblasts and RA synovial fibroblasts. Accordingly, IDO1 mRNA and protein expression, STAT1 phosphorylation and tryptophan metabolism were greatly reduced in OA synovial fibroblasts by hypoxia. MMP-3, IL-6, IL-10 and IFNγ secretion were significantly decreased under hypoxia in synovial fibroblast-Th cell co-cultures, while IL-17A levels were elevated. Supplementation with IFNγ, a well-known inducer of IDO1 expression, could rescue neither IDO1 expression nor Th cell suppression under hypoxic conditions. CONCLUSION: Hypoxia strongly affected the crosstalk between synovial fibroblasts and Th cells. By reducing the efficiency of synovial fibroblasts to restrict Th cell proliferation and by increasing the expression of IL-17A, hypoxia might have implications on the pathophysiology of RA.

Hypoxia Inducible Factor 1α Inhibits the Expression of Immunosuppressive Tryptophan-2,3-Dioxygenase in Glioblastoma.

Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.

Distinct human circulating NKp30+FcεRIγ+CD8+ T cell population exhibiting high natural killer-like antitumor potential.

CD8+ T cells are considered prototypical cells of adaptive immunity. Here, we uncovered a distinct CD8+ T cell population expressing the activating natural killer (NK) receptor NKp30 in the peripheral blood of healthy individuals. We revealed that IL-15 could de novo induce NKp30 expression in a population of CD8+ T cells and drive their differentiation toward a broad innate transcriptional landscape. The adaptor FcεRIγ was concomitantly induced and was shown to be crucial to enable NKp30 cell-surface expression and function in CD8+ T cells. FcεRIγ de novo expression required promoter demethylation and was accompanied by acquisition of the signaling molecule Syk and the "innate" transcription factor PLZF. IL-15-induced NKp30+CD8+ T cells exhibited high NK-like antitumor activity in vitro and were able to synergize with T cell receptor signaling. Importantly, this population potently controlled tumor growth in a preclinical xenograft mouse model. Our study, while blurring the borders between innate and adaptive immunity, reveals a unique NKp30+FcεRIγ+CD8+ T cell population with high antitumor therapeutic potential.

Extracellular vesicles mediate intercellular communication: Transfer of functionally active microRNAs by microvesicles into phagocytes.

Cell activation and apoptosis lead to the formation of extracellular vesicles (EVs) such as exosomes or microvesicles (MVs). EVs have been shown to modulate immune responses; recently, MVs were described to carry microRNA (miRNA) and a role for MVs in the pathogenesis of autoimmune diseases has been discussed. Here we systematically characterized MVs and exosomes according to their release stimuli. The miRNA content of viable or apoptotic human T lymphocytes and the corresponding MVs was analyzed. miRNA, protein and surface marker expression, as well as cytokine release by human monocytes was measured after EV engulfment. Finally, miRNA expression in T lymphocytes and MVs of healthy individuals was compared with those of systemic lupus erythematosus (SLE) patients. We demonstrate that, depending on the stimuli, distinct subtypes of EVs are released, differing in size and carrying a specific RNA profile. We observed an accumulation of distinct miRNAs in MVs after induction of apoptosis and the transfer of functional miRNA by MVs into human monocytes. MVs released from apoptotic cells provoke less of an inflammatory response than those released from viable cells. MiR-155*, miR-34b and miR-34a levels in T lymphocytes and corresponding MVs were deregulated in SLE when compared to healthy individuals.

Microvesicles released by apoptotic human neutrophils suppress proliferation and IL-2/IL-2 receptor expression of resting T helper cells.

Membrane-coated microvesicles (MVs) have been identified as important mediators in intercellular communication. During the process of apoptosis, dying cells dynamically release MVs. Neutrophils are the most abundant type of leukocytes in the circulation. Due to their very short lifespan, it is likely that they are the source of large amounts of apoptotic cell-derived MVs. Here, we show that MVs released by apoptotic human polymorphonuclear neutrophils (apoPMN-MVs), but not the apoptotic neutrophils themselves, selectively suppress the proliferation of CD25 (IL-2Rα)neg CD127 (IL-7Rα)pos Th cells in a dose-dependent manner. In contrast, the proliferation of total T cells is not affected by MVs. Importantly, apoPMN-MVs suppress the secretion of IL-2 as well as the expression of and signaling via the IL-2 receptor (IL-2R) by CD25neg CD127pos Th cells. Addition of IL-7 strongly reduced the suppression of T-cell proliferation by MVs and the addition of IL-2 completely abrogated the suppressive effect. Thus, apoPMN-MVs suppressed a subset of Th cells by downregulating IL-2 and IL-2R expression and signaling. This may represent an important mechanism to prevent the activation and expansion of resting T cells in the absence of sufficient cytokine stimulation, and thereby maintaining immune tolerance.

Synovial Fibroblasts Selectively Suppress Th1 Cell Responses through IDO1-Mediated Tryptophan Catabolism.

The development of rheumatoid arthritis (RA) is linked to functional changes in synovial fibroblasts (SF) and local infiltration of T lymphocytes. Fibroblasts possess the capacity to suppress T cell responses, although the molecular mechanisms of this suppression remain incompletely understood. In this study, we aimed to define the mechanisms by which noninflammatory SF modulate Th cell responses and to determine the immunosuppressive efficacy of RASF. Hence, the influence of SF from osteoarthritis or RA patients on total Th cells or different Th cell subsets of healthy donors was analyzed in vitro. We show that SF strongly suppressed the proliferation of Th cells and the secretion of IFN-γ in a cell contact-independent manner. In cocultures of SF and Th cells, tryptophan was completely depleted within a few days, resulting in eukaryotic initiation factor 2α phosphorylation, TCRζ-chain downregulation, and proliferation arrest. Blocking IDO1 activity completely restored Th cell proliferation, but not IFN-γ production. Interestingly, only the proliferation of Th1 cells, but not of Th2 or Th17 cells, was affected. Finally, RASF had a significantly lower IDO1 expression and a weaker Th cell suppressive capacity compared with osteoarthritis SF. We postulate that the suppression of Th cell growth by SF through tryptophan catabolism may play an important role in preventing inappropriate Th cell responses under normal conditions. However, expansion of Th17 cells that do not induce IDO1-mediated suppression and the reduced capacity of RASF to restrict Th cell proliferation through tryptophan metabolism may support the initiation and propagation of synovitis in RA patients.

Activated human B cells induce inflammatory fibroblasts with cartilage-destructive properties and become functionally suppressed in return.

BACKGROUND: Cross-talk between synovial fibroblasts (SF) and immune cells is suggested to play a crucial role in inflammation and chronification of rheumatoid arthritis (RA). The contribution of B cells in this process is poorly defined. METHODS: Here, primary B cells from healthy donors were polyclonally activated and cocultured with SF of non-synovitic origin from patients with osteoarthritis. RESULTS: In B-SF cocultures the concentrations of interleukin 6 (IL-6) and IL-8 increased manifold compared with single cultures even under physical separation and remained stable for several days after B-cell removal. Intracellular staining confirmed SF as key producers of IL-6 and IL-8, and B cells as main producers of tumour necrosis factor alpha (TNFα) and IL-1ß. Blocking experiments with a combination of anti-TNFα-antibodies and rIL-1RA significantly reduced SF cytokine production by up to 90%, suggesting that B-cell-derived TNFα and IL-1ß were crucial mediators of SF activation. Interestingly, B-cell cytokine production, CD25 expression and proliferation decreased in cocultures by at least 50%, demonstrating a negative regulatory loop towards the activated B cells. Inhibition of activin receptor-like kinase 5, a crucial component of the tumour growth factor ß (TGFß) signalling pathway, partly restored B-cell proliferation, suggesting a contribution of SF-derived TGFß in B-cell suppression. Besides cytokines, B-cell-activated SF also upregulated secretion of matrix metalloproteases such as MMP-3, thereby acquiring potential tissue destructive properties. This was confirmed by their invasion into human cartilage in the severe combined immunodeficiency mouse fibroblast invasion model in vivo. CONCLUSIONS: Interaction with activated B cells leads to conversion of non-arthritic SF into SF with a proinflammatory and aggressive RA-like phenotype, thereby suggesting a new, so far unrecognised role for B cells in RA pathogenesis.

Apoptotic-cell-derived membrane microparticles and IFN-α induce an inflammatory immune response.

A dysregulation in the clearance of apoptotic material is considered a major pathogenetic factor for the emergence of autoimmune diseases. Apoptotic-cell-derived membrane microparticles (AdMPs), which are released from the cell surface during apoptosis, have been implicated in the pathogenesis of autoimmunity. Also of importance are cytokines, such as interferon-α (IFN-α), which is known to be a major player in patients with systemic lupus erythematosus (SLE). This study investigates the combined effect of AdMPs and IFN-α on professional phagocytes. In the presence of IFN-α, phagocytosis of AdMPs by human monocytes was significantly increased in a dose-dependent manner. The combination of AdMPs and raised IFN-α concentrations resulted in an increase in the secretion of pro-inflammatory cytokines and an upregulation of surface molecule expression involved in antigen uptake. In addition, macrophage polarisation was shifted towards a more inflammatory type of cell. The synergism between IFN-α and AdMPs seemed to be mediated by an upregulation of phosphorylated STAT1. Our results indicate that IFN-α, together with AdMPs, amplify the initiation and maintenance of inflammation. This mechanism might especially play a crucial role in disorders with a defective clearance of apoptotic material.

An evolutionarily conserved mutual interdependence between Aire and microRNAs in promiscuous gene expression.

The establishment and maintenance of central tolerance depends to a large extent on the ability of medullary thymic epithelial cells to express a variety of tissue-restricted antigens, the so-called promiscuous gene expression (pGE). Autoimmune regulator (Aire) is to date the best characterised transcriptional regulator known to at least partially coordinate pGE. There is accruing evidence that the expression of Aire-dependent and -independent genes is modulated by higher order chromatin configuration, epigenetic modifications and post-transcriptional control. Given the involvement of microRNAs (miRNAs) as potent post-transcriptional modulators of gene expression, we investigated their role in the regulation of pGE in purified mouse and human thymic epithelial cells (TECs). Microarray profiling of TEC subpopulations revealed evolutionarily conserved cell type and differentiation-specific miRNA signatures with a subset of miRNAs being significantly upregulated during terminal medullary thymic epithelial cell differentiation. The differential regulation of this subset of miRNAs was correlated with Aire expression and some of these miRNAs were misexpressed in the Aire knockout thymus. In turn, the specific absence of miRNAs in TECs resulted in a progressive reduction of Aire expression and pGE, affecting both Aire-dependent and -independent genes. In contrast, the absence of miR-29a only affected the Aire-dependent gene pool. These findings reveal a mutual interdependence of miRNA and Aire.

DNA methylation signatures of the AIRE promoter in thymic epithelial cells, thymomas and normal tissues.

Mutations in the AIRE gene cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which is associated with autoimmunity towards several peripheral organs. The AIRE protein is almost exclusively expressed in medullary thymic epithelial cells (mTEC) and CpG methylation in the promoter of the AIRE gene has been suggested to control its tissue-specific expression pattern. We found that in human AIRE-positive medullary and AIRE-negative cortical epithelium, the AIRE promoter is hypomethylated, whereas in thymocytes, the promoter had high level of CpG methylation. Likewise, in mouse mTECs the AIRE promoter was uniformly hypomethylated. In the same vein, the AIRE promoter was hypomethylated in AIRE-negative thymic epithelial tumors (thymomas) and in several peripheral tissues. Our data are compatible with the notion that promoter hypomethylation is necessary but not sufficient for tissue-specific regulation of the AIRE gene. In contrast, a positive correlation between AIRE expression and histone H3 lysine 4 trimethylation, an active chromatin mark, was found in the AIRE promoter in human and mouse TECs.

Association of an SNP with intrathymic transcription of TSHR and Graves' disease: a role for defective thymic tolerance.

Graves' disease (GD) is the paradigm of an anti-receptor autoimmune disease, with agonistic auto-antibodies against the thyrotropin receptor (TSHR-thyroid-stimulating hormone receptor) being the underlying pathogenic effector mechanism. The TSHR belongs to the category of tissue-restricted antigens, which are promiscuously expressed in the thymus and thereby induce central T cell tolerance. In order to understand the association between TSHR gene polymorphisms and GD, we tested the hypothesis that TSHR gene variants affect susceptibility to GD by influencing levels of TSHR transcription in the thymus. We show that thymic glands from non-autoimmune donors homozygous for the rs179247 SNP predisposing allele of TSHR had significantly fewer TSHR mRNA transcripts than carriers of the protective allele. In addition, in heterozygous individuals, the TSHR predisposing allele was expressed at a lower level than the protective one as demonstrated by allele-specific transcript quantification. This unbalanced allelic expression was detectable in both thymic epithelial cells and thymocytes. Since the level of self-antigen expression is known to influence the threshold of central tolerance, these results are compatible with the notion that defective central tolerance contributes to the pathogenesis of GD, a scenario already implicated in type 1 diabetes mellitus, myasthenia gravis and autoimmune myocarditis.

Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells.

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ("entry site") to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

The thymus medulla slowly yields its secrets.

The past few years have witnessed considerable advances in our understanding of the mechanisms underlying the induction of central tolerance. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. This brief review covers progress of the last two years in deciphering the functional interrelationship among TEC development, promiscuous gene expression, and central tolerance. We discuss new insights into signaling pathways directing the differentiation and homeostasis of mTECs, and new clues to the molecular regulation of promiscuous gene expression (pGE), including the role of the transcriptional regulator autoimmune regulator (AIRE). Furthermore, we emphasize the importance of promiscuous expression of particular tissue-restricted self-antigens in preventing organ-specific autoimmunity and evaluate new data supporting the threshold model of central tolerance.

A critical control element for interleukin-4 memory expression in T helper lymphocytes.

Naive T helper (Th) lymphocytes are induced to express the il4 (interleukin-4) gene by simultaneous signaling through the T cell receptor and the interleukin (IL)-4 receptor. Upon restimulation with antigen, such preactivated Th lymphocytes can reexpress the il4 gene independent of IL-4 receptor signaling. This memory for expression of the il4 gene depends on epigenetic modification of the il4 gene locus and an increased expression of GATA-3, the key transcription factor for Th2 differentiation. Here, we have identified a phylogenetically conserved sequence, the conserved intronic regulatory element, in the first intron of the il4 gene containing a tandem GATA-3 binding site. We show that GATA-3 binds to this sequence in a position- and orientation-dependent manner, in vitro and in vivo. DNA demethylation and histone acetylation of this region occurs early and selectively in differentiating, IL-4-secreting Th2 lymphocytes. Deletion of the conserved element by replacement of the first exon and part of the first intron of the il4 gene with gfp leads to a defect in the establishment of memory for expression of IL-4, in that reexpression of IL-4 still requires costimulation by exogenous IL-4. The conserved intronic regulatory element thus links the initial epigenetic modification of the il4 gene to GATA-3 and serves as a genetic control element for memory expression of IL-4.