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1.  The proteasome inhibitior bortezomib depletes plasma cells and ameliorates clinical manifestations of refractory systemic lupus erythematosus 
Annals of the Rheumatic Diseases  2015;74(7):1474-1478.
Objectives
To investigate whether bortezomib, a proteasome inhibitor approved for treatment of multiple myeloma, induces clinically relevant plasma cell (PC) depletion in patients with active, refractory systemic lupus erythematosus (SLE).
Methods
Twelve patients received a median of two (range 1–4) 21-day cycles of intravenous bortezomib (1.3 mg/m2) with the coadministration of dexamethasone (20 mg) for active SLE. Disease activity was assessed using the SLEDAI-2K score. Serum concentrations of anti–double-stranded DNA (anti-dsDNA) and vaccine-induced protective antibodies were monitored. Flow cytometry was performed to analyse peripheral blood B-cells, PCs and Siglec-1 expression on monocytes as surrogate marker for type-I interferon (IFN) activity.
Results
Upon proteasome inhibition, disease activity significantly declined and remained stable for 6 months on maintenance therapies. Nineteen treatment-emergent adverse events occurred and, although mostly mild to moderate, resulted in treatment discontinuation in seven patients. Serum antibody levels significantly declined, with greater reductions in anti-dsDNA (∼60%) than vaccine-induced protective antibody titres (∼30%). Bortezomib significantly reduced the numbers of peripheral blood and bone marrow PCs (∼50%), but their numbers increased between cycles. Siglec-1 expression on monocytes significantly declined.
Conclusions
These findings identify proteasome inhibitors as a putative therapeutic option for patients with refractory SLE by targeting PCs and type-I IFN activity, but our results must be confirmed in controlled trials.
doi:10.1136/annrheumdis-2014-206016
PMCID: PMC4484251  PMID: 25710470
Systemic Lupus Erythematosus; Autoimmune Diseases; B cells; Treatment; Autoimmunity
2.  Memory CD8+ T cells colocalize with IL-7+ stromal cells in bone marrow and rest in terms of proliferation and transcription 
European Journal of Immunology  2015;45(4):975-987.
It is believed that memory CD8+ T cells are maintained in secondary lymphoid tissues, peripheral tissues, and BM by homeostatic proliferation. Their survival has been shown to be dependent on IL-7, but it is unclear where they acquire it. Here we show that in murine BM, memory CD8+ T cells individually colocalize with IL-7+ reticular stromal cells. The T cells are resting in terms of global transcription and do not express markers of activation, for example, 4-1BB (CD137), IL-2, or IFN-γ, despite the expression of CD69 on about 30% of the cells. Ninety-five percent of the memory CD8+ T cells in BM are in G0 phase of cell cycle and do not express Ki-67. Less than 1% is in S/M/G2 of cell cycle, according to propidium iodide staining. While previous publications have estimated the extent of proliferation of CD8+ memory T cells on the basis of BrdU incorporation, we show here that BrdU itself induces proliferation of CD8+ memory T cells. Taken together, the present results suggest that CD8+ memory T cells are maintained as resting cells in the BM in dedicated niches with their survival conditional on IL-7 receptor signaling.
doi:10.1002/eji.201445295
PMCID: PMC4415462  PMID: 25639669
Bone marrow; CD8 T cell; Gene expression; Interleukin-7; Memory cells
3.  A Ca2+ concentration of 1.5 mM, as present in IMDM but not in RPMI, is critical for maximal response of Th cells to PMA/ionomycin 
European Journal of Immunology  2015;45(4):1270-1273.
doi:10.1002/eji.201445247
PMCID: PMC4407954  PMID: 25545753
CD4+ T cells; Cellular activation; Cytokines; Flow cytometry; PMA/ionomycin
4.  miR-148a is upregulated by Twist1 and T-bet and promotes Th1-cell survival by regulating the proapoptotic gene Bim 
European Journal of Immunology  2015;45(4):1192-1205.
Repeatedly activated T helper 1 (Th1) cells present during chronic inflammation can efficiently adapt to the inflammatory milieu, for example, by expressing the transcription factor Twist1, which limits the immunopathology caused by Th1 cells. Here, we show that in repeatedly activated murine Th1 cells, Twist1 and T-bet induce expression of microRNA-148a (miR-148a). miR-148a regulates expression of the proapoptotic gene Bim, resulting in a decreased Bim/Bcl2 ratio. Inhibition of miR-148a by antagomirs in repeatedly activated Th1 cells increases the expression of Bim, leading to enhanced apoptosis. Knockdown of Bim expression by siRNA in miR-148a antagomir-treated cells restores viability of the Th1 cells, demonstrating that miR-148a controls survival by regulating Bim expression. Thus, Twist1 and T-bet not only control the differentiation and function of Th1 cells, but also their persistence in chronic inflammation.
doi:10.1002/eji.201444633
PMCID: PMC4406154  PMID: 25486906
Bim; miR-148a; T-bet; Th1; Twist1
5.  Long-lived plasma cells are early and constantly generated in New Zealand Black/New Zealand White F1 mice and their therapeutic depletion requires a combined targeting of autoreactive plasma cells and their precursors 
Introduction
Autoantibodies contribute significantly to the pathogenesis of systemic lupus erythematosus (SLE). Unfortunately, the long-lived plasma cells (LLPCs) secreting such autoantibodies are refractory to conventional immunosuppressive treatments. Although generated long before the disease becomes clinically apparent, it remains rather unclear whether LLPC generation continues in the established disease. Here, we analyzed the generation of LLPCs, including autoreactive LLPCs, in SLE-prone New Zealand Black/New Zealand White F1 (NZB/W F1) mice over their lifetime, and their regeneration after depletion.
Methods
Bromodeoxyuridine pulse-chase experiments in mice of different ages were performed in order to analyze the generation of LLPCs during the development of SLE. LLPCs were enumerated by flow cytometry and autoreactive anti-double-stranded DNA (anti-dsDNA) plasma cells by enzyme-linked immunospot (ELISPOT). For analyzing the regeneration of LLPCs after depletion, mice were treated with bortezomib alone or in combination with cyclophosphamide and plasma cells were enumerated 12 hours, 3, 7, 11 and 15 days after the end of the bortezomib cycle.
Results
Autoreactive LLPCs are established in the spleen and bone marrow of SLE-prone mice very early in ontogeny, before week 4 and before the onset of symptoms. The generation of LLPCs then continues throughout life. LLPC counts in the spleen plateau by week 10, but continue to increase in the bone marrow and inflamed kidney. When LLPCs are depleted by the proteasome inhibitor bortezomib, their numbers regenerate within two weeks. Persistent depletion of LLPCs was achieved only by combining a cycle of bortezomib with maintenance therapy, for example cyclophosphamide, depleting the precursors of LLPCs or preventing their differentiation into LLPCs.
Conclusions
In SLE-prone NZB/W F1 mice, autoreactive LLPCs are generated throughout life. Their sustained therapeutic elimination requires both the depletion of LLPCs and the inhibition of their regeneration.
doi:10.1186/s13075-015-0551-3
PMCID: PMC4411657  PMID: 25889236
6.  Role of Blimp-1 in programing Th effector cells into IL-10 producers 
The Journal of Experimental Medicine  2014;211(9):1807-1819.
The transcriptional regulator Blimp-1 is absolutely required for IL-10 production in Th1 cells and limits inflammatory effector T cell responses downstream of IL-12 and IL-27.
Secretion of the immunosuppressive cytokine interleukin (IL) 10 by effector T cells is an essential mechanism of self-limitation during infection. However, the transcriptional regulation of IL-10 expression in proinflammatory T helper (Th) 1 cells is insufficiently understood. We report a crucial role for the transcriptional regulator Blimp-1, induced by IL-12 in a STAT4-dependent manner, in controlling IL-10 expression in Th1 cells. Blimp-1 deficiency led to excessive inflammation during Toxoplasma gondii infection with increased mortality. IL-10 production from Th1 cells was strictly dependent on Blimp-1 but was further enhanced by the synergistic function of c-Maf, a transcriptional regulator of IL-10 induced by multiple factors, such as the Notch pathway. We found Blimp-1 expression, which was also broadly induced by IL-27 in effector T cells, to be antagonized by transforming growth factor (TGF) β. While effectively blocking IL-10 production from Th1 cells, TGF-β shifted IL-10 regulation from a Blimp-1–dependent to a Blimp-1–independent pathway in IL-27–induced Tr1 (T regulatory 1) cells. Our findings further illustrate how IL-10 regulation in Th cells relies on several transcriptional programs that integrate various signals from the environment to fine-tune expression of this critical immunosuppressive cytokine.
doi:10.1084/jem.20131548
PMCID: PMC4144744  PMID: 25073792
7.  Nuclear Factor of Activated T Cells Regulates the Expression of Interleukin-4 in Th2 Cells in an All-or-none Fashion* 
The Journal of Biological Chemistry  2014;289(39):26752-26761.
Background: Not every T helper type 2 (Th2) lymphocyte imprinted to express interleukin-4 (IL-4) does so when activated.
Results: Preventing nuclear translocation of the nuclear factor of activated T cells (NFAT) reduces the number of Th2 lymphocytes reexpressing IL-4.
Conclusion: NFAT is the limiting factor determining digital IL-4 expression in Th2 lymphocytes.
Significance: This might help us to understand the regulation of immunopathology in allergy and asthma.
Th2 memory lymphocytes have imprinted their Il4 genes epigenetically for expression in dependence of T cell receptor restimulation. However, in a given restimulation, not all Th cells with a memory for IL-4 expression express IL-4. Here, we show that in reactivated Th2 cells, the transcription factors NFATc2, NF-kB p65, c-Maf, p300, Brg1, STAT6, and GATA-3 assemble at the Il4 promoter in Th2 cells expressing IL-4 but not in Th2 cells not expressing it. NFATc2 is critical for assembly of this transcription factor complex. Because NFATc2 translocation into the nucleus occurs in an all-or-none fashion, dependent on complete dephosphorylation by calcineurin, NFATc2 controls the frequencies of cells reexpressing Il4, translates analog differences in T cell receptor stimulation into a digital decision for Il4 reexpression, and instructs all reexpressing cells to express the same amount of IL-4. This analog-to-digital conversion may be critical for the immune system to respond to low concentrations of antigens.
doi:10.1074/jbc.M114.587865
PMCID: PMC4175318  PMID: 25037220
Calcineurin; Cytokine; NFAT Transcription Factor; T-cell; T-cell Receptor (TCR)
8.  Cell-Specific Type I IFN Signatures in Autoimmunity and Viral Infection: What Makes the Difference? 
PLoS ONE  2013;8(12):e83776.
Gene expression profiling of peripheral blood mononuclear cells (PBMCs) has revealed a crucial role for type I interferon (IFN) in the pathogenesis of systemic lupus erythematosus (SLE). However, it is unclear how particular leucocyte subsets contribute to the overall type I IFN signature of PBMCs and whole blood samples.Furthermore, a detailed analysis describing the differences in the IFN signature in autoimmune diseases from that observed after viral infection has not been performed to date. Therefore, in this study, the transcriptional responses in peripheral T helper cells (CD4+) and monocyte subsets (CD16− inflammatory and CD16+ resident monocytes) isolated from patients with SLE, healthy donors (ND) immunised with the yellow fever vaccine YFV-17Dand untreated controls were compared by global gene expression profiling.It was striking that all of the transcripts that were regulated in response to viral exposure were also found to be differentially regulated in SLE, albeit with markedly lower fold-change values. In addition to this common IFN signature, a pathogenic IFN-associated gene signature was detected in the CD4+ T cells and monocytes from the lupus patients. IL-10, IL-9 and IL-15-mediated JAK/STAT signalling was shown to be involved in the pathological amplification of IFN responses observed in SLE. Type I IFN signatures identified were successfully applied for the monitoring of interferon responses in PBMCs of an independent cohort of SLE patients and virus-infected individuals. Moreover, these cell-type specific gene signatures allowed a correct classification of PBMCs independent from their heterogenic cellular composition. In conclusion, our data show for the first time that monocytes and CD4 cells are sensitive biosensors to monitor type I interferon response signatures in autoimmunity and viral infection and how these transriptional responses are modulated in a cell- and disease-specific manner.
doi:10.1371/journal.pone.0083776
PMCID: PMC3877094  PMID: 24391825
9.  CD49b/CD69-Dependent Generation of Resting T Helper Cell Memory 
In the absence of antigen, memory T helper (Th) cells are maintained in a resting state. Recently it has been shown that bone marrow (BM) is a major reservoir of resting memory Th cells. In a given immune response, less than 10% of the activated CD4 T cells are recruited to the pool of resting BM memory Th cells. Here we review recent evidence that CD69 and CD49b control homing of memory Th cell precursors to the BM. During the effector phase of an immune response, about 10% of activated CD4 T cells in the spleen express both CD69 and CD49b, and thus qualify as precursors of resting memory Th cells of BM. Loss or blockade of CD69 and CD49b expression on CD4 T cells impairs the generation of resting memory Th cells in the BM. Moreover, in the absence of BM memory Th cells in CD69-deficient mice, T-cell help for B cells is impaired, confirming the central role of BM memory Th cells in the maintenance of immunological memory.
doi:10.3389/fimmu.2013.00183
PMCID: PMC3706785  PMID: 23847623
T helper cells; immunological memory; bone marrow; CD49b; CD69; homing
10.  Memory on the move 
Cellular and Molecular Life Sciences  2012;69(10):1563-1564.
doi:10.1007/s00018-012-0964-y
PMCID: PMC3337994  PMID: 22481435
11.  IL-2 Expression in Activated Human Memory FOXP3+ Cells Critically Depends on the Cellular Levels of FOXP3 as Well as of Four Transcription Factors of  T Cell Activation 
The human CD4+FOXP3+ T cell population is heterogeneous and consists of various subpopulations which remain poorly defined. Anergy and suppression are two main functional characteristics of FOXP3+Treg cells. We used the anergic behavior of FOXP3+Treg cells for a better discrimination and characterization of such subpopulations. We compared IL-2-expressing with IL-2-non-expressing cells within the memory FOXP3+ T cell population. In contrast to IL-2-non-expressing FOXP3+ cells, IL-2-expressing FOXP3+ cells exhibit intermediate characteristics of Treg and Th cells concerning the Treg cell markers CD25, GITR, and Helios. Besides lower levels of FOXP3, they also have higher levels of the transcription factors NFATc2, c-Fos, NF-κBp65, and c-Jun. An approach combining flow cytometric measurements with statistical interpretation for quantitative transcription factor analysis suggests that the physiological expression levels not only of FOXP3 but also of NFATc2, c-Jun, c-Fos, and NF-κBp65 are limiting for the decision whether IL-2 is expressed or not in activated peripheral human memory FOXP3+ cells. These findings demonstrate that concomitant high levels of NFATc2, c-Jun, c-Fos, and NF-κBp65 lead in addition to potential IL-2 expression in those FOXP3+ cells with low levels of FOXP3. We hypothesize that not only the level of FOXP3 expression but also the amounts of the four transcription factors studied represent determining factors for the anergic phenotype of FOXP3+ Treg cells.
doi:10.3389/fimmu.2012.00264
PMCID: PMC3428033  PMID: 22969764
cytokine expression; transcription factors; T cell activation; IL-2 expression; lymphocyte; flow cytometry; human Treg cells; memory Th cells
12.  Development of replication-defective lymphocytic choriomeningitis virus vectors for the induction of potent CD8+ T cell immunity 
Nature medicine  2010;16(3):339-345.
Lymphocytic choriomeningitis virus (LCMV) exhibits natural tropism for dendritic cells and represents the prototypic infection that elicits protective CD8+ T cell (cytotoxic T lymphocyte (CTL)) immunity. Here we have harnessed the immunobiology of this arenavirus for vaccine delivery. By using producer cells constitutively synthesizing the viral glycoprotein (GP), it was possible to replace the gene encoding LCMV GP with vaccine antigens to create replication-defective vaccine vectors. These rLCMV vaccines elicited CTL responses that were equivalent to or greater than those elicited by recombinant adenovirus 5 or recombinant vaccinia virus in their magnitude and cytokine profiles, and they exhibited more effective protection in several models. In contrast to recombinant adenovirus 5, rLCMV failed to elicit vector-specific antibody immunity, which facilitated re-administration of the same vector for booster vaccination. In addition, rLCMV elicited T helper type 1 CD4+ T cell responses and protective neutralizing antibodies to vaccine antigens. These features, together with low seroprevalence in humans, suggest that rLCMV may show utility as a vaccine platform against infectious diseases and cancer.
doi:10.1038/nm.2104
PMCID: PMC3247638  PMID: 20139992
14.  Cytokine imprinting - mechanisms for memory 
Arthritis Research & Therapy  2011;13(Suppl 2):O12.
doi:10.1186/ar3416
PMCID: PMC3194141
15.  Analysis of IL-17+ cells in facet joints of patients with spondyloarthritis suggests that the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response 
Introduction
In this study, we analysed the number of IL-17+ cells in facet joints, in the peripheral blood (PB) and synovial fluid (SF) of spondyloarthritis (SpA) patients and compared these results with those of patients with other rheumatic diseases and controls.
Methods
Immunohistochemical analysis of IL-17+ cells was performed in facet joints of 33 ankylosing spondylitis (AS) patients and compared with data from 20 osteoarthritis (OA) patients. The frequency of IL-17+CD4+ T cells in PB and SF of SpA patients (PB n = 30, SF n = 11), rheumatoid arthritis (RA) patients (PB n = 14, SF n = 7), OA patients (PB n = 10) and healthy controls (PB n = 12) was analysed after stimulation with Staphylococcus aureus Enterotoxin B and phorbol 12-myristate 13-acetate/ionomycin and quantified by flow cytometry.
Results
In AS facet joints, the frequency of IL-17-secreting cells was significantly higher than in samples obtained from OA patients (P < 0.001), with a slight predominance of IL-17+ cells among the mononuclear cells (61.5% ± 14.9%) compared to cells with polysegmental nuclei. Immunofluorescence microscopy revealed that the majority of IL-17+ cells were myeloperoxidase-positive (35.84 ± 13.06/high-power field (HPF) and CD15+ neutrophils (24.25 ± 10.36/HPF), while CD3+ T cells (0.51 ± 0.49/HPF) and AA-1+ mast cells (2.28 ± 1.96/HPF) were less often IL-17-positive. The frequency of IL-17+CD4+ T cells in the PB and SF of SpA patients did not differ significantly compared to RA patients, OA patients or healthy controls.
Conclusions
Our data suggest an important role for IL-17 in the inflammatory processes in AS. However, the innate immune pathway might be of greater relevance than the Th17-mediated adaptive immune response.
doi:10.1186/ar3370
PMCID: PMC3218910  PMID: 21689402
16.  Generation of stable monoclonal antibody-producing BCR+ human memory B cells by genetic programming 
Nature medicine  2009;16(1):123-128.
B cell lymphoma (BCL)6 and Bcl-xL are expressed in germinal center (GC) B cells and enable them to endure the proliferative and mutagenic environment of the GC. By introducing these genes into peripheral blood memory B cells and culturing these cells with factors produced by follicular helper T cells, CD40L and IL-21, we convert them to highly proliferating, cell surface BCR positive, Ig-secreting B cells with features of GC B cells including expression of activation-induced cytidine deaminase. We generated cloned lines of B cells specific for respiratory syncytial virus and used these cells as a source of antibodies that effectively neutralized this virus in vivo. This method provides a new tool to study GC B cell biology, signal transduction through antigen-specific B cell receptors, and for the rapid generation of high affinity human monoclonal antibodies.
doi:10.1038/nm.2071
PMCID: PMC2861345  PMID: 20023635
17.  Short-term memory in gene induction reveals the regulatory principle behind stochastic IL-4 expression 
Combining experiments on primary T cells and mathematical modeling, we characterized the stochastic expression of the interleukin-4 cytokine gene in its physiologic context, showing that a two-step model of transcriptional regulation acting on chromatin rearrangement and RNA polymerase recruitment accounts for the level, kinetics, and population variability of expression.A rate-limiting step upstream of transcription initiation, but occurring at the level of an individual allele, controls whether the interleukin-4 gene is expressed during antigenic stimulation, suggesting that the observed stochasticity of expression is linked to the dynamics of chromatin rearrangement.The computational analysis predicts that the probability to re-express an interleukin-4 gene that has been expressed once is transiently increased. In support, we experimentally demonstrate a short-term memory for interleukin-4 expression at the predicted time scale of several days.The model provides a unifying framework that accounts for both graded and binary modes of gene regulation. Graded changes in expression level can be achieved by controlling transcription initiation, whereas binary regulation acts at the level of chromatin rearrangement and is targeted during the differentiation of T cells that specialize in interleukin-4 production.
Cell populations are typically heterogeneous with respect to protein expression even when clonally derived from a single progenitor. In bacteria and yeast, such heterogeneity has been shown to be due to intrinsically stochastic dynamics of gene expression (Raj and van Oudenaarden, 2008). Thus, cross-population heterogeneity may be an unavoidable by-product of random fluctuations in molecular interactions (Raser and O'Shea, 2004; Pedraza and van Oudenaarden, 2005). The phenotypic variability deriving from it may also be beneficial for cell function, differentiation, or adaptation to changing environments (Chang et al, 2008; Feinerman et al, 2008; Losick and Desplan, 2008). However, little is known about how gene-expression variability is caused in mammalian cells.
Two principal modes of gene regulation have been identified: graded and binary. In the graded mode, transcriptional regulators can tune the level of a gene product in a continuous manner (Hazzalin and Mahadevan, 2002). In the binary mode, the gene is expressed at an invariant level, whereas its probability of being expressed in a given cell is regulated, so that the gene has discrete ‘on' and ‘off' states (Walters et al, 1995; Hume, 2000; Biggar and Crabtree, 2001). In humans and mice, cytokine genes are expressed in a binary manner (Bix and Locksley, 1998; Riviere et al, 1998; Hu-Li et al, 2001; Apostolou and Thanos, 2008). A particularly well-studied case is the interleukin-4 (il4) gene that is critical for antibody-based immune responses. This gene is expressed by antigen-stimulated T cells initially with low probability, so that in most IL-4-positive cells only one allele is active (Bix and Locksley, 1998; Riviere et al, 1998). The expressed allele is not imprinted but chosen stochastically during each cell stimulation (Hu-Li et al, 2001).
Here, we have studied the dynamics of IL-4 expression quantitatively. Primary murine CD4+ T cells have been differentiated uniformly into type-2 T-helper (Th2) cells that express the lineage-specifying transcription factor (TF) Gata-3 and are competent to activate the il4 gene upon challenge with antigen. Using T cells heterozygous for an il4 wild-type allele and an il4 allele with GFP knock-in after the promoter, the alleles are found to be expressed stochastically and in an uncorrelated manner (Figure 2A; Hu-Li et al, 2001). To account for the observed stochastic dynamics of IL-4 expression, we considered a basic model of gene transcription, mRNA translation, turnover, and protein secretion (Figure 2B). However, our experimental estimates of the intracellular life times of IL-4 mRNA and protein (∼1 h) and their absolute numbers (mRNA∼103, protein∼105) rule out random fluctuations in transcription, translation as well as mRNA and protein turnover as an explanation for the observed stochastic properties of IL-4 expression (Thattai and van Oudenaarden, 2001; Paulsson, 2004).
As il4 is known to be strongly regulated at the chromatin level (Ansel et al, 2006), we included in the model a reversible step of chromatin opening that is permissive for transcription (Figure 2C and D). Both chromatin opening and transcription initiation are driven by TFs that are transiently activated during the antigen stimulus, with NFAT1 playing a prominent role (Agarwal et al, 2000; Avni et al, 2002; Guo et al, 2004). The model accounts for the kinetics of NFAT1 TF activity (Figure 2E) (Loh et al, 1996). Using a best-fit procedure for estimating the kinetics of the chromatin transition and TF activity from experimental data, we found that the model accurately reproduces the distribution of IL-4 expression within the cell population over the entire time course of a stimulation (Figure 3A). At the same time, it accounts for the measured kinetics of IL-4 mRNA, intracellular and secreted protein (Figure 3B). Additional data show that the model can also explain IL-4 expression at different stages of Th2 differentiation and upon pharmacological inhibition of NFAT1 activity. In each case, the model predicts a slow and stochastic chromatin opening (Step 1 in Figure 2C) that is the limiting step for the activation of the gene.
The slowness of chromatin opening inferred by the model implies an extended lifetime of the open chromatin state (several days), which lasts longer than TF activity during antigenic stimulation (several hours). This indicates that acute IL-4 expression is terminated by the cessation of TF activity (Step 2 in Figure 2C), rather than by the closing of the chromatin (Step 1). In support of this prediction, we observed an elevated fraction of IL-4-producing cells after secondary stimulations administered within a few days of the primary stimulus. Consistent with the model, this elevation disappeared with a half-life of ∼3 days (Figure 4B). To test whether this ‘short-term memory' for activation of the il4 gene is indeed due to the IL-4 producers in the primary stimulation, we sorted stimulated Th2 cells into viable IL-4-producing and non-producing fractions using the cytokine secretion assay (Ouyang et al, 2000) and cultured them separately for different resting periods. The probability of IL-4 re-expression in the positive-sorted cells was consistently larger than in negative-sorted cells and decreased progressively over several days (Figure 4C). By contrast, the sorted IL-4 negative cells exhibited a constant induction probability indistinguishable from the unsorted population. This behavior was not due to differential cell proliferation in the sorted populations or different success of Th2 differentiation. Moreover, using heterozygous il4-wild-type/il4-gfp cells, and sorting for expression of the wild-type allele, we observed that expression of the il4-gfp allele was similar in IL-4-positive and negative sorted fractions. Taken together, these findings imply that stochastic, slow chromatin changes at individual il4 genes govern the binary expression pattern of this cytokine.
In conclusion, we propose an experimentally based model of inducible gene expression where strong stochasticity arises from slow (hours to days) chromatin opening and closing transitions, rather than being due to small numbers of mRNA or protein molecules or transcriptional bursting (Raj et al, 2006). This rate-limiting step upstream of transcription initiation (which may entail several interacting epigenetic processes) naturally gives rise to a binary expression pattern of the gene. By contrast, regulation at the level of transcription initiation can have a graded effect on the expression level. We provide evidence that both binary and graded regulation can occur for the il4 gene. Physiological regulation of il4 seems to be mainly binary, thus enabling a dose–response within a population while producing an unequivocal all-or-none signal at the single-cell level.
Although cell-to-cell variability has been recognized as an unavoidable consequence of stochasticity in gene expression, it may also serve a functional role for tuning physiological responses within a cell population. In the immune system, remarkably large variability in the expression of cytokine genes has been observed in homogeneous populations of lymphocytes, but the underlying molecular mechanisms are incompletely understood. Here, we study the interleukin-4 gene (il4) in T-helper lymphocytes, combining mathematical modeling with the experimental quantification of expression variability and critical parameters. We show that a stochastic rate-limiting step upstream of transcription initiation, but acting at the level of an individual allele, controls il4 expression. Only a fraction of cells reaches an active, transcription-competent state in the transient time window determined by antigen stimulation. We support this finding by experimental evidence of a previously unknown short-term memory that was predicted by the model to arise from the long lifetime of the active state. Our analysis shows how a stochastic mechanism acting at the chromatin level can be integrated with transcriptional regulation to quantitatively control cell-to-cell variability.
doi:10.1038/msb.2010.13
PMCID: PMC2872609  PMID: 20393579
cytokines; cytokine secretion assay; epigenetic regulation; gene expression; stochastic model
18.  SiPaGene: A new repository for instant online retrieval, sharing and meta-analyses of GeneChip® expression data 
BMC Genomics  2009;10:98.
Background
Microarray expression profiling is becoming a routine technology for medical research and generates enormous amounts of data. However, reanalysis of public data and comparison with own results is laborious. Although many different tools exist, there is a need for more convenience and online analysis with restriction of access and user specific sharing options. Furthermore, most of the currently existing tools do not use the whole range of statistical power provided by the MAS5.0/GCOS algorithms.
Description
With a current focus on immunology, infection, inflammation, tissue regeneration and cancer we developed a database platform that can load preprocessed Affymetrix GeneChip expression data for immediate access. Group or subgroup comparisons can be calculated online, retrieved for candidate genes, transcriptional activity in various biological conditions and compared with different experiments. The system is based on Oracle 9i with algorithms in java and graphical user interfaces implemented as java servlets. Signals, detection calls, signal log ratios, change calls and corresponding p-values were calculated with MAS5.0/GCOS algorithms. MIAME information and gene annotations are provided via links to GEO and EntrezGene. Users access via https protocol their own, shared or public data. Sharing is comparison- and user-specific with different levels of rights. Arrays for group comparisons can be selected individually. Twenty-two different group comparison parameters can be applied in user-defined combinations on single or multiple group comparisons. Identified genes can be reviewed online or downloaded. Optimized selection criteria were developed and reliability was demonstrated with the "Latin Square" data set. Currently more than 1,000 arrays, 10,000 pairwise comparisons and 500 group comparisons are presented with public or restricted access by different research networks or individual users.
Conclusion
SiPaGene is a repository and a high quality tool for primary analysis of GeneChips. It exploits the MAS5.0/GCOS pairwise comparison algorithm, enables restricted access and user specific sharing. It does not aim for a complete representation of all public arrays but for high quality analysis with stepwise integration of reference signatures for detailed meta-analyses. Development of additional tools like functional annotation networks based on expression information will be future steps towards a systematic biological analysis of expression profiles.
doi:10.1186/1471-2164-10-98
PMCID: PMC2657156  PMID: 19265543
19.  Autoregulation of Th1-mediated inflammation by twist1 
The Journal of Experimental Medicine  2008;205(8):1889-1901.
The basic helix-loop-helix transcriptional repressor twist1, as an antagonist of nuclear factor κB (NF-κB)–dependent cytokine expression, is involved in the regulation of inflammation-induced immunopathology. We show that twist1 is expressed by activated T helper (Th) 1 effector memory (EM) cells. Induction of twist1 in Th cells depended on NF-κB, nuclear factor of activated T cells (NFAT), and interleukin (IL)-12 signaling via signal transducer and activator of transcription (STAT) 4. Expression of twist1 was transient after T cell receptor engagement, and increased upon repeated stimulation of Th1 cells. Imprinting for enhanced twist1 expression was characteristic of repeatedly restimulated EM Th cells, and thus of the pathogenic memory Th cells characteristic of chronic inflammation. Th lymphocytes from the inflamed joint or gut tissue of patients with rheumatic diseases, Crohn's disease or ulcerative colitis expressed high levels of twist1. Expression of twist1 in Th1 lymphocytes limited the expression of the cytokines interferon-γ, IL-2, and tumor necrosis factor-α, and ameliorated Th1-mediated immunopathology in delayed-type hypersensitivity and antigen-induced arthritis.
doi:10.1084/jem.20072468
PMCID: PMC2525589  PMID: 18663125
20.  Long-lived virus-reactive memory T cells generated from purified cytokine-secreting T helper type 1 and type 2 effectors 
Many vaccination strategies and immune cell therapies aim at increasing the numbers of memory T cells reactive to protective antigens. However, the differentiation lineage and therefore the optimal generation conditions of CD4 memory cells remain controversial. Linear and divergent differentiation models have been proposed, suggesting CD4 memory T cell development from naive precursors either with or without an effector-stage intermediate, respectively. Here, we address this question by using newly available techniques for the identification and isolation of effector T cells secreting effector cytokines. In adoptive cell transfers into normal, nonlymphopenic mice, we show that long-lived virus-specific memory T cells can efficiently be generated from purified interferon γ–secreting T helper (Th) type 1 and interleukin (IL)-4– or IL-10–secreting Th2 effectors primed in vitro or in vivo. Importantly, such effector-derived memory T cells were functional in viral challenge infections. They proliferated vigorously, rapidly modulated IL-7 receptor expression, exhibited partial stability and flexibility of their cytokine patterns, and exerted differential effects on virus-induced immunopathology. Thus, cytokine-secreting effectors can evade activation-induced cell death and develop into long-lived functional memory cells. These findings demonstrate the efficiency of linear memory T cell differentiation and encourage the design of vaccines and immune cell therapies based on differentiated effector T cells.
doi:10.1084/jem.20071855
PMCID: PMC2234365  PMID: 18195073
21.  Digital NFATc2 Activation per Cell Transforms Graded T Cell Receptor Activation into an All-or-None IL-2 Expression 
PLoS ONE  2007;2(9):e935.
The expression of interleukin-2 (IL-2) is a key event in T helper (Th) lymphocyte activation, controlling both, the expansion and differentiation of effector Th cells as well as the activation of regulatory T cells. We demonstrate that the strength of TCR stimulation is translated into the frequency of memory Th cells expressing IL-2 but not into the amount of IL-2 per cell. This molecular switch decision for IL-2 expression per cell is located downstream of the cytosolic Ca2+ level. Here we show that in a single activated Th cell, NFATc2 activation is digital but NF-κB activation is graded after graded T cell receptor (TCR) signaling. Subsequently, NFATc2 translocates into the nucleus in an all-or-none fashion per cell, transforming the strength of TCR-stimulation into the number of nuclei positive for NFATc2 and IL-2 transcription. Thus, the described NFATc2 switch regulates the number of Th cells actively participating in an immune response.
doi:10.1371/journal.pone.0000935
PMCID: PMC1978524  PMID: 17895976
22.  Selecting B cells and plasma cells to memory 
Humoral immunity appears to be based on immunological memory provided by memory plasma cells, which secrete protective antibodies, and memory B cells, which react to antigen challenge by differentiating into plasma cells. How these differentiation pathways relate to each other, how cells are selected into these memory populations, and how these populations are maintained remains enigmatic.
doi:10.1084/jem.20050218
PMCID: PMC2213048  PMID: 15728231
23.  The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4+CD25+ T cells 
Arthritis Research & Therapy  2005;7(2):R291-R301.
It is now generally accepted that CD4+CD25+ Treg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4+CD25+ cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4+CD25+ cells in the inflamed joint as compared with CD4+CD25- cells. These data imply a significant role for CD4+CD25+ Treg cells in the control of chronic arthritis. However, transferred Treg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of Treg cell transfer in potential therapeutic applications.
doi:10.1186/ar1484
PMCID: PMC1065322  PMID: 15743476
arthritis; regulatory T cells
24.  Short-lived Plasmablasts and Long-lived Plasma Cells Contribute to Chronic Humoral Autoimmunity in NZB/W Mice 
The Journal of Experimental Medicine  2004;199(11):1577-1584.
The current view holds that chronic autoimmune diseases are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs designed to interfere with this activation the production of autoantibodies often persists and contributes to progression of the immunopathology. In the present study, we analyzed the life span of (auto)antibody-secreting cells in the spleens of NZB × NZW F1 (NZB/W) mice, a murine model of systemic lupus erythematosus. The number of splenic ASCs increased in mice aged 1–5 mo and became stable thereafter. Less than 60% of the splenic (auto)antibody-secreting cells were short-lived plasmablasts, whereas 40% were nondividing, long-lived plasma cells with a half-life of >6 mo. In NZB/W mice and D42 Ig heavy chain knock-in mice, a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce (auto)antibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases.
doi:10.1084/jem.20040168
PMCID: PMC2211779  PMID: 15173206
plasma cell; autoimmunity; SLE; antibodies; anti-DNA
25.  CD152 (CTLA-4) Determines the Unequal Resistance of Th1 and Th2 Cells against Activation-induced Cell Death by a Mechanism Requiring PI3 Kinase Function 
Survival of antigen-experienced T cells is essential for the generation of adaptive immune responses. Here, we show that the genetic and antibody-mediated inactivation of CD152 (cytotoxic T lymphocyte antigen 4) in T helper (Th) effector cells reduced the frequency of nonapoptotic cells in a completely Fas/Fas ligand (FasL)–dependent manner. CD152 cross-linking together with stimulation of CD3 and CD28 on activated Th2 cells prevented activation-induced cell death (AICD) as a result of reduced Fas and FasL expression. Apoptosis protection conferred by CD152 correlated with the up-regulation of Bcl-2 and was mediated by phosphatidylinositol 3 kinase, which prevented FasL expression through the inhibitory phosphorylation of Forkhead transcription factor FKHRL1. We show that signals induced by CD152 act directly on activated T lymphocytes and, due to its differential surface expression on activated Th1 and Th2 cells, induce resistance to AICD mainly in Th2 cells.
doi:10.1084/jem.20031058
PMCID: PMC2212725  PMID: 15007096
costimulation; apoptosis; survival; signal transduction; FasL

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