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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The delineation of the in vivo role of GATA-3 in human T cell differentiation is a critical step in the understanding of molecular mechanisms directing human immune responses. We examined T cell differentiation and T cell–mediated effector functions in individuals lacking one functional GATA-3 allele. CD4 T cells from GATA-3+/− individuals expressed significantly reduced levels of GATA-3, associated with markedly decreased T helper cell (Th)2 frequencies in vivo and in vitro. Moreover, Th2 cell–mediated effector functions, as assessed by serum levels of Th2-dependent immunoglobulins (Igs; IgG4, IgE), were dramatically decreased, whereas the Th1-dependent IgG1 was elevated compared with GATA-3+/+ controls. Concordant with these data, silencing of GATA-3 in GATA-3+/+ CD4 T cells with small interfering RNA significantly reduced Th2 cell differentiation. Moreover, GATA-3 mRNA levels increased under Th2-inducing conditions and decreased under Th1-inducing conditions. Taken together, the data strongly suggest that GATA-3 is an important transcription factor in regulating human Th2 cell differentiation in vivo.

The deciphering of the sequence of the human genome has raised the expectation of unravelling the specific role of each gene in physiology and pathology. High-throughput technologies for gene expression profiling provide the first practical basis for applying this information. In rheumatology, with its many diseases of unknown pathogenesis and puzzling inflammatory aspects, these advances appear to promise a significant advance towards the identification of leading mechanisms of pathology. Expression patterns reflect the complexity of the molecular processes and are expected to provide the molecular basis for specific diagnosis, therapeutic stratification, long-term monitoring and prognostic evaluation. Identification of the molecular networks will help in the discovery of appropriate drug targets, and permit focusing on the most effective and least toxic compounds. Current limitations in screening technologies, experimental strategies and bioinformatic interpretation will shortly be overcome by the rapid development in this field. However, gene expression profiling, by its nature, will not provide biochemical information on functional activities of proteins and might only in part reflect underlying genetic dysfunction. Genomic and proteomic technologies will therefore be complementary in their scientific and clinical application.

The studies performed to date analyzed the overall participation of the inducible costimulator (ICOS) in model diseases, but did not yield information on the nature and function of ICOS-expressing T cells in vivo. We examined ICOS+ T cells in the secondary lymphoid organs of nonmanipulated mice, in the context of an “unbiased” immune system shaped by environmental antigens. Using single cell analysis, ICOSlow cells were found to be loosely associated with the early cytokines interleukin (IL)-2, IL-3, IL-6, and interferon (IFN)-γ. ICOSmedium cells, the large majority of ICOS+ T cells in vivo, were very tightly associated with the synthesis of the T helper type 2 (Th2) cytokines IL-4, IL-5, and IL-13, and these cells exhibited potent inflammatory effects in vivo. In contrast, ICOShigh T cells were highly and selectively linked to the anti-inflammatory cytokine IL-10. Overall, these data seem to indicate that ICOS cell surface density serves as a regulatory mechanism for the release of cytokines with different immunological properties. Further in vivo functional experiments with in vitro–activated T cells strongly suggested that the ICOS+ population, although representing in vivo only around 10% of T cells bearing early or late activation markers, nevertheless encompasses virtually all effector T cells, a finding with major diagnostic and therapeutic implications.

During ageing thymic function declines and is unable to meet the demand for peripheral T helper (Th) cell replenishment. Therefore, population maintenance of naive Th cells must be at least partly peripherally based. Such peripheral postthymic expansion of recent thymic emigrants (RTEs) during ageing consequently should lead to loss or dilution of T cell receptor excision circles (TRECs) from a subset of naive T cells. We have identified two subsets of naive Th cells in human adult peripheral blood characterized by a striking unequal content of TRECs, indicating different peripheral proliferative histories. TRECs are highly enriched in peripheral naive CD45RA+ Th cells coexpressing CD31 compared with peripheral naive CD45RA+ Th cells lacking CD31 expression, in which TRECs can hardly be detected. Furthermore we show that CD31−CD45RA+ Th cells account for increasing percentages of the naive peripheral Th cell pool during ageing but retain phenotypic and functional features of naive Th cells. As CD31 is lost upon T cell receptor (TCR) engagement in vitro, we hypothesize that TCR triggering is a prerequisite for homeostatically driven peripheral postthymic expansion of human naive RTEs. We describe here the identification of peripherally expanded naive Th cells in human adult blood characterized by the loss of CD31 expression and a highly reduced TREC content.

Although interleukin (IL)-12 and IL-4 polarize naive CD4+ T cells toward T helper cell type 1 (Th1) or Th2 phenotypes, it is not known whether cytokines instruct the developmental fate in uncommitted progenitors or select for outgrowth of cells that have stochastically committed to a particular fate. To distinguish these instructive and selective models, we used surface affinity matrix technology to isolate committed progenitors based on cytokine secretion phenotype and developed retroviral-based tagging approaches to directly monitor individual progenitor fate decisions at the clonal and population levels. We observe IL-4–dependent redirection of phenotype in cells that have already committed to a non–IL-4–producing fate, inconsistent with predictions of the selective model. Further, retroviral tagging of naive progenitors with the Th2-specific transcription factor GATA-3 provided direct evidence for instructive differentiation, and no evidence for the selective outgrowth of cells committed to either the Th1 or Th2 fate. These data would seem to exclude selection as an exclusive mechanism in Th1/Th2 differentiation, and support an instructive model of cytokine-driven transcriptional programming of cell fate decisions.

Autoimmune diseases that are resistant to conventional treatment cause severe morbidity and even mortality. In the present study we demonstrate that complete remissions can be achieved in refractory polychondritis and systemic lupus erythematosus (SLE), even at advanced stage, with the use of autologous stem-cell transplantation (SCT). Remissions persisted after reconstitution of the immune system. In the treatment of advanced systemic sclerosis (SSc), stable disease may be achieved with autologous SCT.

Introduction:

Patients with persistently active autoimmune diseases are considered to be candidates for autologous SCT. We performed a phase 1/2 study in a limited number of patients who were refractory to conventional immunosuppressive treatment. Following a period of uncontrolled disease activity for at least 6 months, autologous SCT was performed, after in vivo immunoablation and ex vivo depletion of mononuclear cells.

Aims:

To investigate feasibility, toxicity and efficacy of the treatment, and the incidence of emergent infections.

Methods:

Seven patients (aged between 23 and 48 years) were included in the single-centre trial: one had relapsing polychondritis, three had treatment-refractory SLE and three patients had SSc. Stem-cell mobilization was achieved by treatment with moderate-dose cyclophosphamide (2 g/m2; in terms of myelotoxic side effects or myelosuppression) and granulocyte colony-stimulating factor (G-CSF). CD34- cells of the leukapheresis products were removed by high-gradient magnetic cell sorting. After stem-cell collection, immunoablation was performed with high-dose cyclophosphamide (200 mg/kg body weight) and antithymocyte globulin (ATG; 90 mg/kg body weight). Autologous SCT was followed by reconstitution of the immune system, which was monitored by six-parameter flow cytometry and standard serology. The trial fulfilled the European League Against Rheumatism (EULAR) and the European Group for Blood and Marrow Transplantation (EBMT) guidelines for blood and bone marrow stem-cell transplants in autoimmune disease.

Results:

Among the seven patients studied, the patient with relapsing polychondritis and the patients with SLE were successfully treated and remained in complete remission during a follow up of 10-21 months. Remission persisted despite reconstitution of the immune system, resulting in high numbers of effector-/memory-type T-helper lymphocytes and increasing populations in the naïve T-cell compartment. Before autologous SCT, one of the patients with SLE had a long-lasting secondary antiphospholipid syndrome, with high anticardiolipin antibodies and thromboembolic events. After autologous SCT the antiphospholipid antibodies became negative, and no thrombosis occurred during follow up. Two of the patients with SSc were unaffected by treatment with autologous SCT for 6 or 13 months. The other patient with SSc died 2 days after autologous SCT because of cardiac failure.

During stem-cell mobilization with G-CSF, flares of autoimmune disease were seen in the patient with polychondritis and in one patient with SLE. The strategy utilized for depletion of CD34- cells led to a reduction by 4.5-5 log of contaminating CD3+ cells in the transplant. T-cell add-back was required in the patient with polychondritis and in one patient with SLE to provide a dose of 1×104 CD3+ cells/kg body weight for the transplant.

Discussion:

In vivo immunoablation in combination with autologous SCT after ex vivo depletion of CD34- cells can block the autoimmune process in relapsing polychondritis or SLE without incidence of severe infections. The remissions were achieved in patients with advanced disease that was refractory to previous intensive immunosuppressive therapy. The present results do not indicate that large-scale contamination of the stem-cell transplant with autoreactive cells after selection for CD34+cells occurred. After the preparative regimen, the application of G-CSF was avoided, because induction of flares of the autoimmune disease were noticed during the mobilization of stem cells. In SSc patients, distinct remissions were not observable after autologous SCT; the serological and clinical status did not improve. Follow-up periods of more than 12 months may be required to identify successful treatment with autologous SCT in SSc patients. Among the various autoimmune diseases the efficacy of autologous SCT appears to be dependent on the underlying pathophysiology. The results of the present phase 1/2 study suggest that patients with advanced stage SSc should not be treated with autologous SCT, until the reasons for the lack of response and the possible mortality due to cardiac complications are identified. The observation of flares of autoimmune disease after application of G-CSF emphasizes the need for critical evaluation of the role of G-CSF in immunoablative regimens.

T helper (Th) lymphocytes, when reactivated, recall expression of those cytokines they had been instructed to express in earlier activations, even in the absence of specific cytokine-inducing factors. In cells that memorize their expression, the cytokine genes are modified by chromatin rearrangement and demethylation, suggesting that they have been somatically imprinted. Here we show, by using inhibitors blocking the cell cycle in various stages, that for the instruction of a Th cell to express interleukin (IL)-4 or IL-10 upon restimulation, entry of the cell into the S phase of the first cell cycle after initial activation is required. Separation of the IL-4 receptor (IL-4R) and T cell antigen receptor (TCR) signals in time, demonstrates that this instruction is dependent on concomitant signaling from both receptors. In Th cells, inhibited to progress into the first S phase after activation, the IL-4R and TCR signals can be memorized for at least 1 d, priming the T cell to become instructed for expression of IL-4 upon restimulation, when entering the S phase after release of the cell cycle block. The requirement of the initial S phase of T cell activation, for instruction of Th cells to express IL-4 or IL-10 upon restimulation points to the decisive role of epigenetic modification of cytokine genes as a molecular correlate of the memory to express particular cytokines.

Antibody class switching is mediated by somatic recombination between switch regions of the immunoglobulin heavy chain gene locus. Targeting of recombination to particular switch regions is strictly regulated by cytokines through the induction of switch transcripts starting 5′ of the repetitive switch regions. However, switch transcription as such is not sufficient to target switch recombination. This has been shown in mutant mice, in which the I-exon and its promoter upstream of the switch region were replaced with heterologous promoters. Here we show that, in the murine germline targeted replacement of the endogenous γ1 promoter, I-exon, and I-exon splice donor site by heterologous promoter and splice donor sites directs switch recombination in activated B lymphocytes constitutively to the γ1 switch region. In contrast, switch recombination to IgG1 is inhibited in mutant mice, in which the replacement does not include the heterologous splice donor site. Our data unequivocally demonstrate that targeting of switch recombination to IgG1 in vivo requires processing of the Iγ1 switch transcripts. Either the processing machinery or the processed transcripts are involved in class switch recombination.