Jorg van Loosdregt

Functional human regulatory T cells fail to control autoimmune inflammation due to PKB/c-akt hyperactivation in effector cells

During the last decade research has focused on the application of FOXP3(+) regulatory T cells (Tregs) in the treatment of autoimmune disease. However, thorough functional characterization of these cells in patients with chronic autoimmune disease, especially at the site of inflammation, is still missing. Here we studied Treg function in patients with juvenile idiopathic arthritis (JIA) and observed that Tregs from the peripheral blood as well as the inflamed joints are fully functional. Nevertheless, Treg-mediated suppression of cell proliferation and cytokine production by effector cells from the site of inflammation was severely impaired, because of resistance to suppression. This resistance to suppression was not caused by a memory phenotype of effector T cells or activation status of antigen presenting cells. Instead, activation of protein kinase B (PKB)/c-akt was enhanced in inflammatory effector cells, at least partially in response to TNFα and IL-6, and inhibition of this kinase restored responsiveness to suppression. We are the first to show that PKB/c-akt hyperactivation causes resistance of effector cells to suppression in human autoimmune disease. Furthermore, these findings suggest that for a Treg enhancing strategy to be successful in the treatment of autoimmune inflammation, resistance because of PKB/c-akt hyperactivation should be targeted as well.

Rapid Temporal Control of Foxp3 Protein Degradation by Sirtuin-1

Maintenance of Foxp3 protein expression in regulatory T cells (Treg) is crucial for a balanced immune response. We have previously demonstrated that Foxp3 protein stability can be regulated through acetylation, however the specific mechanisms underlying this observation remain unclear. Here we demonstrate that SIRT1 a member of the lysine deacetylase Sirtuin (SIRT) family, but not the related SIRTs 2–7, co-localize with Foxp3 in the nucleus. Ectopic expression of SIRT1, but not SIRTs 2–7 results in decreased Foxp3 acetylation, while conversely inhibition of endogenous SIRT activity increased Foxp3 acetylation. We show that SIRT1 inhibition decreases Foxp3 poly-ubiquitination, thereby increasing Foxp3 protein levels. Co-transfection of SIRT1 with Foxp3 results in increased Foxp3 proteasomal degradation, while SIRT inhibition increases FOXP3 transcriptional activity in human Treg. Taken together, these data support a central role for SIRT1 in the regulation of Foxp3 protein levels and thereby in regulation of Treg suppressive capacity. Pharmacological modulation of SIRT1 activity in Treg may therefore provide a novel therapeutic strategy for controlling immune responses.

Ex Vivo–Expanded but Not In Vitro–Induced Human Regulatory T Cells Are Candidates for Cell Therapy in Autoimmune Diseases Thanks to Stable Demethylation of the FOXP3 Regulatory T Cell–Specific Demethylated Region

Regulatory T cell (Treg) therapy is a promising approach for transplant rejection and severe autoimmunity. Unfortunately, clinically meaningful Treg numbers can be obtained only upon in vitro culture. Functional stability of human expanded (e)Tregs and induced (i)Tregs has not been thoroughly addressed for all proposed protocols, hindering clinical translation. We undertook a systematic comparison of eTregs and iTregs to recommend the most suitable for clinical implementation, and then tested their effectiveness and feasibility in rheumatoid arthritis (RA). Regardless of the treatment, iTregs acquired suppressive function and FOXP3 expression, but lost them upon secondary restimulation in the absence of differentiation factors, which mimics in vivo reactivation. In contrast, eTregs expanded in the presence of rapamycin (rapa) retained their regulatory properties and FOXP3 demethylation upon restimulation with no stabilizing agent. FOXP3 demethylation predicted Treg functional stability upon secondary TCR engagement. Rapa eTregs suppressed conventional T cell proliferation via both surface (CTLA-4) and secreted (IL-10, TGF-β, and IL-35) mediators, similarly to ex vivo Tregs. Importantly, Treg expansion with rapa from RA patients produced functionally stable Tregs with yields comparable to healthy donors. Moreover, rapa eTregs from RA patients were resistant to suppression reversal by the proinflammatory cytokine TNF-α, and were more efficient in suppressing synovial conventional T cell proliferation compared with their ex vivo counterparts, suggesting that rapa improves both Treg function and stability. In conclusion, our data indicate Treg expansion with rapa as the protocol of choice for clinical application in rheumatological settings, with assessment of FOXP3 demethylation as a necessary quality control step.

Early adipogenesis is regulated through USP7-mediated deubiquitination of the histone acetyltransferase TIP60

Transcriptional coregulators, including the acetyltransferase Tip60, have a key role in complex cellular processes such as differentiation. Whereas post-translational modifications have emerged as an important mechanism to regulate transcriptional coregulator activity, the identification of the corresponding demodifying enzymes has remained elusive. Here we show that the expression of the Tip60 protein, which is essential for adipocyte differentiation, is regulated through polyubiquitination on multiple residues. USP7, a dominant deubiquitinating enzyme in 3T3-L1 adipocytes and mouse adipose tissue, deubiquitinates Tip60 both in intact cells and in vitro and increases Tip60 protein levels. Furthermore, inhibition of USP7 expression and activity decreases adipogenesis. Transcriptome analysis reveals several cell cycle genes to be co-regulated by both Tip60 and USP7. Knockdown of either factor results in impaired mitotic clonal expansion, an early step in adipogenesis. These results reveal deubiquitination of a transcriptional coregulator to be a key mechanism in the regulation of early adipogenesis.

A circulating reservoir of pathogenic-like CD4+ T cells shares a genetic and phenotypic signature with the inflamed synovial micro-environment

Objectives Systemic immunological processes are profoundly shaped by the micro-environments where antigen recognition occurs. Identifying molecular signatures distinctive of such processes is pivotal to understand pathogenic immune responses and manipulate them for therapeutic purposes. Unfortunately, direct investigation of peripheral tissues, enriched in pathogenic T cells, is often impossible or imposingly invasive in humans. Conversely, blood is easily accessible, but pathogenic signatures are diluted systemically as a result of the strict compartmentalisation of immune responses. In this work, we aimed at defining immune mediators shared between the bloodstream and the synovial micro-environment, and relevant for disease activity in autoimmune arthritis. Methods CD4+ T cells from blood and synovium of patients with juvenile idiopathic arthritis (JIA) were immunophenotyped by flow cytometry. The TCR repertoire of a circulating subset showing similarity with the synovium was analysed through next-generation sequencing of TCR β-chain CDR3 to confirm enrichment in synovial clonotypes. Finally, clinical relevance was established by monitoring the size of this subset in the blood of patients with JIA and rheumatoid arthritis (RA). Results We identified a small subset of circulating CD4+ T cells replicating the phenotypical signature of lymphocytes infiltrating the inflamed synovium. These circulating pathogenic-like lymphocytes (CPLs) were enriched in synovial clonotypes and they exhibited strong production of pro-inflammatory cytokines. Importantly, CPLs were expanded in patients with JIA, who did not respond to therapy, and also correlated with disease activity in patients with RA. Conclusions CPLs provide an accessible reservoir of pathogenic cells recirculating into the bloodstream and correlating with disease activity, to be exploited for diagnostic and research purposes.

Post-translational modification networks regulating FOXP3 function

Forkhead box (FOX)P3 is a requisite transcription factor for the development and maintenance of immunosuppressive function of regulatory T (Treg) cells, and therefore for immune homeostasis. Post-translational modifications (PTMs) can transiently alter the functionality of transcription factors, and recent evidence reveals that FOXP3 can be regulated by various PTMs including acetylation, ubiquitination, and phosphorylation. Here, we review the current understanding of how these modifications control FOXP3, including regulation of DNA binding, transactivation potential, and proteasomal degradation. We place these findings in the context of the biology of Treg cells, and discuss both limitations in translating biochemical findings into in vivo functions and the opportunities presented by a better understanding of the molecular mechanisms that can transiently control FOXP3 activity in response to environmental cues.

Hydroxychloroquine preferentially induces apoptosis of CD45RO+ effector T cells by inhibiting autophagy: A possible mechanism for therapeutic modulation of T cells

Although hydroxychloroquine is used for treatment of numerous autoimmune disorders the mechanism is unclear. We here demonstrate that hydroxychloroquine preferentially induces apoptosis of CD45RO+ memory and effector T cells by inhibiting the survival pathway of autophagy.

Increased autophagy in CD4(+) T cells of rheumatoid arthritis patients results in T-cell hyperactivation and apoptosis resistance.

Rheumatoid arthritis (RA) is an autoimmune disease hallmarked by aberrant cellular homeostasis, resulting in hyperactive CD4+ T cells that are more resistant to apoptosis. Both hyperactivation and resistance to apoptosis may contribute to the pathogenicity of CD4+ T cells in the autoimmune process. A better knowledge of the mechanisms determining such impaired homeostasis could contribute significantly to both the understanding and the treatment of the disease. Here we investigated whether autophagy, is dysregulated in CD4+ T cells of RA patients, resulting in disturbed T‐cell homeostasis. We demonstrate that the rate of autophagy is significantly increased in CD4+ T cells from RA patients, and that increased autophagy is also a feature of in vitro activated CD4+ T cells. The increased apoptosis resistance observed in CD4+ T cells from RA patients was significantly reversed upon autophagy inhibition. These mechanisms may contribute to RA pathogenesis, as autophagy inhibition reduced both arthritis incidence and disease severity in a mouse collagen induced arthritis mouse model. Conversely, in Atg5flox/flox‐CD4‐Cre+ mice, in which all T cells are autophagy deficient, T cells showed impaired activation and proliferation. These data provide novel insight into the pathogenesis of RA and underscore the relevance of autophagy as a promising therapeutic target.

A sensitive protocol for FOXP3 epigenetic analysis in scarce human samples

CD4+CD25high regulatory T (Treg) cells are key players in the maintenance of peripheral immune tolerance [1]. Stable expression of the FOXP3 transcription factor is essential for Treg cells’ ability to suppress the immune responses of conventional T (Tconv) cells [2]. FOXP3 stability in murine Treg cells has been linked to FOXP3 locus demethylation at the CNS2 [3, 4], also called the Treg-specific demethylated region (TSDR) [5]. In contrast to FOXP3 expression [6, 7], complete demethylation of the TSDR [3], in addition to other genomic regions [8], is a true hallmark of human and mouse Treg cells, and its role in the early Treg lineage specification is currently under intense scrutiny in the mouse [3, 8, 9]. Unfortunately, the investigation of TSDR demethylation to define whether FOXP3+ T cells are bona fide Treg cells in translational research settings has so far been inaccessible due to limitations in clinical sample amount. Indeed, methods currently available to inspect the TSDR at single-CpG resolution [5] lack sensitivity due to bottlenecks at the amplification stage (Fig. 1A). Other methods originally aimed at counting Treg cells in unsegregated populations [10] can be adapted to study the regulatory lineage commitment of sorted cells. However, because these methods separately amplify methylated and unmethylated TSDRs, the sample amount requirement is a function of the degree of TSDR methylation, growing exponentially at the two ends of the range, where one of the two species becomes limiting. In this work, we report an inexpensive, single-CpG resolution, PCR-based protocol with very low requirements on sample amount and robust to aldehyde-based fixation, features making it of immediate relevance for sample-limited research settings. Our optimized protocol enhances the sensitivity of existing techniques [5] while retaining specificity. We introduced nested PCR, touchdown preamplification, primer tailing, and a two-step sequencing cycle (detailed protocol available as Supporting Information). The nested PCR targets 15 commonly investigated CpGs sites [5, 10], thereby allowing for direct comparison of results with previous literature. However, we found that measurements of the first CpG dinucleotide are typically noisier due to proximity to the sequencing primer, and we recommend excluding this first CpG dinucleotide, unless several technical replicates are performed. The touchdown preamplification step was introduced not to compromise specificity with the enhanced sensitivity. Moreover, we added 5′ tails to the inner primers in order to (i) increase their length, which allows raising the annealing temperature, thereby minimizing spurious amplification; (ii) introduce C and G nucleotides, which are rarer in amplicons from bisulphiteconverted templates, thereby increasing DNA complexity and, consequently, the specificity of primer annealing; (iii) extend the amplicon at the 5′ end, which allows to shift the sequencing primer upstream, thereby improving base resolution at the 5′ end; and (iv) allow the sequencing primer to anneal only to the inner-PCR product. Finally, the two-step sequencing cycle yields a better signal balance when compared with the standard three-step denaturation/annealing/extension cycle. Electropherograms were analyzed with ESME to quantitatively determine the methylated/unmethylated CpG ratio [11]. We favored Sanger sequencing for its widespread availability, but pyrosequencing is also an attractive option. Our method was able to successfully generate TSDR amplicons from much lower amounts of template than singleround PCR techniques (Fig. 1A). The TSDR methylation profiles of CD25low/− Tconv cells and CD25highCD127low/− Treg cells generated by our protocol are in line with published data [5, 8–10], demonstrating its accuracy (i.e. proximity to the true value) in measuring highly methylated (Tconv cells), highly demethylated (male Treg cells), and partially demethylated (female Treg cells, due to FOXP3 methylation on the inactive X chromosome) samples (Fig. 1B). Accuracy was linearly preserved across the whole range of TSDR methylation, indicating no amplification bias of either methylated or unmethylated TSDR templates (Fig. 1C). In addition, to carefully characterize the lower limit of detection, we calculated the percentage error (a normalized measure of the difference between observed and expected value, i.e. an estimate of accuracy) and the coefficient of variation (a normalized measure of data dispersion around the mean, i.e. an estimate of precision) of technical replicates across decreasing amounts of template gDNA. We were able to amplify down to 1.25 ng bisulphiteconverted gDNA with 100% success rates

Acetylation of C/EBPε is a prerequisite for terminal neutrophil differentiation

C/EBPε, a member of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, is exclusively expressed in myeloid cells and regulates transition from the promyelocytic stage to the myelocytic stage of neutrophil development, being indispensable for secondary and tertiary granule formation. Knowledge concerning the functional role of C/EBPε posttranslational modifications is limited to studies concerning phosphorylation and sumoylation. In the current study, using ectopic expression and ex vivo differentiation of CD34(+) hematopoietic progenitor cells, we demonstrate that C/EBPε is acetylated, which was confirmed by mass spectrometry analysis, identifying 4 acetylated lysines in 3 distinct functional domains. Regulation of C/EBPε acetylation levels by the p300 acetyltransferase and the sirtuin 1 deacetylase controls transcriptional activity, which can at least in part be explained by modulation of DNA binding. During neutrophil development, acetylation of lysines 121 and 198 were found to be crucial for terminal neutrophil differentiation and the expression of neutrophil-specific granule proteins, including lactoferrin and collagenase. Taken together, our data illustrate a critical role for acetylation in the functional regulation of C/EBPε activity during terminal neutrophil development.