Maura Rossetti

Enforced IL-10 Expression Confers Type 1 Regulatory T Cell (Tr1) Phenotype and Function to Human CD4+ T Cells

Type 1 regulatory T (Tr1) cells are an inducible subset of CD4+ Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4+ T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP+ LV-IL-10-transduced human CD4+ T (CD4LV-IL-10) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4LV-IL-10 T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4LV-IL-10 T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4+ T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.Type 1 regulatory T (Tr1) cells are an inducible subset of CD4(+) Tr cells characterized by high levels of interleukin (IL)-10 production and regulatory properties. Several protocols to generate human Tr1 cells have been developed in vitro. However, the resulting population includes a significant fraction of contaminating non-Tr1 cells, representing a major bottleneck for clinical application of Tr1 cell therapy. We generated an homogeneous IL-10-producing Tr1 cell population by transducing human CD4(+) T cells with a bidirectional lentiviral vector (LV) encoding for human IL-10 and the marker gene, green fluorescent protein (GFP), which are independently coexpressed. The resulting GFP(+) LV-IL-10-transduced human CD4(+) T (CD4(LV-IL-10)) cells expressed, upon T-cell receptor (TCR) activation, high levels of IL-10 and concomitant low levels of IL-4, and markers associated with IL-10. Moreover, CD4(LV-IL-10) T cells displayed typical Tr1 features: the anergic phenotype, the IL-10, and transforming growth factor (TGF)-β dependent suppression of allogeneic T-cell responses, and the ability to suppress in a cell-to-cell contact independent manner in vitro. CD4(LV-IL-10) T cells were able to control xeno graft-versus-host disease (GvHD), demonstrating their suppressive function in vivo. These results show that constitutive over-expression of IL-10 in human CD4(+) T cells leads to a stable cell population that recapitulates the phenotype and function of Tr1 cells.

Granulocyte-colony stimulating factor drives the in vitro differentiation of human dendritic cells that induce anergy in naïve T cells.

G‐CSF is a modulator of T‐cell and DC functions. Previous reports show that monocytes from G‐CSF‐treated (post‐G) healthy donors differentiate into tolerogenic DC in vitro in the presence of autologous serum, containing high levels of IL‐10 and IFN‐α, and in turn induce type 1 Treg (Tr1) cells. However, the direct effect of G‐CSF on DC differentiation was not investigated. Here, we show that monocytes differentiated in the presence of exogenous G‐CSF (G‐DC) remain CD14+CD1a−, but acquire a DC‐like morphology, express CD83 and CD86 and low levels of the tolerogenic markers Ig‐like transcript (ILT)4 and HLA‐G. G‐DC spontaneously produce IL‐10 and, upon stimulation, low levels of IL‐12. G‐DC display low stimulatory capacity and induce anergy in naïve T cells, but do not confer suppressive function. Therefore, in vitro differentiation of monocyte‐derived DC in the presence of G‐CSF can replicate some but not all features of post‐G DC. These findings indicate that the tolerogenic properties of G‐CSF do not exclusively reside in its direct effect on DC, which in turn induce T‐cell anergy, but also in its ability to generate a tolerogenic milieu in vivo, which is necessary for Tr1 cell induction and cannot be replicated in vitro.

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.

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.

HIV-1-Derived Lentiviral Vectors Directly Activate Plasmacytoid Dendritic Cells, Which in Turn Induce the Maturation of Myeloid Dendritic Cells

Lentiviral vectors (LV) can induce type I interferon (IFN I) production from murine plasmacytoid dendritic cells (pDC), but not myeloid (my)DC. Here, we investigated whether this mechanism is conserved in human DC. MyDC and pDC were isolated from peripheral blood and transduced with increasing vector concentrations. Compared with in vitro differentiated monocyte-derived DC, the transduction efficiency of peripheral blood DC was low (ranging from <1% to 45%), with pDC showing the lowest susceptibility to LV transduction. Phenotype and function of myDC were not directly modified by LV transduction; by contrast, pDC produced significant levels of IFN-α and tumor necrosis factor-α. pDC activation was dependent on functional vector particles and was mediated by Toll-like receptor 7/9 triggering. Coculture of myDC with pDC in the presence of LV resulted in myDC activation, with CD86 up-regulation and interleukin-6 secretion. These findings demonstrate that the induction of transgene-specific immunity is triggered by an innate immune response with pDC activation and consequent myDC maturation, a response that closely resembles the one induced by functional viruses. This information is important to design strategies aimed at using LV in humans for gene therapy, where adverse immune responses must be avoided, or for cancer immunotherapy, where inducing immunity is the goal.

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.

TCR repertoire sequencing identifies synovial Treg cell clonotypes in the bloodstream during active inflammation in human arthritis

Objectives The imbalance between effector and regulatory T (Treg) cells is crucial in the pathogenesis of autoimmune arthritis. Immune responses are often investigated in the blood because of its accessibility, but circulating lymphocytes are not representative of those found in inflamed tissues. This disconnect hinders our understanding of the mechanisms underlying disease. Our goal was to identify Treg cells implicated in autoimmunity at the inflamed joints, and also readily detectable in the blood upon recirculation. Methods We compared Treg cells of patients with juvenile idiopathic arthritis responding or not to therapy by using: (i) T cell receptor (TCR) sequencing, to identify clonotypes shared between blood and synovial fluid; (ii) FOXP3 Treg cell-specific demethylated region DNA methylation assays, to investigate their stability and (iii) flow cytometry and suppression assays to probe their tolerogenic functions. Results We found a subset of synovial Treg cells that recirculated into the bloodstream of patients with juvenile idiopathic and adult rheumatoid arthritis. These inflammation-associated (ia)Treg cells, but not other blood Treg cells, expanded during active disease and proliferated in response to their cognate antigens. Despite the typical inflammatory-skewed balance of immune mechanisms in arthritis, iaTreg cells were stably committed to the regulatory lineage and fully suppressive. A fraction of iaTreg clonotypes were in common with pathogenic effector T cells. Conclusions Using an innovative antigen-agnostic approach, we uncovered a population of bona fide synovial Treg cells readily accessible from the blood and selectively expanding during active disease, paving the way to non-invasive diagnostics and better understanding of the pathogenesis of autoimmunity.

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

Human CD4+CD3− Innate-Like T Cells Provide a Source of TNF and Lymphotoxin-αβ and Are Elevated in Rheumatoid Arthritis

Innate lymphoid cells encompass a diverse array of lymphocyte subsets with unique phenotype that initiate inflammation and provide host defenses in specific microenvironments. In this study, we identify a rare human CD4+CD3− innate-like lymphoid population with high TNF expression that is enriched in blood from patients with rheumatoid arthritis. These CD4+CD3− cells belong to the T cell lineage, but the lack of AgR at the cell surface renders them nonresponsive to TCR-directed stimuli. By developing a culture system that sustains survival, we show that CD4+CD3− innate-like T cells display IL-7–dependent induction of surface lymphotoxin-αβ, demonstrating their potential to modify tissue microenvironments. Furthermore, expression of CCR6 on the CD4+CD3− population defines a CD127high subset that is highly responsive to IL-7. This CD4+CD3− population is enriched in the peripheral blood from rheumatoid arthritis patients, suggesting a link to their involvement in chronic inflammatory disease.