Roberto Spreafico

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.

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.

Exhaustion-associated regulatory regions in CD8+ tumor-infiltrating T cells

Significance Cancer cells can be recognized and attacked by CD8+ cytolytic T cells, but tumor-infiltrating T cells often become functionally incompetent (“exhausted”) and fail to destroy tumor cells. We show that T-cell exhaustion requires antigen recognition by tumor-infiltrating T cells. By examining the transcriptional and chromatin accessibility profiles of antigen-reactive and -unreactive tumor-infiltrating cells, we confirm our previous conclusion that the transcription factor NFAT promotes CD8+ T-cell exhaustion and we identify Nr4a transcription factors as new targets for future investigation. We show that anti–PD-L1 treatment, a clinically relevant checkpoint blockade therapy that counteracts T-cell exhaustion, has modest but functionally important effects on gene expression in exhausted cells, without causing major changes in patterns of chromatin accessibility. T-cell exhaustion is a progressive loss of effector function and memory potential due to persistent antigen exposure, which occurs in chronic viral infections and cancer. Here we investigate the relation between gene expression and chromatin accessibility in CD8+ tumor-infiltrating lymphocytes (TILs) that recognize a model tumor antigen and have features of both activation and functional exhaustion. By filtering out accessible regions observed in bystander, nonexhausted TILs and in acutely restimulated CD8+ T cells, we define a pattern of chromatin accessibility specific for T-cell exhaustion, characterized by enrichment for consensus binding motifs for Nr4a and NFAT transcription factors. Anti–PD-L1 treatment of tumor-bearing mice results in cessation of tumor growth and partial rescue of cytokine production by the dysfunctional TILs, with only limited changes in gene expression and chromatin accessibility. Our studies provide a valuable resource for the molecular understanding of T-cell exhaustion in cancer and other inflammatory settings.

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.

Loss of the BBSome perturbs endocytic trafficking and disrupts virulence of Trypanosoma brucei

Significance We combine genetics, biochemistry, cell biology, and proteomics to define Bardet–Biedl Syndrome complex (BBSome) composition, location, and function in the deadly parasite Trypanosoma brucei. BBSome mutants have reduced infectivity in mice, and quantitative proteomics identified parasite surface proteome changes that may underlie reduced virulence. To our knowledge, this work presents the first comprehensive study of the BBSome in any microbial pathogen. T. brucei is also among the earliest organisms to have diverged from other eukaryotes, showing deep evolutionary origins of the BBSome. Localization to membranes and vesicles at the flagellar pocket, together with functional analyses and interaction with clathrin and ubiquitin, supports a model whereby the BBSome functions in postendocytic sorting of select surface proteins. Cilia (eukaryotic flagella) are present in diverse eukaryotic lineages and have essential motility and sensory functions. The cilium’s capacity to sense and transduce extracellular signals depends on dynamic trafficking of ciliary membrane proteins. This trafficking is often mediated by the Bardet–Biedl Syndrome complex (BBSome), a protein complex for which the precise subcellular distribution and mechanisms of action are unclear. In humans, BBSome defects perturb ciliary membrane protein distribution and manifest clinically as Bardet–Biedl Syndrome. Cilia are also important in several parasites that cause tremendous human suffering worldwide, yet biology of the parasite BBSome remains largely unexplored. We examined BBSome functions in Trypanosoma brucei, a flagellated protozoan parasite that causes African sleeping sickness in humans. We report that T. brucei BBS proteins assemble into a BBSome that interacts with clathrin and is localized to membranes of the flagellar pocket and adjacent cytoplasmic vesicles. Using BBS gene knockouts and a mouse infection model, we show the T. brucei BBSome is dispensable for flagellar assembly, motility, bulk endocytosis, and cell viability but required for parasite virulence. Quantitative proteomics reveal alterations in the parasite surface proteome of BBSome mutants, suggesting that virulence defects are caused by failure to maintain fidelity of the host–parasite interface. Interestingly, among proteins altered are those with ubiquitination-dependent localization, and we find that the BBSome interacts with ubiquitin. Collectively, our data indicate that the BBSome facilitates endocytic sorting of select membrane proteins at the base of the cilium, illuminating BBSome roles at a critical host–pathogen interface and offering insights into BBSome molecular mechanisms.

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.

Training the 21st Century Immunologist

Immunology, along with other fields of biology, is undergoing a revolution. Here we discuss the challenges and opportunities presented by considering the dynamical systems properties of the immune system, and harnessing the power of data-rich technologies. We present specific recommendations for changing graduate programs to incorporate training that will enable students to actively participate in the analyses of complex data and their biological system, and urge that we move from viewing quantitative and computational biology as interdisciplinary, to recognizing these as intrinsic to the discipline of immunology going forward.