Alessio Mazzoni

Distinctive features of classic and nonclassic (Th17 derived) human Th1 cells

T helper17 (Th17) lymphocytes represent a third arm of the CD4+ T‐cell effector responses, in addition to Th1 and Th2 cells. Th17 cells have been found to exhibit high plasticity because they rapidly shift into the Th1 phenotype in inflammatory sites. In humans, Th1 cells derived from Th17 cells express CD161, whereas classic Th1 cells do not; these Th17‐derived Th1 cells have been termed nonclassic Th1 cells. In this study, we examined similarities and differences between classic and nonclassic human Th1 cells by assessing a panel of T‐cell clones, as well as CD161+ or CD161− CD4+ T cells derived ex vivo from the circulation of healthy subjects or the synovial fluid of patients with juvenile idiopathic arthritis. The results show that nonclassic Th1 cells can be identified based on CD161 expression, as well as the consistent expression of retinoic acid orphan receptor C, IL‐17 receptor E, CCR6, and IL‐4‐induced gene 1, which are all virtually absent in classic Th1 cells. The possibility to distinguish these two‐cell subsets by using such a panel of markers may allow the opportunity to better establish the respective pathogenic roles of classic and nonclassic (Th17 derived) Th1 cells in different chronic inflammatory disorders.

Demethylation of the RORC2 and IL17A in Human CD4 + T Lymphocytes Defines Th17 Origin of Nonclassic Th1 Cells

Th17-derived Th1 lymphocytes, termed nonclassic, differ from classic Th1 cells because of the presence of retinoic acid orphan receptor (ROR)C2 and the surface expression of CD161 and CCR6. We demonstrate in this article that nonclassic Th1 cells, like Th17 cells, have a marked RORC2 and IL17A demethylation, whereas classic Th1 cells exhibit a complete methylation of these genes. The analysis of RORC2 DNA methylation in the CD4+CD161+ and CD4+CD161− naive Th subsets from umbilical cord blood surprisingly revealed comparable hypermethylation levels. PCR analysis at the single-cell level revealed that RORC2 mRNA was expressed by none of the CD4+CD161− and present only in a minority of CD4+CD161+ naive Th cells. These findings provide two important novel observations on the physiology of human Th17 cells: 1) they confirm at the epigenetic level the origin of nonclassic Th1 cells from Th17 cells, also identifying in the RORC2 and IL17A methylation status a novel tool for their distinction from classic Th1 cells, and 2) they demonstrate that RORC2-expressing cells are only a minority in the subset of CD4+CD161+ naive Th cells, which are known to contain all Th17 cell precursors.

Human circulating group 2 innate lymphoid cells can express CD154 and promote IgE production

Background: Protection against helminths consists of adaptive responses by TH2 cells and innate responses by group 2 innate lymphoid cells (ILC2s), with these latter being well characterized in mice but less so in human subjects. Objective: We sought to characterize human circulating ILC2s and compare their functional profile with that of autologous TH2 cells. Methods: Circulating ILC2s and TH2 cells were isolated by means of fluorescence‐activated cell sorting and magnetic cell sorting and expanded in vitro. ILC2s were then stimulated with phorbol 12‐myristate 13‐acetate plus ionomycin, IL‐25 plus IL‐33 (IL‐25/IL‐33), or a mixture of Toll‐like receptor ligands to evaluate their ability to produce cytokines, express CD154, and induce IgE production by autologous B cells. Cytokines and transcription factor gene methylation were assessed. Results: ILC2s expressed GATA‐3, retinoic acid orphan receptor (RORC) 2, and ROR&agr;; were able to produce IL‐5, IL‐13, and IL‐4; and, accordingly, were characterized by demethylation of IL4, IL13, IL5, GATA3, and RORC2, whereas the IFNG, IFNG promoter, and TBX21 regions of interest were methylated. ILC2s expressed TLR1, TLR4, and TLR6, and TLR stimulation induced IL‐5 and IL‐13 production. Moreover, ILC2s expressed CD154 in response to phorbol 12‐myristate 13‐acetate plus ionomycin, IL‐25/IL‐33, or a mixture of TLR ligands. Stimulated ILC2s also induced IgM, IgG, IgA, and IgE production by B cells. Finally, circulating ILC2s from atopic patients were not different in numbers and frequency but expressed higher IL‐4 levels than those from nonatopic subjects. Conclusion: This study provides the first evidence that human ILC2s can express CD154 and stimulate the production of IgE by B lymphocytes through IL‐25/IL‐33 stimulation or TLR triggering.

IL‐4‐induced gene 1 maintains high Tob1 expression that contributes to TCR unresponsiveness in human T helper 17 cells

Human Th17 cells have a limited proliferative capacity compared to other T‐cell subsets. We have shown that human Th17 cells display impaired IL‐2 production due to IL‐4‐induced gene 1 (IL4I1) upregulation. Here, we show that in human Th17 cells, IL4I1 also maintains high levels of Tob1, a member of the Tob/BTG (B‐cell traslocation gene) antiproliferative protein family, which prevents cell‐cycle progression mediated by TCR stimulation. Indeed, Th17 cells exhibited higher levels of Tob1 than Th1 cells in both resting and TCR‐activated conditions. Accordingly, the expression of positive regulators of the cell cycle (cyclin A, B, C, and E and Cdk2), as well as of Skp2, which promotes Tob1 degradation, was lower in Th17 cells than in Th1 cells. Tob1 expression in human Th17 cells correlated with both RAR (retinoic acid receptor)‐related orphan receptor C (RORC) and IL4I1 levels. However, RORC was not directly involved in the regulation of Tob1 expression, whereas IL4I1 silencing in Th17 cells induced a substantial decrease of Tob1 expression. These data suggest that IL4I1 upregulation in human Th17 cells limits their TCR‐mediated expansion not only by blocking the molecular pathway involved in the activation of the IL‐2 promoter, but also by maintaining high levels of Tob1, which impairs entry into the cell cycle.

Th1-Induced CD106 Expression Mediates Leukocytes Adhesion on Synovial Fibroblasts from Juvenile Idiopathic Arthritis Patients

This study tested the hypothesis that subsets of human T helper cells can orchestrate leukocyte adhesion to synovial fibroblasts (SFbs), thus regulating the retention of leukocytes in the joints of juvenile idiopathic arthritis (JIA) patients. Several cell types, such as monocytes/macrophages, granulocytes, T and B lymphocytes, SFbs and osteoclasts participate in joint tissue damage JIA. Among T cells, an enrichment of classic and non-classic Th1 subsets, has been found in JIA synovial fluid (SF), compared to peripheral blood (PB). Moreover, it has been shown that IL-12 in the SF of inflamed joints mediates the shift of Th17 lymphocytes towards the non-classic Th1 subset. Culture supernatants of Th17, classic and non-classic Th1 clones, have been tested for their ability to stimulate proliferation, and to induce expression of adhesion molecules on SFbs, obtained from healthy donors. Culture supernatants of both classic and non-classic Th1, but not of Th17, clones, were able to induce CD106 (VCAM-1) up-regulation on SFbs. This effect, mediated by tumor necrosis factor (TNF)-α, was crucial for the adhesion of circulating leukocytes on SFbs. Finally, we found that SFbs derived from SF of JIA patients expressed higher levels of CD106 than those from healthy donors, resembling the phenotype of SFbs activated in vitro with Th1-clones supernatants. On the basis of these findings, we conclude that classic and non-classic Th1 cells induce CD106 expression on SFbs through TNF-α, an effect that could play a role in leukocytes retention in inflamed joints.

Chitinase 3-like-1 is produced by human Th17 cells and correlates with the level of inflammation in juvenile idiopathic arthritis patients

BackgroundCHI3L1 is a chitinase-like protein without enzymatic activity, produced by activated macrophages, chondrocytes, neutrophils. Recent studies on arthritis, asthma, and inflammatory bowel diseases suggest that chitinases are important in inflammatory processes and tissue remodeling, but their production by human T cells, has never been reported.MethodsA microarray analysis of gene expression profile was performed on Th17 and classic Th1 cell clones and CHI3L1 was found among the up-regulated genes on Th17 cells. Different types of helper T cell clones (TCCs) were then evaluated by Real Time PCR (RT-PCR) for CHI3L1 mRNA expression; protein expression was investigated in cell lysates by western blotting and in cultures supernatants by ELISA. ELISA was also used to measure CHI3L1 in the serum and in the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients.ResultsAt mRNA level CHI3L1 was highly expressed by Th17, Th17/Th1, non classic Th1 and even in Th17/Th2 cell clones, whereas it was virtually absent in CD161− classic Th1 and Th2 TCCs. CHI3L1 was also detected in cell culture supernatants of Th17 and Th17-derived cells but not of classic Th1. Moreover CHI3L1 was higher in the SF than in serum of JIA patients, and it positively correlated with the frequency of Th17 and non-classic Th1 cells in SF. CHI3L1 in SF also positively correlated with the C reactive protein (CRP) serum levels, and with the levels of some proinflammatory cytokines, such as IL-6 and p40, which is the common subunit of IL12 and IL23.ConclusionsHere we describe for the first time CHI3L1 production by T cells owing the Th17 family. Moreover the positive correlation found between the frequency of Th17 and Th17-derived cell subsets and CHI3L1 levels in SF of JIA patients, in agreement with the suggested role of these cells in inflammatory process, candidates CHI3L1 as a possible biological target in JIA treatment.

The SIICA School of Immunology 2017: a gathering for NGS (next generation scientists)

On 26th May 2017, just a few days before the kickoff of the national conference of the Italian Society of Immunology, Clinical Immunology and Allergology (SIICA), SIICA (a member society of EFIS www.efis.org) opened its doors to young scientists by hosting its 1st School of Immunology. This builds on SIICA’s tradition of actively educating junior scientists by way of dedicated meetings (e.g. the annual international retreat of PhD students in immunology that will take place on October 6–7th in Verona, http://www.siica.org/siica/node/155) and by sponsoring the participation of junior scientists at congresses. It also complements the work of other EFIS member societies [1–3] and EFIS-EJI sponsored educational activities [4]. Some international outreach programmes target even younger budding scientists by engaging them at school level [5]. The location of SIICA’s School was Bari, a magnificent city in Apulia facing the Adriatic sea, offering wonderful examples of Romanesque architecture and breathtaking landscapes. It was a peaceful place to teach/learn immunology and, of course, to enjoy the local amenities and the terrific food. The school was intended for PhD students, but participation was also extended to young postdocs and anyone seeking a deeper understanding of immunology. Based on their knowledge and their expectations, the students had the chance to choose between two classes: “basic”, suited for either students in the first year of their PhD program or immunology novices; and “advanced”, for those who already had a strong immunological background and wanted to stay up-to-date with cuttingedge immunology. The initial success of the school was evidenced by the good response obtained during the registration phase. Indeed, more than 80 students arrived eager to learn for the afternoon session on the first day of school. Playing a great role in this success story were the outstanding teachers from all around Europe, including James P. Di Santo (Paris, France), Andreas Radbruch (Berlin, Germany), Oreste Acuto (Oxford, UK), Giuseppe Pantaleo (Lausanne, Switzerland) and Winfried F. Pickl (Wien, Austria), as well as a panel of exoert Italian Immunologists (see the complete list at http://www.siica.org/ siica/school-of-immunology), which made the program rich, varied and appealing. After two intense days of lessons, the Saturday night fever around the streets of the old town which led to the last day of the school (Sunday, 28th), was preceded by the candlelight lecture delivered by Alberto Mantovani (Milan, Italy) The end of the lessons was followed by a learning test and the filling out of a questionnaire to gauge participant satisfaction. For many there was no time for after-school recreation as more than 30 students followed-up their learning program at the summer school by participating in the national congress of the Society (http://www.siica2017.org/) and becoming SIICA members.

Immunosuppressive Activity of Abatacept on Circulating T Helper Lymphocytes from Juvenile Idiopathic Arthritis Patients

Background: Abatacept is used in the treatment of juvenile idiopathic arthritis (JIA) patients, but the activity of the drug on T helper cell function is not yet fully known. Methods: The ability of abatacept to affect cytokine production in vitro and the proliferative response to both recall antigens and polyclonal stimulation was firstly assessed in healthy donors. Then, 10 JIA patients who were due to start abatacept treatment were recruited and longitudinally evaluated during the first 90 days of therapy. Both their clinical response to the treatment and in vitro analysis aimed to assess the proliferative response to recall antigens and the proportions of circulating T helper subsets. Results: Abatacept reduced the proliferative response to recall antigens and the production of proinflammatory cytokines such as IFN-γ and TNF-α in healthy donors in vitro. It was also efficient in improving symptoms and reducing parameters of inflammation in JIA patients. Abatacept reduced the proliferative response to recall antigens, and this effect was significant soon after drug infusion (2 days). Regarding the proportions of circulating CD4+ T lymphocytes, only a reduction in the frequencies of circulating Treg cells was observed. Conclusions: Abatacept in vitro inhibits proliferation and cytokine production in healthy donors, and reduces parameters of inflammation in vivo in JIA patients. The reduction of the proliferative response to recall antigens induced by abatacept was evident only soon after drug administration, suggesting that its immunosuppressive activity is maintained only for a short time.

Sphingosine Kinases promote IL-17 expression in human T lymphocytes

Sphingosine 1-phosphate (S1P) has a role in many cellular processes. S1P is involved in cell growth and apoptosis, regulation of cell trafficking, production of cytokines and chemokines. The kinases SphK1 and SphK2 (SphKs) phosphorilate Sphingosine (Sph) to S1P and several phosphatases revert S1P to sphingosine, thus assuring a balanced pool that can be depleted by a Sphingosine lyase in hexadecenal compounds and aldehydes. There are evidences that SphK1 and 2 may per se control cellular processes. Here, we report that Sph kinases regulate IL-17 expression in human T cells. SphKs inhibition impairs the production of IL-17, while their overexpression up-regulates expression of the cytokine through acetylation of IL-17 promoter. SphKs were up-regulated also in PBMCs of patients affected by IL-17 related diseases. Thus, S1P/S1P kinases axis is a mechanism likely to promote IL-17 expression in human T cells, representing a possible therapeutic target in human inflammatory diseases.

Eomes controls the development of Th17-derived (non-classic) Th1 cells during chronic inflammation

It is well accepted that Th17 cells are a highly plastic cell subset that can be easily directed toward the Th1 phenotype in vitro and also in vivo during inflammation. However, there is an ongoing debate regarding the reverse plasticity (conversion from Th1 to Th17). We show here that ectopic ROR‐γt expression can restore or initiate IL‐17 expression by non‐classic or classic Th1 cells, respectively, while common pro‐Th17 cytokine cocktails are ineffective. This stability of the Th1 phenotype is at least partially due to the presence of a molecular machinery governed by the transcription factor Eomes, which promotes IFN‐γ secretion while inhibiting the expression of ROR‐γt and IL‐17. By using a mouse model of T cell‐dependent colitis we demonstrate that Eomes controls non‐classic Th1 cell development also in vivo and promotes their pathogenic potential. Eomes expression associates to a highly inflammatory phenotype also in patients with juvenile idiopathic arthritis. Indeed, it favors the acquisition of a cytotoxic signature, and promotes the development of IFN‐γ+GM‐CSF+ cells that have been described to be pathogenic in chronic inflammatory disorders.