Publications by authors named "Jens Geginat"

52 Publications

Ex vivo microRNA and gene expression profiling of human Tr1-like cells suggests a role for miR-92a and -125a in the regulation of EOMES and IL-10R.

Eur J Immunol 2021 Sep 15. Epub 2021 Sep 15.

Istituto Nazionale Genetica Molecolare INGM 'Romeo ed Enrica Invernizzi', Milan, Italy.

Ex vivo gene expression and miRNA profiling of Eomes Tr1-like cells suggested that they represent a differentiation stage that is intermediate between Th1-cells and cytotoxic CD4 T-cells. Several microRNAs were downregulated in Eomes Tr1-like cells that might inhibit Tr1-cell differentiation. In particular, miR-92a targeted Eomes, while miR-125a inhibited IFN-g and IL-10R expression.
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http://dx.doi.org/10.1002/eji.202149315DOI Listing
September 2021

Clonally expanded EOMES Tr1-like cells in primary and metastatic tumors are associated with disease progression.

Nat Immunol 2021 06 20;22(6):735-745. Epub 2021 May 20.

Niguarda Cancer Center, Grande Ospedale Metropolitano Niguarda, Milan, Italy.

Regulatory T (T) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4 T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3 T and eomesodermin homolog (EOMES) type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES Tr1-like cells, but not T cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of T cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.
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http://dx.doi.org/10.1038/s41590-021-00930-4DOI Listing
June 2021

The induction and function of the anti-inflammatory fate of T17 cells.

Nat Commun 2020 07 3;11(1):3334. Epub 2020 Jul 3.

Department of General, Visceral and Thoracic Surgery, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany.

T17 cells exemplify environmental immune adaptation: they can acquire both a pathogenic and an anti-inflammatory fate. However, it is not known whether the anti-inflammatory fate is merely a vestigial trait, or whether it serves to preserve the integrity of the host tissues. Here we show that the capacity of T17 cells to acquire an anti-inflammatory fate is necessary to sustain immunological tolerance, yet it impairs immune protection against S. aureus. Additionally, we find that TGF-β signalling via Smad3/Smad4 is sufficient for the expression of the anti-inflammatory cytokine, IL-10, in T17 cells. Our data thus indicate a key function of T17 cell plasticity in maintaining immune homeostasis, and dissect the molecular mechanisms explaining the functional flexibility of T17 cells with regard to environmental changes.
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http://dx.doi.org/10.1038/s41467-020-17097-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7335205PMC
July 2020

Immunological Variables Associated With Clinical and Endoscopic Response to Vedolizumab in Patients With Inflammatory Bowel Diseases.

J Crohns Colitis 2020 Sep;14(9):1190-1201

Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca' Granda, Ospedale Maggiore Policlinico, Milan, Italy.

Background And Aims: Vedolizumab [VDZ] is a monoclonal antibody directed against the α4β7 integrin heterodimer, approved for patients with inflammatory bowel diseases [IBD]. This study aimed at identifying immunological variables associated with response to vedolizumab in patients with ulcerative colitis [UC] and Crohn's disease [CD].

Methods: This is a phase IV explorative prospective interventional trial. IBD patients received open-label VDZ at Weeks 0, 2, 6, and 14. Patients with a clinical response at Week 14 were maintained with VDZ up to Week 54. At Weeks 0 and 14, their peripheral blood was obtained and endoscopy with biopsies was performed. The Week 14 clinical response and remission, Week 54 clinical remission, and Week 14 endoscopic response were evaluated as endpoints of the study. The expression of surface markers, chemokine receptors, and α4β7 heterodimer in peripheral blood and lamina propria lymphocytes was assessed by flow cytometry. A panel of soluble mediators was assessed in sera at baseline and at Week 14 by 45-plex.

Results: A total of 38 IBD patients [20 UC, 18 CD] were included in the study. At Week 14, the clinical response and remission rates were 87% and 66%, respectively. Higher baseline levels of circulating memory Th1 cells were strongly associated with clinical response at Week 14 [p = 0.0001], whereas reduced baseline levels of lamina propria memory Th17 and Th1/17 cells were associated with endoscopic response. Immunological clusters were found to be independently associated with vedolizumab outcomes at multivariable analysis. A panel of soluble markers, including IL17A, TNF, CXCL1, CCL19 for CD and G-CSF and IL7 for UC, associated with vedolizumab-induced Week 54 clinical remission.

Conclusions: The results of this exploratory study uncovered a panel of circulating and mucosal immunological variables associated with response to treatment with vedolizumab.
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http://dx.doi.org/10.1093/ecco-jcc/jjaa035DOI Listing
September 2020

Introduction to the Special Issue: Interleukin-10 "The surprising twists and turns of an anti-inflammatory cytokine on its way to the clinic".

Authors:
Jens Geginat

Semin Immunol 2019 08 6;44:101343. Epub 2019 Nov 6.

Fondazione Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi", Via F. Sforza 35, I-20122 Milano, Mi, Italy. Electronic address:

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http://dx.doi.org/10.1016/j.smim.2019.101343DOI Listing
August 2019

Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition).

Eur J Immunol 2019 Oct;49(10):1457-1973

Flow Cytometry Laboratory, Institute of Molecular Toxicology and Pharmacology, Helmholtz Zentrum München, German Research Center for Environmental Health, München, Germany.

These guidelines are a consensus work of a considerable number of members of the immunology and flow cytometry community. They provide the theory and key practical aspects of flow cytometry enabling immunologists to avoid the common errors that often undermine immunological data. Notably, there are comprehensive sections of all major immune cell types with helpful Tables detailing phenotypes in murine and human cells. The latest flow cytometry techniques and applications are also described, featuring examples of the data that can be generated and, importantly, how the data can be analysed. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid, all written and peer-reviewed by leading experts in the field, making this an essential research companion.
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http://dx.doi.org/10.1002/eji.201970107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350392PMC
October 2019

Novel biomarkers for primary biliary cholangitis to improve diagnosis and understand underlying regulatory mechanisms.

Liver Int 2019 11 15;39(11):2124-2135. Epub 2019 May 15.

Istituto Nazionale Genetica Molecolare, Padiglione Romeo ed Enrica Invernizzi, IRCCS Ospedale Maggiore Policlinico, Milan, Italy.

Background And Aims: Primary biliary cholangitis is an autoimmune biliary disease characterized by injury of bile ducts, eventually leading to cirrhosis and death. In most cases, anti-mitochondrial antibodies and persistently elevated serum alkaline phosphatase are the basis for the serological diagnosis. Anti-nuclear antibodies are also useful and may indicate a more aggressive diseases course. In patients in which anti-mitochondrial antibodies are not detected, an accurate diagnosis requires liver histology. This study aims at identifying specific biomarkers for the serological diagnosis of primary biliary cholangitis.

Methods: Sera from patients affected by primary biliary cholangitis, primary sclerosing cholangitis, hepatitis C virus (with and without cryoglobulinemia), hepatocarcinoma and healthy donors were tested on a protein array representing 1658 human proteins. The most reactive autoantigens were confirmed by DELFIA analysis on expanded cohorts of the same mentioned serum classes, and on autoimmune hepatitis sera, using anti-PDC-E2 as reference biomarker.

Results: Two autoantigens, SPATA31A3 and GARP, showed high reactivity with primary biliary cholangitis sera, containing or not anti-mitochondrial antibodies. Their combination with PDC-E2 allowed to discriminate primary biliary cholangitis from all tested control classes with high sensitivity and specificity. We found that GARP expression is upregulated upon exposure to biliary salts in human cholangiocytes, an event involving EGFR and insulin pathways. GARP expression was also detected in biliary duct cells of PBC patients.

Conclusions: This study highlighted SPATA31A3 and GARP as new biomarkers for primary biliary cholangitis and unravelled molecular stimuli underlying GARP expression in human cholangiocytes.
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http://dx.doi.org/10.1111/liv.14128DOI Listing
November 2019

Molecular and functional heterogeneity of IL-10-producing CD4 T cells.

Nat Commun 2018 12 21;9(1):5457. Epub 2018 Dec 21.

I. Department of Medicine, University Medical Center Hamburg-Eppendorf, 20246, Hamburg, Germany.

IL-10 is a prototypical anti-inflammatory cytokine, which is fundamental to the maintenance of immune homeostasis, especially in the intestine. There is an assumption that cells producing IL-10 have an immunoregulatory function. However, here we report that IL-10-producing CD4 T cells are phenotypically and functionally heterogeneous. By combining single cell transcriptome and functional analyses, we identified a subpopulation of IL-10-producing Foxp3 CD4 T cells that displays regulatory activity unlike other IL-10-producing CD4 T cells, which are unexpectedly pro-inflammatory. The combinatorial expression of co-inhibitory receptors is sufficient to discriminate IL-10-producing CD4 T cells with regulatory function from others and to identify them across different tissues and disease models in mice and humans. These regulatory IL-10-producing Foxp3 CD4 T cells have a unique transcriptional program, which goes beyond the regulation of IL-10 expression. Finally, we found that patients with Inflammatory Bowel Disease demonstrate a deficiency in this specific regulatory T-cell subpopulation.
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http://dx.doi.org/10.1038/s41467-018-07581-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303294PMC
December 2018

Eomesodermin controls a unique differentiation program in human IL-10 and IFN-γ coproducing regulatory T cells.

Eur J Immunol 2019 01 29;49(1):96-111. Epub 2018 Nov 29.

INGM-National Institute of Molecular Genetics "Romeo ed Enrica Invernizzi", Milan, Italy.

Whether human IL-10-producing regulatory T cells ("Tr1") represent a distinct differentiation lineage or an unstable activation stage remains a key unsolved issue. Here, we report that Eomesodermin (Eomes) acted as a lineage-defining transcription factor in human IFN-γ/IL-10 coproducing Tr1-like cells. In vivo occurring Tr1-like cells expressed Eomes, and were clearly distinct from all other CD4 T-cell subsets, including conventional cytotoxic CD4 T cells. They expressed Granzyme (Gzm) K, but had lost CD40L and IL-7R expression. Eomes antagonized the Th17 fate, and directly controlled IFN-γ and GzmK expression. However, Eomes binding to the IL-10 promoter was not detectable in human CD4 T cells, presumably because critical Tbox binding sites of the mouse were not conserved. A precommitment to a Tr1-like fate, i.e. concominant induction of Eomes, GzmK, and IFN-γ, was promoted by IL-4 and IL-12-secreting myeloid dendritic cells. Consistently, Th1 effector memory cells contained precommitted Eomes GzmK T cells. Stimulation with T-cell receptor (TCR) agonists and IL-27 promoted the generation of Tr1-like effector cells by inducing switching from CD40L to IL-10. Importantly, CD4 Eomes T-cell subsets were present in lymphoid and nonlymphoid tissues, and their frequencies varied systemically in patients with inflammatory bowel disease and graft-versus-host disease. We propose that Eomes Tr1-like cells are effector cells of a unique GzmK-expressing CD4 T-cell subset.
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http://dx.doi.org/10.1002/eji.201847722DOI Listing
January 2019

Pathogenicity of In Vivo Generated Intestinal Th17 Lymphocytes is IFNγ Dependent.

J Crohns Colitis 2018 Jul;12(8):981-992

Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.

Background And Aims: T helper 17 [Th17] cells are crucially involved in the immunopathogenesis of inflammatory bowel diseases in humans. Nevertheless, pharmacological blockade of interleukin 17A [IL17A], the Th17 signature cytokine, yielded negative results in patients with Crohn's disease [CD], and attempts to elucidate the determinants of Th17 cells' pathogenicity in the gut have so far proved unsuccessful. Here, we aimed to identify and functionally validate the pathogenic determinants of intestinal IL-17-producing T cells.

Methods: In vivo-generated murine intestinal IL-17-producing T cells were adoptively transferred into immunodeficient Rag1-/- recipients to test their pathogenicity. Human IL-17, IFNγ/IL-17, and IFNγ actively secreting T cell clones were generated from lamina propria lymphocytes of CD patients. The pathogenic activity of intestinal IL-17-producing T cells against the intestinal epithelium was evaluated.

Results: IL-17-producing cells with variable colitogenic activity can be generated in vivo using different experimental colitis models. The pathogenicity of IL-17-secreting cells was directly dependent on their IFNγ secretion capacity, as demonstrated by the reduced colitogenic activity of IL-17-secreting cells isolated from IFNγ-/- mice. Moreover, IFNγ production is a distinguished attribute of CD-derived lamina propria Th17 cells. IFNγ secretion by CD-derived IL-17-producing intestinal clones is directly implicated in the epithelial barrier disruption through the modulation of tight junction proteins.

Conclusions: Intestinal Th17 cell pathogenicity is associated with IFNγ production, which directly affects intestinal permeability through the disruption of epithelial tight junctions.
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http://dx.doi.org/10.1093/ecco-jcc/jjy051DOI Listing
July 2018

Successful sequential therapy with rituximab and belimumab in patients with active systemic lupus erythematosus: a case series.

Clin Exp Rheumatol 2018 Jul-Aug;36(4):643-647. Epub 2018 Feb 27.

Lupus Clinic, Division of Rheumatology, ASST Pini, Department of Clinical Sciences and Community Health, University of Milan; Immunorheumatology Research Laboratory, Istituto Auxologico Italiano, IRCCS, Milan, Italy.

Objectives: B cells play an important role in the initiation and progression of systemic lupus erythematosus (SLE). Accordingly, B cell-targeted therapy has been suggested as a new rational approach for treating lupus. Belimumab, a human monoclonal antibody directed against B lymphocyte stimulator (BLyS), was reported as the first biological treatment effective in reducing mild-to-moderate SLE disease activity by using different scoring systems and endpoints. Conversely clinical trials with rituximab, a chimeric monoclonal antibody directed against the CD20 expressed by B cells, have failed to achieve primary endpoints in spite of a number of reports showing its beneficial effects. Anecdotal reports have described the sequential use of rituximab and belimumab as a more effective treatment than using the individual drugs alone, without compromising safety.

Methods: We report a case series of three patients with active SLE refractory to conventional therapies, who underwent treatment with rituximab followed by belimumab as maintenance therapy.

Results: We observed a beneficial effect after sequential treatment with rituximab and belimumab. All patients achieved long-standing remission and could reduce or discontinue corticosteroids. Concomitantly, after rituximab administration we observed a rise in BLyS levels, which were dramatically reduced after belimumab introduction.

Conclusions: The modulation of plasma BLyS kinetics in patients undergoing sequential treatment with rituximab and belimumab may represent a possible rationale behind the effectiveness of this combined therapy.
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September 2018

Intestinal IFN-γ-producing type 1 regulatory T cells coexpress CCR5 and programmed cell death protein 1 and downregulate IL-10 in the inflamed guts of patients with inflammatory bowel disease.

J Allergy Clin Immunol 2018 11 31;142(5):1537-1547.e8. Epub 2018 Jan 31.

INGM-National Institute of Molecular Genetics "Romeo ed Enrica Invernizzi" Milan, Milan, Italy. Electronic address:

Background: IL-10 is an anti-inflammatory cytokine required for intestinal immune homeostasis. It mediates suppression of T-cell responses by type 1 regulatory T (T1) cells but is also produced by CD25 regulatory T (Treg) cells.

Objective: We aimed to identify and characterize human intestinal T1 cells and to investigate whether they are a relevant cellular source of IL-10 in patients with inflammatory bowel diseases (IBDs).

Methods: CD4 T cells isolated from the intestinal lamina propria of human subjects and mice were analyzed for phenotype, cytokine production, and suppressive capacities. Intracellular IL-10 expression by CD4 T-cell subsets in the inflamed guts of patients with IBD (Crohn disease or ulcerative colitis) was compared with that in cells from noninflamed control subjects. Finally, the effects of proinflammatory cytokines on T-cell IL-10 expression were analyzed, and IL-1β and IL-23 responsiveness was assessed.

Results: Intestinal T1 cells could be identified by coexpression of CCR5 and programmed cell death protein 1 (PD-1) in human subjects and mice. CCR5PD-1 T1 cells expressed IFN-γ and efficiently suppressed T-cell proliferation and transfer colitis. Intestinal IFN-γ T1 cells, but not IL-7 receptor-positive T cells or CD25 Treg cells, showed lower IL-10 expression in patients with IBDs. T1 cells were responsive to IL-23, and IFN-γ T1 cells downregulated IL-10 with IL-1β and IL-23. Conversely, CD25 Treg cells expressed higher levels of IL-1 receptor but showed stable IL-10 expression.

Conclusions: We provide the first ex vivo characterization of human intestinal T1 cells. Selective downregulation of IL-10 by IFN-γ T1 cells in response to proinflammatory cytokines is likely to drive excessive intestinal inflammation in patients with IBDs.
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http://dx.doi.org/10.1016/j.jaci.2017.12.984DOI Listing
November 2018

Maintenance of memory CD8 T cells: Divided over division.

Eur J Immunol 2017 11;47(11):1875-1879

INGM, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy.

Once generated during an infection, memory CD8 T cells can provide long-lasting protection against reinfection with an intracellular pathogen, but the longevity of this defense depends on the ability of these pathogen-specific memory cells to be maintained. It is generally believed that the bone marrow plays an important role in this respect, where memory CD8 T cells receive reinvigorating signals from cytokines that induce homeostatic proliferation. However, in the current issue of the European Journal of Immunology, Siracusa et al. (Eur. J. Immunol. 2017. 47: 1900-1905) argue against this dogma, as they provide evidence that CD8 memory T cells in murine bone marrow are not proliferating, but largely quiescent, which protects them from elimination by the cytostatic drug Cyclophosphamide. Interestingly, this is in sharp contrast to the proliferating cell counterparts in the spleen, which are eliminated by this treatment. Here, we will discuss the impact of these results, how they relate to opposing findings by others in the field, and what the relevance of these findings is for humans and clinical applications.
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http://dx.doi.org/10.1002/eji.201747249DOI Listing
November 2017

The Enigmatic Role of Viruses in Multiple Sclerosis: Molecular Mimicry or Disturbed Immune Surveillance?

Trends Immunol 2017 07 23;38(7):498-512. Epub 2017 May 23.

INGM, Istituto Nazionale Genetica Molecolare 'Romeo ed Enrica Invernizzi', Milan, Italy; DISCCO, Department of Clinical Sciences and Community Health, University of Milan, Milan, Italy.

Multiple sclerosis (MS) is a T cell driven autoimmune disease of the central nervous system (CNS). Despite its association with Epstein-Barr Virus (EBV), how viral infections promote MS remains unclear. However, there is increasing evidence that the CNS is continuously surveyed by virus-specific T cells, which protect against reactivating neurotropic viruses. Here, we discuss how viral infections could lead to the breakdown of self-tolerance in genetically predisposed individuals, and how the reactivations of viruses in the CNS could induce the recruitment of both autoaggressive and virus-specific T cell subsets, causing relapses and progressive disability. A disturbed immune surveillance in MS would explain several experimental findings, and has important implications for prognosis and therapy.
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http://dx.doi.org/10.1016/j.it.2017.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185415PMC
July 2017

Differences in serum and synovial CD4+ T cells and cytokine profiles to stratify patients with inflammatory osteoarthritis and rheumatoid arthritis.

Arthritis Res Ther 2017 05 19;19(1):103. Epub 2017 May 19.

DISCCO-Department of Clinical Science and Community Health Università degli Studi di Milano, 20122, Milan, Italy.

Background: The aim was to investigate CD4T-cell subsets, immune cells and their cytokine profiles in blood and synovial compartments in rheumatoid arthritis (RA) and inflammatory osteoarthritis (OA) to define specific immune signatures.

Methods: Peripheral blood, synovial fluid (SF) and synovial membranes (SM) of RA and OA patients were analyzed. CD4T-cell subset frequencies were determined by flow cytometry, and cytokine concentrations in serum and SF were measured by ELISA.

Results: In peripheral blood, OA patients had altered frequencies of regulatory T-cell subsets, and higher frequencies of Th17 and of Th1/17 cells than RA patients. In the synovial compartment of OA patients, conventional Th17 cells were largely excluded, while Th1/17 cells were enriched and more frequent than in RA patients. Conversely, in the synovial compartment of RA patients, regulatory T cells and Tfh cells were enriched and more frequent then in OA patients. IL-17 and Blys were increased both in serum and SF of RA patients, and correlated with autoantibodies and disease activity. Notably, Blys levels were already significantly elevated in RA patients with low disease activity score in 28 joints (DAS28) and without autoantibody positivity.

Conclusions: Although patients with inflammatory OA have immune activation in the synovial compartment, they display different T-cell subset frequencies and cytokine profiles. Soluble mediators such as Blys might help to discriminate mild clinical forms of RA from inflammatory OA particularly at the onset of the disease.
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http://dx.doi.org/10.1186/s13075-017-1305-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437517PMC
May 2017

CD4 T Helper Cell Plasticity in Infection, Inflammation, and Autoimmunity.

Mediators Inflamm 2017 29;2017:7083153. Epub 2017 Jan 29.

Gastroenterology and Endoscopy Unit, Fondazione IRCCS Ca' Granda Ospedale Maggiore Policlinico, Milan, Italy.

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http://dx.doi.org/10.1155/2017/7083153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303579PMC
September 2018

Recognition of viral and self-antigens by T1 and T1/T17 central memory cells in patients with multiple sclerosis reveals distinct roles in immune surveillance and relapses.

J Allergy Clin Immunol 2017 Sep 22;140(3):797-808. Epub 2017 Feb 22.

INGM, Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", Milan, Italy. Electronic address:

Background: Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) that is caused by autoreactive T cells and associated with viral infections. However, the phenotype of pathogenic T cells in peripheral blood remains to be defined, and how viruses promote MS is debated.

Objective: We aimed to identify and characterize potentially pathogenic autoreactive T cells, as well as protective antiviral T cells, in patients with MS.

Methods: We analyzed CD4 helper T-cell subsets from peripheral blood or cerebrospinal fluid for cytokine production, gene expression, plasticity, homing potentials, and their reactivity to self-antigens and viral antigens in healthy subjects and patients with MS. Moreover, we monitored their frequencies in untreated and fingolimod- or natalizumab-treated patients with MS.

Results: T1/T17 central memory (T1/T17) cells were selectively increased in peripheral blood of patients with relapsing-remitting MS with a high disease score. T1/T17 cells were closely related to conventional T17 cells but had more pathogenic features. In particular, they could shuttle between lymph nodes and the CNS and produced encephalitogenic cytokines. The cerebrospinal fluid of patients with active MS was enriched for CXCL10 and contained mainly CXCR3-expressing T1 and T1/T17 subsets. However, while T1 cells responded consistently to viruses, T1/T17 cells reacted strongly with John Cunningham virus in healthy subjects but responded instead to myelin-derived self-antigens in patients with MS. Fingolimod and natalizumab therapies efficiently targeted autoreactive T1/T17 cells but also blocked virus-specific T1 cells.

Conclusions: We propose that autoreactive T1/T17 cells expand in patients with MS and promote relapses after bystander recruitment to the CNS, whereas T1 cells perform immune surveillance. Thus the selective targeting of T1/T17 cells could inhibit relapses without causing John Cunningham virus-dependent progressive multifocal encephalomyelitis.
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http://dx.doi.org/10.1016/j.jaci.2016.11.045DOI Listing
September 2017

Extracellular MicroRNA Signature of Human Helper T Cell Subsets in Health and Autoimmunity.

J Biol Chem 2017 02 11;292(7):2903-2915. Epub 2017 Jan 11.

the IRCCS Istituto di Ricovero e Cura a Carattere Scientifico MultiMedica, 20138 Milan, Italy

Upon T cell receptor stimulation, CD4 T helper (Th) lymphocytes release extracellular vesicles (EVs) containing microRNAs. However, no data are available on whether human CD4 T cell subsets release EVs containing different pattern of microRNAs. The present work aimed at filling this gap by assessing the microRNA content in EVs released upon T cell receptor stimulation of Th1, Th17, and T regulatory (Treg) cells. Our results indicate that EVs released by Treg cells are significantly different compared with those released by the other subsets. In particular, miR-146a-5p, miR-150-5p, and miR-21-5p are enriched, whereas miR-106a-5p, miR-155-5p, and miR-19a-3p are depleted in Treg-derived EVs. The identified EV-associated microRNA signature was increased in serum of autoimmune patients with psoriasis and returned to healthy levels upon effective treatment with etanercept, a biological drug targeting the TNF pathway and suppressing inflammation. Moreover, Gene Set Enrichment Analysis showed an over-representation of genes relevant for T cell activation, such as CD40L, IRAK1, IRAK2, STAT1, and c-Myb in the list of validated targets of Treg-derived EV miRNAs. At functional level, Treg-derived (but not Th1/Th17-derived) EVs inhibited CD4 T cell proliferation and suppressed two relevant targets of miR-146a-5p: STAT1 and IRAK2. In conclusion, our work identified the miRNAs specifically released by different human CD4 T cell subsets and started to unveil the potential use of their quantity in human serum to mark the pathological elicitation of these cells and their biological effect in cell to cell communication during the adaptive immune response.
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http://dx.doi.org/10.1074/jbc.M116.769893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5314185PMC
February 2017

Transcriptional Landscape of Human Tissue Lymphocytes Unveils Uniqueness of Tumor-Infiltrating T Regulatory Cells.

Immunity 2016 11;45(5):1135-1147

Istituto Nazionale Genetica Molecolare INGM 'Romeo ed Enrica Invernizzi,' Milan 20122, Italy; Department of Clinical Sciences and Community Health, Università degli Studi di Milano, Milano 20122, Italy. Electronic address:

Tumor-infiltrating regulatory T lymphocytes (Treg) can suppress effector T cells specific for tumor antigens. Deeper molecular definitions of tumor-infiltrating-lymphocytes could thus offer therapeutic opportunities. Transcriptomes of T helper 1 (Th1), Th17, and Treg cells infiltrating colorectal or non-small-cell lung cancers were compared to transcriptomes of the same subsets from normal tissues and validated at the single-cell level. We found that tumor-infiltrating Treg cells were highly suppressive, upregulated several immune-checkpoints, and expressed on the cell surfaces specific signature molecules such as interleukin-1 receptor 2 (IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8 chemokine, which were not previously described on Treg cells. Remarkably, high expression in whole-tumor samples of Treg cell signature genes, such as LAYN, MAGEH1, or CCR8, correlated with poor prognosis. Our findings provide insights into the molecular identity and functions of human tumor-infiltrating Treg cells and define potential targets for tumor immunotherapy.
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http://dx.doi.org/10.1016/j.immuni.2016.10.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5119953PMC
November 2016

Reverse plasticity: TGF-β and IL-6 induce Th1-to-Th17-cell transdifferentiation in the gut.

Eur J Immunol 2016 10;46(10):2306-2310

Istituto Nazionale di Genetica Molecolare Romeo ed Enrica Invernizzi, Milan, Italy.

Th17 cells are a heterogeneous population of pro-inflammatory T cells that have been shown to mediate immune responses against intestinal bacteria. Th17 cells are highly plastic and can transdifferentiate to Th1/17 cells or unconventional Th1 cells, which are highly pathogenic in animal models of immune-mediated diseases such as inflammatory bowel diseases. A recent European Journal of Immunology article by Liu et al. (Eur. J. Immunol. 2015. 45:1010-1018) showed, surprisingly, that Th1 cells have a similar plasticity, and could transdifferentiate to Th17 cells. Thus, IFN-γ-producing Th1 effector cells specific for an intestinal microbial antigen were shown to acquire IL-17-producing capacities in the gut in a mouse model of colitis, and in response to TGF-β and IL-6 in vitro. TGF-β induced Runx1, and together with IL-6 was shown to render the ROR-γt and IL-17 promoters in Th1 cells accessible for Runx1 binding. In this commentary, we discuss how this unexpected plasticity of Th1 cells challenges our view on the generation of Th1/17 cells with the capacity to co-produce IL-17 and IFN-γ, and consider possible implications of this Th1-to-Th17-cell conversion for therapies of inflammatory bowel diseases and protective immune responses against intracellular pathogens.
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http://dx.doi.org/10.1002/eji.201646618DOI Listing
October 2016

Uncontrolled IL-17 Production by Intraepithelial Lymphocytes in a Case of non-IPEX Autoimmune Enteropathy.

Clin Transl Gastroenterol 2016 Jul 14;7(7):e182. Epub 2016 Jul 14.

Department of Pathophysiology and Transplantation, University of Milan, Milan, Italy.

Objectives: To provide a functional and phenotypic characterization of immune cells infiltrating small intestinal mucosa during non-IPEX autoimmune enteropathy (AIE), as to gain insights on the pathogenesis of this clinical condition.

Methods: Duodenal biopsies from a patient with AIE at baseline and following drug-induced remission were analyzed by immunohistochemistry, immunofluorescence, and flow cytometry, and results were compared with those obtained from patients with active celiac disease, ileal Crohn's disease and healthy controls. Lamina propria (LP) and intraepithelial (IELs) lymphocytes from AIE and controls were analyzed for mechanisms regulating cytokine production. Foxp3 expression and suppressive functions of LP regulatory T cells (Tregs) were analyzed.

Results: The quantitative deficit of Foxp3 expression in Tregs in AIE associates with unrestrained IL-17 production by IELs. Interleukin (IL)-17-producing IELs were rare in the uninflamed duodenum and in the ileum of Crohn's disease patients, and disappeared upon drug-induced AIE remission. IL-17 upregulation in CD4(+)IELs and CD4(+)LP T cells had different requirements for pro-inflammatory cytokines. Moreover, transforming growth factor-β (TGF-β) selectively enhanced IL-17 production by CD8(+)IELs. Intriguingly, although Foxp3(low)Tregs in AIE were poorly suppressive, they could upregulate GARP-LAP/TGF-β surface expression and enhanced IL-17 production selectively by CD8(+)IELs. Finally, phosphorylated Smad2/3 was detectable in duodenal CD8(+) lymphocytes in active AIE in situ, indicating that they received signals from the TGF-β receptor in vivo.

Conclusions: AIE is characterized by the appearance of unconventional IL-17-producing IELs, which could be generated locally by pro-inflammatory cytokines and TGF-β. These results suggest that Foxp3(+)Tregs and Treg-derived TGF-β regulate IL-17 production by IELs in the small intestine and in AIE.
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http://dx.doi.org/10.1038/ctg.2016.41DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5543485PMC
July 2016

The Adipose Mesenchymal Stem Cell Secretome Inhibits Inflammatory Responses of Microglia: Evidence for an Involvement of Sphingosine-1-Phosphate Signalling.

Stem Cells Dev 2016 07 27;25(14):1095-107. Epub 2016 Jun 27.

1 Laboratory of Experimental Neurosurgery and Cell Therapy, Neurosurgery Unit, Fondazione IRCCS Ca'Granda Ospedale Maggiore Policlinico, University of Milan , Milan, Italy .

Central nervous system (CNS) inflammation is primarily driven by microglial cells which secrete proinflammatory cytokines and undergo proliferation upon activation, as it occurs in neurodegenerative diseases. Uncontrolled or prolonged CNS inflammation is potentially harmful and can result in cellular damage. Recently, many studies have focused on human adipose tissue as an attractive source of cytokines with immunosuppressive properties that potentially modulate inflammation. Our study aimed to evaluate if different methods of human tissue collection could affect adipose mesenchymal stem cell (ADSC)-derived cytokine secretion and investigate the effects of ADSC secretome in modulating microglia activation and the possible implication of sphingosine-1-phosphate (S1P) in these effects. Our results demonstrate that the conditioned medium (CM) of ADSCs isolated by two different processing methods (lipoaspirate and Lipogems) significantly inhibited the lipopolysaccharide (LPS)-induced effects on microglia activation, including microglial expression of CD68, cytokine secretion, proliferation, and migration. Pulse studies with radiolabeled sphingosine demonstrated that LPS treatment of resting microglia induced a significant increase of both cellular and extracellular S1P. Moreover, and of relevance, FTY720, a functional antagonist of S1P receptor, inhibited the multiple LPS-induced proinflammatory effects on microglia, and S1P suppressed the anti-inflammatory effect of ADSC-CM. This suggests that LPS-mediated microglial activation is countered by ADSC-CM through the modulation of sphingosine kinase/S1P signalling.
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http://dx.doi.org/10.1089/scd.2015.0268DOI Listing
July 2016

IL-10 promotes homeostatic proliferation of human CD8(+) memory T cells and, when produced by CD1c(+) DCs, shapes naive CD8(+) T-cell priming.

Eur J Immunol 2016 07 17;46(7):1622-32. Epub 2016 May 17.

Istituto Nazionale di Genetica Molecolare, "Romeo ed Enrica Invernizzi", Milan, Italy.

IL-10 is an anti-inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8(+) CTL, IL-10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL-10 produced by human APC subsets in T-cell responses. IL-10 production was restricted to CD1c(+) DCs and CD14(+) monocytes. Interestingly, it was differentially regulated, since R848 induced IL-10 in DCs, but inhibited IL-10 in monocytes. Autocrine IL-10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high-affinity peptides. Nevertheless, it completely blocked cross-priming and priming with low-affinity peptides of a self/tumor-antigen. IL-10 also inhibited CD1c(+) DC-induced CD4(+) T-cell priming and enhanced Foxp3 induction, but was insufficient to induce T-cell IL-10 production. CD1c(+) DC-derived IL-10 had also no effect on DC-induced secondary expansions of memory CTL. However, IL-15-driven, TCR-independent proliferation of memory CTL was enhanced by IL-10. We conclude that DC-derived IL-10 selects high-affinity CTL upon priming. Moreover, IL-10 preserves established CTL memory by enhancing IL-15-dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL-10 promotes CTL responses in humans.
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http://dx.doi.org/10.1002/eji.201546136DOI Listing
July 2016

The light and the dark sides of Interleukin-10 in immune-mediated diseases and cancer.

Cytokine Growth Factor Rev 2016 08 8;30:87-93. Epub 2016 Mar 8.

Department of Immunobiology, Yale University School of Medicine, New Haven, Connecticut, USA; Howard Hughes Medical Institute, New Haven, Connecticut, USA.

Interleukin-10 (IL-10) is known to be a tolerogenic cytokine since it inhibits pro-inflammatory cytokine production and T cell stimulatory capacities of myeloid cells, such as macrophages and dendritic cells. In particular, it has a non-redundant tolerogenic role in intestinal immune homeostasis, since mice and patients with genetic defects in the IL-10/IL-10R pathway develop spontaneously colitis in the presence of a normal intestinal flora. However, IL-10 is also a growth and differentiation factor for B-cells, can promote autoantibody production and has consequently a pathogenic role in systemic lupus erythematosus. Moreover, IL-10 can promote cytotoxic T-cell (CTL) responses and this immunogenic activity might be relevant in type-1 diabetes and anti-tumor immune responses. This review summarizes these paradoxic effects of IL-10 on different types of immune responses, and proposes that different cellular sources of IL-10, in particular IL-10-secreting helper and regulatory T-cells, have different effects on B-cell and CTL responses. Based on this concept we discuss the rationales for targeting the IL-10 pathway in immune-mediated diseases and cancer.
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http://dx.doi.org/10.1016/j.cytogfr.2016.02.003DOI Listing
August 2016

Immunity to Pathogens Taught by Specialized Human Dendritic Cell Subsets.

Front Immunol 2015 13;6:527. Epub 2015 Oct 13.

Istituto Nazionale di Genetica Molecolare "Romeo ed Enrica Invernizzi" (INGM) , Milan , Italy ; DISCCO, Department of Clinical Sciences and Community Health, University of Milano , Milan , Italy.

Dendritic cells (DCs) are specialized antigen-presenting cells (APCs) that have a key role in immune responses because they bridge the innate and adaptive arms of the immune system. They mature upon recognition of pathogens and upregulate MHC molecules and costimulatory receptors to activate antigen-specific CD4(+) and CD8(+) T cells. It is now well established that DCs are not a homogeneous population but are composed of different subsets with specialized functions in immune responses to specific pathogens. Upon viral infections, plasmacytoid DCs (pDCs) rapidly produce large amounts of IFN-α, which has potent antiviral functions and activates several other immune cells. However, pDCs are not particularly potent APCs and induce the tolerogenic cytokine IL-10 in CD4(+) T cells. In contrast, myeloid DCs (mDCs) are very potent APCs and possess the unique capacity to prime naive T cells and consequently to initiate a primary adaptive immune response. Different subsets of mDCs with specialized functions have been identified. In mice, CD8α(+) mDCs capture antigenic material from necrotic cells, secrete high levels of IL-12, and prime Th1 and cytotoxic T-cell responses to control intracellular pathogens. Conversely, CD8α(-) mDCs preferentially prime CD4(+) T cells and promote Th2 or Th17 differentiation. BDCA-3(+) mDC2 are the human homologue of CD8α(+) mDCs, since they share the expression of several key molecules, the capacity to cross-present antigens to CD8(+) T-cells and to produce IFN-λ. However, although several features of the DC network are conserved between humans and mice, the expression of several toll-like receptors as well as the production of cytokines that regulate T-cell differentiation are different. Intriguingly, recent data suggest specific roles for human DC subsets in immune responses against individual pathogens. The biology of human DC subsets holds the promise to be exploitable in translational medicine, in particular for the development of vaccines against persistent infections or cancer.
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http://dx.doi.org/10.3389/fimmu.2015.00527DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4603245PMC
November 2015

Signal Strength and Metabolic Requirements Control Cytokine-Induced Th17 Differentiation of Uncommitted Human T Cells.

J Immunol 2015 Oct 16;195(8):3617-27. Epub 2015 Sep 16.

Istituto Nazionale di Genetica Molecolare Romeo ed Enrica Invernizzi, 20122 Milan, Italy;

IL-17 production defines Th17 cells, which orchestrate immune responses and autoimmune diseases. Human Th17 cells can be efficiently generated with appropriate cytokines from precommitted precursors, but the requirements of uncommitted T cells are still ill defined. In standard human Th17 cultures, IL-17 production was restricted to CCR6(+)CD45RA(+) T cells, which expressed CD95 and produced IL-17 ex vivo, identifying them as Th17 memory stem cells. Uncommitted naive CD4(+) T cells upregulated CCR6, RORC2, and IL-23R expression with Th17-promoting cytokines but in addition required sustained TCR stimulation, late mammalian target of rapamycin (mTOR) activity, and HIF-1α to produce IL-17. However, in standard high-density cultures, nutrients like glucose and amino acids became progressively limiting, and mTOR activity was consequently not sustained, despite ongoing TCR stimulation and T cell proliferation. Sustained, nutrient-dependent mTOR activity also induced spontaneous IL-22 and IFN-γ production, but these cytokines had also unique metabolic requirements. Thus, glucose promoted IL-12-independent Th1 differentiation, whereas aromatic amino acid-derived AHR ligands were selectively required for IL-22 production. The identification of Th17 memory stem cells and the stimulation requirements for induced human Th17/22 differentiation have important implications for T cell biology and for therapies targeting the mTOR pathway.
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http://dx.doi.org/10.4049/jimmunol.1501016DOI Listing
October 2015

Th17 cells transdifferentiate into regulatory T cells during resolution of inflammation.

Nature 2015 Jul 29;523(7559):221-5. Epub 2015 Apr 29.

1] Department of Immunobiology, School of Medicine, Yale University, New Haven, 06520, USA [2] Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520, USA.

Inflammation is a beneficial host response to infection but can contribute to inflammatory disease if unregulated. The Th17 lineage of T helper (Th) cells can cause severe human inflammatory diseases. These cells exhibit both instability (they can cease to express their signature cytokine, IL-17A) and plasticity (they can start expressing cytokines typical of other lineages) upon in vitro re-stimulation. However, technical limitations have prevented the transcriptional profiling of pre- and post-conversion Th17 cells ex vivo during immune responses. Thus, it is unknown whether Th17 cell plasticity merely reflects change in expression of a few cytokines, or if Th17 cells physiologically undergo global genetic reprogramming driving their conversion from one T helper cell type to another, a process known as transdifferentiation. Furthermore, although Th17 cell instability/plasticity has been associated with pathogenicity, it is unknown whether this could present a therapeutic opportunity, whereby formerly pathogenic Th17 cells could adopt an anti-inflammatory fate. Here we used two new fate-mapping mouse models to track Th17 cells during immune responses to show that CD4(+) T cells that formerly expressed IL-17A go on to acquire an anti-inflammatory phenotype. The transdifferentiation of Th17 into regulatory T cells was illustrated by a change in their signature transcriptional profile and the acquisition of potent regulatory capacity. Comparisons of the transcriptional profiles of pre- and post-conversion Th17 cells also revealed a role for canonical TGF-β signalling and consequently for the aryl hydrocarbon receptor (AhR) in conversion. Thus, Th17 cells transdifferentiate into regulatory cells, and contribute to the resolution of inflammation. Our data suggest that Th17 cell instability and plasticity is a therapeutic opportunity for inflammatory diseases.
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http://dx.doi.org/10.1038/nature14452DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4498984PMC
July 2015

The long intergenic noncoding RNA landscape of human lymphocytes highlights the regulation of T cell differentiation by linc-MAF-4.

Nat Immunol 2015 Mar 26;16(3):318-325. Epub 2015 Jan 26.

Istituto Nazionale Genetica Molecolare "Romeo ed Enrica Invernizzi", 20122 Milano, Italy.

Long noncoding RNAs are emerging as important regulators of cellular functions, but little is known of their role in the human immune system. Here we investigated long intergenic noncoding RNAs (lincRNAs) in 13 subsets of T lymphocytes and B lymphocytes by next-generation sequencing-based RNA sequencing (RNA-seq analysis) and de novo transcriptome reconstruction. We identified over 500 previously unknown lincRNAs and described lincRNA signatures. Expression of linc-MAF-4, a chromatin-associated lincRNA specific to the TH1 subset of helper T cells, was inversely correlated with expression of MAF, a TH2-associated transcription factor. Downregulation of linc-MAF-4 skewed T cell differentiation toward the TH2 phenotype. We identified a long-distance interaction between the genomic regions of the gene encoding linc-MAF-4 and MAF, where linc-MAF-4 associated with the chromatin modifiers LSD1 and EZH2; this suggested that linc-MAF-4 regulated MAF transcription through the recruitment of chromatin modifiers. Our results demonstrate a key role for lincRNA in T lymphocyte differentiation.
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http://dx.doi.org/10.1038/ni.3093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4333215PMC
March 2015
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