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Human Regulatory T Cells Mediate Transcriptional Modulation of Dendritic Cell Function Human Regulatory T Cells Mediate Transcriptional Modulation of Dendritic Cell Function This information is current as Emily Mavin, Lindsay Nicholson, Syed Rafez Ahmed, Fei of September 29, 2021. Gao, Anne Dickinson and Xiao-nong Wang J Immunol published online 28 November 2016 http://www.jimmunol.org/content/early/2016/11/26/jimmun ol.1502487 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2016/11/26/jimmunol.150248 Material 7.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2016 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published November 28, 2016, doi:10.4049/jimmunol.1502487 The Journal of Immunology Human Regulatory T Cells Mediate Transcriptional Modulation of Dendritic Cell Function Emily Mavin,*,1 Lindsay Nicholson,*,1 Syed Rafez Ahmed,* Fei Gao,† Anne Dickinson,* and Xiao-nong Wang* Regulatory T cells (Treg) attenuate dendritic cell (DC) maturation and stimulatory function. Current knowledge on the functional impact of semimature DC is limited to CD4+ T cell proliferation and cytokine production. Little is known about the molecular basis underpinning the functional effects of Treg-treated DC (Treg-DC). We present novel evidence that Treg-DC skewed CD4+ naive T cell polarization toward a regulatory phenotype and impaired CD8+ T cell allo-reactive responses, including their ability to induce target tissue damage in a unique in vitro human graft-versus-host disease skin explant model. Microarray analysis clustered Treg-DC as a discrete population from mature-DC and immature-DC, with 51 and 93 genes that were significantly over- or underexpressed, respectively, compared with mature-DC. Quantitative real-time PCR analysis revealed an intermediate Downloaded from expression level of CD38, CD83, CD80 and CD86 mRNA in Treg-DC, lower than mature-DC, higher than immature-DC. We also observed an attenuation of NF-kB pathway, an upstream regulator of the aforementioned genes, concomitant with reduced expression of two NF-kB-signaling related genes RELB and NFkBIZ, in the Treg-DC, together with an increased expression of Wnt5a, a negative regulator of DC differentiation. We further confirmed that the Treg-DC–mediated skewed CD4+ naive T cell polarization resulted from decreased IL-12 secretion by Treg-DC, which may be post-transcriptionally modulated by decreased expression of microRNA-155 in Treg-DC. To our knowledge, this is the first study demonstrating a transcriptional modulation of http://www.jimmunol.org/ DC function by human Treg, partially via attenuation of the NF-kB signaling pathway and upregulation of Wnt5a, suggesting Treg may interfere with DC reprogramming during maturation, thereby modulating DC function. The Journal of Immunology, 2017, 198: 000–000. egulatory T cells are of central importance for the main- However, previous studies have only reported a very narrow range tenance of peripheral tolerance and regulation of cellular of functional outcomes in terms of the effect of Treg-treated DC R immune responses. Despite originally being recognized for on target T cells, which were primarily limited to the ability of their suppressive function directly exerted on effector T cells (1–3), Treg-cultured DC to stimulate CD4+ T cell proliferation (4–6). by guest on September 29, 2021 it is now clear that regulatory T cells (Treg) can block effective Furthermore, little is known about the molecular basis underlying T cell priming by influencing dendritic cell (DC) functions. A the Treg-mediated modulation of DC function. Conflicting results number of studies have reported that DC can be arrested at a have been reported in murine studies about whether Treg modu- semimature status following coculture with Treg, expressing low late DC costimulatory molecule expression at the transcriptional levels of costimulatory molecules and high levels of HLA-DR, or posttranscriptional level (7, 8). Research on the modulation of rendering them deficient in initiating T cell proliferation (4, 5). DC function by human Treg is scarce. It is not known if human Treg can modulate DC phenotypic and functional properties at the transcriptional level. Our study aimed to extend limited knowl- *Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, NE2 edge in the functional impacts of Treg-treated DC on target T cells, 4HH, United Kingdom; and †Institute of Neuroscience, Newcastle University, New- castle upon Tyne, NE7 1RU, United Kingdom particularly in the context of allograft transplant complications 1E.M. and L.N. contributed equally to this work. where Treg have shown a promising therapeutic potential. We also ORCIDs: 0000-0002-2132-9011 (S.R.A.); 0000-0003-1797-0479 (F.G.); 0000-0002- sought to gain further insight into the molecular mechanisms by 7356-7636 (A.D.). which Treg modulate DC function. To address these research aims, Received for publication November 25, 2015. Accepted for publication October 21, we have generated monocyte-derived DCs with or without Treg 2016. coculture. The DCs were then purified from the coculture by FACS This work was supported by funding from CellEurope, the British Society for Hae- sorting following LPS challenge and used for subsequent functional matology, and the Wellcome Trust Institutional Strategic Support Fund. and molecular analysis. The functional impacts of Treg-treated DC The microarray data presented in this article have been submitted to the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) under accession number were investigated by examining their ability to uptake Ag, skew + + GSE72893. CD4 naive T cell polarization and elicit CD8 T cell alloreactive Address correspondence and reprint requests to Dr. Xiao-nong Wang, Haemato- responses including the induction of graft-versus-host target tissue logical Sciences, Newcastle University, Leech Building, The Medical School, damage using an in vitro human graft-versus-host disease (GvHD) Framlington Place, Newcastle upon Tyne NE2 4HH, U.K. E-mail address: X.N. [email protected] skin explant model. The Treg-treated DC were further subjected to The online version of this article contains supplemental material. gene expression profiling using the Illumina Human HT-12 micro- array. The transcript data analysis was focused on selected genes Abbreviations used in this article: CBA, cytometric bead array; DC, dendritic cell; GvHD, graft-versus-host disease; imm-DC, immature DC; mat-DC, mature DC; with known functions in regulating DC maturation, stimulatory func- miR-155, microRNA-155; qRT-PCR, quantitative real-time PCR; Treg, regulatory tion and cytokine production, as well as the genes involved in NF- T cell; Treg-DC, Treg-treated DC. kB signaling pathway activation to identify the molecular basis Copyright Ó 2016 by The American Association of Immunologists, Inc. 0022-1767/16/$30.00 underlying the observed functional impact. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1502487 2 HUMAN Treg MODULATE DC FUNCTION AT THE TRANSCRIPTIONAL LEVEL Materials and Methods IFN-g, IL-4, and FOXP3 as well as mRNA expression of TBX21 (T-bet), GATA-3 and FOXP3. Following restimulation with CD3/CD28 T cell ex- All samples were collected from healthy volunteers with informed consent pander beads at a 1:1 ratio for 24 h cytokine secretion of IFN-g, IL-4, and and approval from the local research ethics committee. Unless otherwise IL-10 in the culture supernatants was measured with CBA (BD Biosci- stated, all cell populations were isolated from leukocyte reduction system ences). For IL-12 neutralization experiments, human anti-IL-12 and cor- cones (National Blood Services, Newcastle, U.K.). responding polyclonal goat IgG (R&D Systems) were added at 5 mg/ml to + + Cell isolation and culture the coculture of CD4 naive T cells and mat-DC on day 0. The naive CD4 polarization was assessed following stimulation and resting as previously General culture medium (RF10) consisted of RPMI 1640 with 100 IU/ml mentioned. Following polarization of CD4+ naive T cells by Treg-DC, the penicillin, 100 mg/ml streptomycin, 2 mM L-glutamine, and 10% heat- FOXP3-expressing T cells were isolated by FACS sorting based on inactivated FCS (Invitrogen). Naturally occurring Treg were isolated by CD25highCD127low gating. The function of Treg-DC induced Treg was negative enrichment of CD4+ T cells (RosetteSep; StemCell Tech) fol- then assessed by their ability to suppress CD8+ T cell activation and pro- lowed by immuno-magnetic positive selection of CD25high cells (RoboSep; liferation in an allogeneic MLR setting as previously described (10). StemCell Tech). Treg were then activated and expanded for up to 3 wk by culturing with CD3/CD28 Dynabeads (Invitrogen), 500 IU/ml IL-2 Skin explant assay (Roche) and 100 nM rapamycin (Sigma) prior to use, as previously de- A well-established in vitro human GvHD skin explant model was used to scribed (9). All Treg were well characterized phenotypically and func- assess the ability of Treg-treated DC to induce cutaneous tissue damage tionally prior to use as described previously (10). Mature and immature mediated by alloreactive T cell responses (11). Punch skin biopsies and DCs (mat-DC and imm-DC) were generated from magnetically isolated + peripheral blood monocyte-derived DC were obtained from the same CD14 monocytes (Miltenyi) cultured with IL-4 and GM-CSF (50 ng/ml; healthy donor.
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