Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are Abundant

102 Pages·2017·4.67 MB·English

by  Ahmed N. Hegazy

Accepted Manuscript
Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal
Microbiota Are Abundant in Healthy Individuals and Function is Altered During
Inflammation
Ahmed N. Hegazy, Nathaniel R. West, Michael J.T. Stubbington, Emily Wendt, Kim
I.M. Suijker, Angeliki Datsi, Sebastien This, Camille Danne, Suzanne Campion,
Sylvia H. Duncan, Benjamin M.J. Owens, Holm H. Uhlig, Andrew McMichael,
Andreas Bergthaler, Sarah A. Teichmann, Satish Keshav, Fiona Powrie
PII: S0016-5085(17)35979-6
DOI: 10.1053/j.gastro.2017.07.047
Reference: YGAST 61339
To appear in: Gastroenterology
Accepted Date: 25 July 2017
Please cite this article as: Hegazy AN, West NR, Stubbington MJT, Wendt E, Suijker KIM, Datsi A,
This S, Danne C, Campion S, Duncan SH, Owens BMJ, Uhlig HH, McMichael A, Oxford IBD Cohort
Investigators, 
 Bergthaler A, Teichmann SA, Keshav S, Powrie F, Circulating and Tissue-resident CD4+
T Cells With Reactivity to Intestinal Microbiota Are Abundant in Healthy Individuals and Function is
Altered During Inflammation, Gastroenterology (2017), doi: 10.1053/j.gastro.2017.07.047.
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CFSE
A Memory CD4+ T cells labelling
MACS/FACS (CD3+ CD4+ CFSE
CD45RA- ) dilution
Expansion
3-7 days
Co-culture C
MACS Incubation with 100 ****
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103 103 103 103 Day 3 w of 40 T
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ICOS 111100002345 105.00.11102 103 104 1.69105 111100002345 106.00.27102 103 104 1015 111100002345 107.00.15102 103 104 1015 111100002345 400.00.43102 103 104 110.59 Day 6 CFno m0icrobSe. Ma. utRruebuesrIculosiPs
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CFSE
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s (%) 15 ******** 20 ******** 10 ******** 40 ******** 15 ******** U15 ****** 25 ********
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Supplementary Figure S4
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Manuscript Number: GASTRO-D-16-01275R1

Title: Circulating and Tissue-resident CD4+ T Cells With Reactivity to Intestinal Microbiota Are
Abundant in Healthy Individuals and Function is Altered During Inflammation
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Authors: Ahmed N. Hegazy1,2,10, Nathaniel R. West1,2,10, Michael J. T. Stubbington3,4, Emily
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Wendt1, Kim I. M. Suijker2, Angeliki Datsi1, Sebastien This1, Camille Danne2, RSuzanne Campion5,
Sylvia H. Duncan6, Benjamin M. J. Owens1, Holm H. Uhlig1,7, Andrew McMichael5, Oxford IBD
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Cohort Investigators8, (cid:1)Andreas Bergthaler9, Sarah A. Teichmann3,4, Satish Keshav1, Fiona
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Powrie1,2 U

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Affiliations: A
1Translational Gastroenterology Unit, Nuffield DepMartment of Clinical Medicine, Experimental
Medicine Division, John Radcliffe Hospital, University of Oxford, UK

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2Kennedy Institute of Rheumatology, Nuffield Department of Orthopaedics, Rheumatology and
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Musculoskeletal Sciences, University of Oxford, UK(cid:1)
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3European Molecular Biology Laboratory-European Bioinformatics Institute, Hinxton, UK
4Wellcome Trust Sanger InstitutPe, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
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5Nuffield Department of Medicine Research Building, University of Oxford, Oxford, UK
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6Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, UK
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7Department of Paediatrics, University of Oxford, Oxford, UK
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8Individual investigators are listed in the acknowledgement section
9CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna,
Austria
10Co-first authors
Grant support: ANH was supported by an EMBO long-term fellowship and a Marie Curie
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fellowship. NRW was supported by a CRI Irvington Post-doctoral Fellowship. BMJO was
supported by an Oxford-UCB Pharma Postdoctoral Fellowship. MJTS and SAT were supported by
ERC grant ThSWITCH and ThDEFINE (260507). SC and AM were supported by the Center for
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HIV/AIDS Vaccine Immunology and Immunogen Discovery (grant UM1-AI100645). The Rowett
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Institute of Nutrition and Health receives financial support from the Scottish Government Rural and
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Environmental Sciences and Analytical Services (SG-RESAS). Foundation Louis Jeantet,
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Wellcome Trust (Investigator award 095688/Z/11/Z), and ERC (ERC/HN/2013/21) supported FP
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and this project. HHU is supported by the Crohn’s & Colitis Foundation of America (CCFA), The
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Leona M. and Harry B. Helmsley Charitable Trust. SD receive financial support from the Scottish
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Government Rural and Environmental Sciences and Analytical Services (RESAS).
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Abbreviations: Antigen presenting cells (APCs), Brefeldin A (BFA), Carboxyfluorescein
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succinimidyl ester (CFSE), Central memory (CM), Chemokine ligand (CCL), Chemokine receptor
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(CCR), Crohn’s disease (CD), Effector memory (EM), Fluorescence minus one (FMO),

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Gastrointestinal tract (GIT), GATA-binding factor-3 (GATA-3), Inflammatory bowel disease
(IBD), Interferon-gamma (IFN-g ), InterlEeukin (IL-), Lamina propria mononuclear cells (LPMCs),
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Lipopolysaccharide (LPS), Magnetic cell separation (MACS), Major histocompatibility complex
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(MHC), Peripheral blood mononuclear cell (PBMC), Phytohaemagglutinin (PHA), RAR-related
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orphan receptor gamma t (RORg t), Regulatory T cells (Treg), Staphylococcus enterotoxin B (SEB),
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T cell receptor (TCR), T helper (Th), T-box-expressed-in-T-cells (T-bet), Tumour necrosis factor
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alpha (TNF-a ), Ulcerative colitis (UC), Violet proliferation dye (VPD).
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Corresponding author: Prof. Fiona Powrie, FRS, Kennedy Institute of Rheumatology, University of
Oxford, Roosevelt Drive, Headington, Oxford, OX3 7FY, UK
Email: [email protected]; Tel: [phone]

Disclosures: These authors disclose the following: F.P. has received research support or
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consultancy fees from Eli Lilly, Merck, GSK, Janssen, Compugen, UCB, and MedImmune. S.K.
has received consulting fees and research support from ChemoCentryx Inc. and GSK in the past.
The remaining authors declare no conflict of interest. HHU has project collaboration with Eli Lilly
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and UCB Pharma related to this project. Dr Keshav has provided consultancy services for a number
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of pharmaceutical and healthcare companies including Abbvie, Actavis Allergan, Astra-Zeneca,
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Boehringer Ingelheim, ChemoCentryx, Dr Falk Pharma, Ferring, Gilead, GSK, Merck, Mitsubishi
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Tanabe Pharma, Pharmacosmos, Pfizer, Takeda, and Vifor Pharma, and received research support
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from Abbvie, ChemoCentryx, GSK, and Merck.
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Transcript Profiling: None
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Writing Assistance: None
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Author Contributions: ANH, NRW designed, performed, and analysed experiments. ANH, NRW,
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and FP conceived and designed the project, interpreted data, and wrote the manuscript. MJTS
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analysed TCR sequencing data. EW, KS, AD, ST, SC, BMJO, CD, SHD, AB were involved in

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acquisition of data, data analysis and interpretation of data. SAT, SK, HHU, AM provided essential
materials and were involved in dataE interpretation and discussions. Oxford IBD Cohort
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Investigators provided IBD patient samples and ethical approval for the project. The Oxford IBD
Cohort Investigators are: Dr. CParolina Arancibia, Dr. Adam Bailey, Dr. Ellie Barnes, Dr. (cid:1)Beth
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Bird-Lieberman, Dr. Oliver Brain, Dr. Barbara Braden, Dr. Jane Collier, Dr. James East, Dr. Lucy
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Howarth, Dr. Satish Keshav, Dr. Paul Klenerman, Dr. Simon Leedham, Dr. Rebecca (cid:1)Palmer, Dr.
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Fiona Powrie, Dr. Astor Rodrigues, Dr. Alison Simmons, Dr. Peter Sullivan, Dr. Simon Travis, Dr.
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Holm Uhlig. (cid:1)
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Abstract (250 words):
Background & Aims: Interactions between commensal microbes and the immune system are
tightly regulated and maintain intestinal homeostasis, but little is known about these interactions in
humans. We investigated responses of human CD4+ T cells to the intestinal microbiota. We
measured the abundance of T cells in circulation and intestinal tissues that respond to Tintestinal
microbes and determined their clonal diversity. We also assessed their functional phenotypes and
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effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in
patients with chronic intestinal inflammation. I
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Methods: We collected samples of peripheral blood mononuclear cells (PBMC) and intestinal
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tissues from healthy individuals (controls, n=13–30) and patients with inflammatory bowel diseases
(IBD, total n=119; 59 with UC and 60 with Crohn’s disease). We used 2 independent assays
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(CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal
bacterial species (Escherichia coli, Lactobacillus acidophilus, BUifidobacterium animalis subsp.
lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus
obeum, Salmonella typhimurium and Clostridium difficile) tNo quantify, expand, and characterize
microbe-reactive CD4+ T cells. We sequenced T-cell receptor vb genes in expanded microbe-
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reactive T-cell lines to determine their clonal diversity. We examined the effects of microbe-
reactive CD4+ T cells on intestinal stromal and epithelial cell lines. Cytokines, chemokines, and
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gene expression patterns were measured by flow cytometry and quantitative PCR.
Results: Circulating and gut-resident CD4+ T cells from controls responded to bacteria at
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frequencies of 40–4000 per million for each bacterial species tested. Microbiota-reactive CD4+ T
cells were mainly of a memory phenotype, present in PBMCs and intestinal tissue, and had a
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diverse T-cell receptor vb repertoire. These cells were functionally heterogeneous, produced barrier
protective cytokines, and stimulateTd intestinal stromal and epithelial cells via interleukin 17A
(IL17A), interferon gamma, and tumor necrosis factor. In patients with IBD, microbiota-reactive
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CD4+ T cells were reduced in the blood compared to intestine; T-cell responses we detected had an
increased frequency of IL17A production compared to responses of T cells from blood or intestinal
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tissues of controls.
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Conclusions: In an analysis of PBMC and intestinal tissues from patients with IBD vs controls, we
found that reactivitCy to intestinal bacteria is a normal property of the human CD4+ T-cell repertoire,
and does not necessarily indicate disrupted interactions between immune cells and the commensal
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microbiota. T-cell responses to commensals might support intestinal homeostasis, by producing
barrier-protective cytokines and providing a large pool of T cells that react to pathogens.

KEY WORDS: IFNG, TNF, immune regulation; activation

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Introduction
Vast numbers of microbes populate the gastrointestinal (GI) tract and contribute to digestion,
epithelial barrier integrity, and the development of appropriately educated mucosal immunity1.
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Intestinal immune responses are tightly regulated to allow protective immunity against pathogens
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while limiting responses to dietary antigens and innocuous microbes. The ‘mucosal firewall’
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prevents systemic dissemination of microbes by confining microbial antigens tRo the gut-associated
lymphoid tissue (GALT)2. In the GALT, dendritic cells drive regulatory CT cell differentiation in
response to dietary antigens and commensal bacteria3. Nevertheless, vast numbers of potentially
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commensal-reactive effector and memory T cells populate intesUtinal mucosae4. Recent evidence
suggests that in mice, tolerance to commensal-deriveNd antigens may be lost during
pathogen-induced epithelial damage and subsequent tAransient exposure to commensals5,6. In
humans, circulating memory T cells recognise pMeptides derived from gut bacteria and can
cross-react to pathogens, which may confer immunological advantage during subsequent new

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infections7,8. While this process may be beneficial during homeostasis, deranged responses to
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commensals may promote inflammatory conditions such as inflammatory bowel disease (IBD).
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IBD (including Crohn’s disease (CD) and ulcerative colitis (UC)) results from a prolonged
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disturbance of gut homeostasis, the precise aetiology of which is uncertain. One hypothesis is that,
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in genetically susceptible individuals, IBD may be triggered by intestinal dysbiosis that promotes
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aberrant immune stimulation9. Indeed, in mouse models of colitis, intestinal microbiota promote
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inflammation in part by stimulating microbiota-reactive CD4+ T cells5,10. Whether this drives IBD
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in humans, however, remains unknown.
Although CD4+ T cell responses to intestinal bacteria are known to occur in humans11-13,
several aspects of this topic are largely uncharacterised, including (a) the frequency of human T
cells in the gut and periphery that are reactive to phylogenetically distinct intestinal microbes; (b)
the T cell receptor diversity and clonotype sharing of these T cells; (c) the functional phenotype of
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gut microbe-reactive T cells and their impact on tissue-resident cell populations; and (d) how
microbe-reactive T cells change during chronic intestinal inflammation. To address this knowledge
gap, we extensively characterised CD4+ T cell responses to intestinal microbiota in healthy
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individuals and IBD patients.
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Using two independent assays, we observed that for almost all enteric bacteria examined,
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bacteria-reactive CD4+ T cells were present at a frequency of 40 to 500 per million CD4+ T cells in
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adult peripheral blood. Bacteria-reactive T cells were also prevalent in the gut mucosa, with
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prominent enrichment for proteobacteria-reactivity. Microbiota-responsive T cells showed a diverse
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TCRVb repertoire and potently stimulated inflammatory responUses by intestinal epithelial and
stromal cells. Intriguingly, T cells from IBD patients displayed a normal spectrum of microbial
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responses, but expressed high amounts of IL-17A, Aconsistent with increased amounts of
Th17-polarising cytokines in inflamed intestinal tisMsue. Collectively, these data demonstrate that
microbiota-reactive CD4+ T cells are prevalent and normal constituents of the human immune

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system that are functionally altered during IBD pathogenesis.
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Materials and Methods
Human Samples and Cell Isolation. Leukoreduction chambers from healthy individuals were
obtained from the National Blood Service (Bristol, UK). Peripheral EDTA blood samples were
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obtained from IBD patients attending the John Radcliffe Hospital Gastroenterology unit or from
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healthy in-house volunteers. IBD patients (total, n=119; UC, n=59; CD, n=60) diagnosed by
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endoscopic, histological and radiological criteria were recruited for the study. Healthy volunteers
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(n=30) without any known underlying acute or chronic pathological condition served as control
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donors. Demographic and clinical characteristics of IBD patients are summarized in Supplementary
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Tables 5, 6, and 7. All donors provided informed, written consent. The NHS Research Ethics
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System provided ethical approval (reference numbers 09/H0606/5 for IBD patients and 11/YH/0020
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for controls; OCHRe ref 15/A237 for cord blood samples). For details regarding cell isolation, see
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Supplemental Experimental Procedures.
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CD154-based Detection of Antigen-Specific T Cells. CD154 detection was done as previously

described14,15. Briefly, cells were plated at 5Dx106/cm2 for 7-12h with heat-inactivated bacteria. 5
µg/ml brefeldin A (BFA, Sigma AldrichE) was added at 2 h. After 8-12 h, cells were harvested and
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treated as described in the Intracellular Cytokine, CD154 And Transcription Factor Staining section
(see below). For MACS enricPhment of CD4+CD154+ T cells, see Supplemental Experimental
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Procedures.
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Antigen-specific Recall Response (CFSE dilution assay) And T Cell Culture. Memory CD4+
CD45RO+ CD45RCA- T cells were enriched from PBMC with untouched memory CD4+ T cell
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enrichment kit (Miltenyi Biotec), sorted to >97% purity on a FACS ARIA III (BD) using CD45RA
and CD45RO expression, and were labelled with CFSE or Violet proliferation dye (VPD,
Invitrogen). CD14+ monocytes were isolated from PBMC using anti-CD14 microbeads (Miltenyi
Biotec), irradiated (45 Gy) and then pre-incubated for 3 h with bacterial lysates before T-cell
co-culture. T cells were co-cultured with the irradiated autologous monocytes at a ratio of 2:1 for 5-7
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days. Cells were cultured in RPMI-1640 supplemented with 2 mM glutamine, 1% (v/v) non-essential
amino acids, 1% (v/v) sodium pyruvate, penicillin (50 U/ml), streptomycin (50 mg/ml; all from
Invitrogen) and 5% (v/v) human serum (National Blood Service, Bristol, UK). CD14+ monocytes
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were irradiated (45 Gy) and then pre-incubated for 3 h with bacterial lysates before T-cell co-culture.
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For MHCII blockade, 10 µg/ml of a pan-HLA class-II blocking antibody (HLA-DR, DP, DQ;
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(Tü39)) was added 30 minutes before T-cell co-culture. T cell lines were generated by sorting
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CFSElow ICOShigh CD4+ T cells after seven days of stimulation and expanding them with IL-2 (300
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U/ml) and anti-CD3/CD28 beads (beads/T cell ratio, 1:4, Dynals) for 10-14 days. Supernatants were
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collected from 1x106 CD4+ T cells stimulated for 24h with PMA (5 ng/ml) and ionomycin (500
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ng/ml; Sigma).
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Flow Cytometry and Cell Sorting. PBMCs and LPMCs were stained according to standard
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protocols. For details, see Supplemental Experimental Procedures.
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Intracellular Cytokine, CD154, And Transcription Factor Staining. For intracellular cytokine

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staining, cells were stained with fixable viability dye eFluor® 780 (eBioscience) and surface
markers, fixed with 2% formaldehydeE (Merck), and stained for cytokines in MACS buffer
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containing 0.05% saponin (Sigma-Aldrich). Transcription factor expression was analysed using the
FoxP3 staining buffer set (eBiosPcience) according to manufacturer’s instructions.
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Stimulation of Intestinal Cell Lines. CCD18Co (non-transformed human colon fibroblasts,
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ATCC), and LIM1863 (human colon epithelial cells; a kind gift of Dr. Robert Whitehead, Ludwig
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Institute for Cancer Research) cells were cultured in humidified incubators with 5% CO at 37°C in
2
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DMEM or RPMI media (Sigma) with 10% FCS (Sigma) and 100 U penicillin/0.1 mg/ml
streptomycin. Cells were stimulated with 5% T cell supernatants for 24h. Cytokine neutralization
was achieved by supernatant pre-incubation for 1-2h with 10 µg/ml anti–IL17A (eBio64CAP17),
anti–IFN-g (B27), anti–TNF-a (Remicade), and anti–IL22 (IL22JOP).
Statistics. Statistical analyses were performed with GraphPad Prism v6.0 for Macintosh
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