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.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. ACCEPTED MANUSCRIPT CFSE Memory CD4+ T cells labelling (CD3+ CD4+ MACS/FACS CFSE A CD45RA- ) dilution Expansion 3-7 days Co-culture C MACS Incubation with 100 **** Monocytes bacterial lysates CD14+ **** 80 S. typhimurium F. prausnitzii C. difficile S. aureus cells (%) 5 5 5 B 5 10 10 10 + 10 0.26 0.2 0.25 0.431 60
4 4 4 104 10 10 10 0.84 0.21 0.34 1.07
3 3 3 103 10 10 10 40 Day 3 of CD4 low
0 0 0 0 20 0.01 0.07 0.05 0.03
2 3 4 5 2 3 4 5 2 3 4 5 CFSE 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 1.69 5 5 5 ICOS 105 15.1 10 16.2 1 10 17.1 1 10 40.4 1.9 0
4 4 4 104 10 10 10
3 3 3 103 10 10 10 Day 6 S. aureus
2 2 2 no microbe 102 10 10 10 0 0 0 0 M. tuberculosis 0.77 4.39 1.72 0.9 3 4 5 0 103 104 105 0 103 104 105 0 103 104 105 0 10 10 10 CFSE
D E. coli S. typhimurium B. animalis L. acidophilus F. prausnitzii C. difficile S. aureus 15 **** 20 **** 10 **** 40 **** 15 **** 15 *** 25 **** **** **** **** **** **** *** **** 8 20 15 30 cells (%)
+ 10 10 10 6 15 10 20 4 10
of CD4 5 5 5 10 low ns 5 ns ns ns ns ns ns 2 5
0 0 0 0 0 0 0 CFSE + + + + + + +
microbe microbe microbe microbe microbe microbe -MHC II -MHC II -MHC II -MHC II -MHC II -MHC II microbe-MHC no II microbe microbeα no microbe microbeα no microbe microbeα no microbe microbeα no microbeMANUSCRIPT microbeα no microbe microbeα no microbe α E E. coli B. animalis L. acidophilus F 5 80 5 10 100 10 17 0.7 13.2 0.6 9.8 0.5 *** ***
**** **** **** **** **** **** **** **** **** **** **** **** **** **** **** ****
104 cells
103 + 80 cells (%) cells (%) + + 60
CD25 4 0 10 CD4 60 CD4 3.6 78.7 2.22 83.9 4.4 85.4 CD4 low 3 4 5 3 4 5 0 103 104 105 0 10 10 10 0 10 10 10 low low 40 105 8.58 0.632 8.4 0.5 7.4 1.2 40 104 3 (Geo mean) 10
103 20 of CFSE of CFSE 20 + +
OX40 0
12 78.8 6.9 84.2 6.5 85 ICOS of CFSE 2 3 4 5 3 4 5 3 4 5 0 0 10 10 10 0 10 10 10 0 10 10 10 10 CD25 0 OX40
high high high CFSE E. coli E. coli E. coli CFSE CFSE CFSE C. difficile C. difficile C. difficile B. animalis B. animalis B. animalis F. prausnitzii F. prausnitzii F. prausnitzii S. typhimuriumL. acidophilus S. typhimuriumL. acidophilus S. typhimuriumL. acidophilus Shared clonotypes, TCRVβ Donor 1 Donor 2 Donor 3 (%) G H 10 105 E. coli s
e S. typhimurium 8 p 104 y ACCEPTEDB. animalis lono t 3 6 c
10
f L. acidophilus o r
e F. prausnitzii 4 b 102 m
u C. difficile N 1 2 10 SEB rium u PHA SEB E. coli PHA 0 phim C. difficile B. animalis F. prausnitzii S. ty L. acidophilus rium rium rium u SEB PHA u SEB PHA u SEB PHA E. coli E. coli E. coli C. difficile C. difficile C. difficile phim B. animalis phim B. animalis phim B. animalis F. prausnitzii F. prausnitzii F. prausnitzii S. ty L. acidophilus S. ty L. acidophilus S. ty L. acidophilus Supplementary Figure S4 ACCEPTED MANUSCRIPT
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
Authors: Ahmed N. Hegazy 1,2,10 , Nathaniel R. West 1,2,10 , Michael J. T. Stubbington 3,4 , Emily
Wendt 1, Kim I. M. Suijker 2, Angeliki Datsi 1, Sebastien This 1, Camille Danne 2, Suzanne Campion 5,
Sylvia H. Duncan 6, Benjamin M. J. Owens 1, Holm H. Uhlig 1,7, Andrew McMichael 5, Oxford IBD
Cohort Investigators 8, Andreas Bergthaler 9, Sarah A. Teichmann 3,4 , Satish Keshav 1, Fiona
Powrie 1,2
Affiliations:
1Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, Experimental
Medicine Division, John Radcliffe Hospital, University of Oxford, UK 2Kennedy Institute of Rheumatology, Nuffield Departm MANUSCRIPTent of Orthopaedics, Rheumatology and Musculoskeletal Sciences, University of Oxford, UK
3European Molecular Biology Laboratory-European Bioinformatics Institute, Hinxton, UK
4Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, UK
5Nuffield Department of Medicine Research Building, University of Oxford, Oxford, UK
6Microbial Ecology Group, Rowett Institute of Nutrition and Health, University of Aberdeen, UK
7Department of Paediatrics, University of Oxford, Oxford, UK
8Individual investigators are listed in the acknowledgement section 9CeMM ResearchACCEPTED Center for Molecular Medicine of the Austrian Academy of Sciences, Vienna, Austria
10 Co-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
HIV/AIDS Vaccine Immunology and Immunogen Discovery (grant UM1-AI100645). The Rowett
Institute of Nutrition and Health receives financial support from the Scottish Government Rural and
Environmental Sciences and Analytical Services (SG-RESAS). Foundation Louis Jeantet,
Wellcome Trust (Investigator award 095688/Z/11/Z), and ERC (ERC/HN/2013/21) supported FP and this project. HHU is supported by the Crohn’s & Colitis Foundation of America (CCFA), The
Leona M. and Harry B. Helmsley Charitable Trust. SD receive financial support from the Scottish
Government Rural and Environmental Sciences and Analytical Services (RESAS).
Abbreviations: Antigen presenting cells (APCs), Brefeldin A (BFA), Carboxyfluorescein succinimidyl ester (CFSE), Central memory (CM), Chemokine ligand (CCL), Chemokine receptor
(CCR), Crohn’s disease (CD), Effector memory (EM), Fluorescence minus one (FMO), Gastrointestinal tract (GIT), GATA-binding factor-3 MANUSCRIPT (GATA-3), Inflammatory bowel disease (IBD), Interferon-gamma (IFN-γ), Interleukin (IL-), Lamina propria mononuclear cells (LPMCs),
Lipopolysaccharide (LPS), Magnetic cell separation (MACS), Major histocompatibility complex
(MHC), Peripheral blood mononuclear cell (PBMC), Phytohaemagglutinin (PHA), RAR-related orphan receptor gamma t (ROR γt), Regulatory T cells (Treg), Staphylococcus enterotoxin B (SEB),
T cell receptor (TCR), T helper (Th), T-box-expressed-in-T-cells (T-bet), Tumour necrosis factor alpha (TNF-α), Ulcerative colitis (UC), Violet proliferation dye (VPD).
Corresponding author: Prof. Fiona Powrie, FRS, Kennedy Institute of Rheumatology, University of Oxford, RooseveltACCEPTED Drive, Headington, Oxford, OX3 7FY, UK Email: [email protected]; Tel: +44 (0)1865 612 659
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 and UCB Pharma related to this project. Dr Keshav has provided consultancy services for a number of pharmaceutical and healthcare companies including Abbvie, Actavis Allergan, Astra-Zeneca,
Boehringer Ingelheim, ChemoCentryx, Dr Falk Pharma, Ferring, Gilead, GSK, Merck, Mitsubishi
Tanabe Pharma, Pharmacosmos, Pfizer, Takeda, and Vifor Pharma, and received research support from Abbvie, ChemoCentryx, GSK, and Merck.
Transcript Profiling: None
Writing Assistance: None
Author Contributions: ANH, NRW designed, performed, and analysed experiments. ANH, NRW, and FP conceived and designed the project, interpreted data, and wrote the manuscript. MJTS analysed TCR sequencing data. EW, KS, AD, ST, SC, BMJO, CD, SHD, AB were involved in acquisition of data, data analysis and interpretation ofMANUSCRIPT data. SAT, SK, HHU, AM provided essential materials and were involved in data interpretation and discussions. Oxford IBD Cohort
Investigators provided IBD patient samples and ethical approval for the project. The Oxford IBD
Cohort Investigators are: Dr. Carolina Arancibia, Dr. Adam Bailey, Dr. Ellie Barnes, Dr. Beth
Bird-Lieberman, Dr. Oliver Brain, Dr. Barbara Braden, Dr. Jane Collier, Dr. James East, Dr. Lucy
Howarth, Dr. Satish Keshav, Dr. Paul Klenerman, Dr. Simon Leedham, Dr. Rebecca Palmer, Dr.
Fiona Powrie, Dr. Astor Rodrigues, Dr. Alison Simmons, Dr. Peter Sullivan, Dr. Simon Travis, Dr.
Holm Uhlig. ACCEPTED
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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 intestinal microbes and determined their clonal diversity. We also assessed their functional phenotypes and effects on intestinal resident cell populations, and studied alterations in microbe-reactive T cells in patients with chronic intestinal inflammation.
Methods : We collected samples of peripheral blood mononuclear cells (PBMC) and intestinal 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 (CD154 detection and carboxy-fluorescein succinimidyl ester dilution assays) and 9 intestinal bacterial species ( Escherichia coli, Lactobacillus acidophilus, Bifidobacterium animalis subsp . lactis, Faecalibacterium prausnitzii, Bacteroides vulgatus, Roseburia intestinalis, Ruminococcus obeum, Salmonella typhimurium and Clostridium difficile ) to quantify, expand, and characterize microbe-reactive CD4 + T cells. We sequenced T-cell receptor v β genes in expanded microbe- 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 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 frequencies of 40–4000 per million for each bacteriMANUSCRIPTal species tested. Microbiota-reactive CD4 + T cells were mainly of a memory phenotype, present in PBMCs and intestinal tissue, and had a diverse T-cell receptor v β repertoire. These cells were functionally heterogeneous, produced barrier protective cytokines, and stimulated intestinal stromal and epithelial cells via interleukin 17A (IL17A), interferon gamma, and tumor necrosis factor. In patients with IBD, microbiota-reactive 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 tissues of controls.
Conclusions: In an analysis of PBMC and intestinal tissues from patients with IBD vs controls, we found that reactivity 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 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. ACCEPTED 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 immunity 1.
Intestinal immune responses are tightly regulated to allow protective immunity against pathogens while limiting responses to dietary antigens and innocuous microbes. The ‘mucosal firewall’ prevents systemic dissemination of microbes by confining microbial antigens to the gut-associated lymphoid tissue (GALT) 2. In the GALT, dendritic cells drive regulatory T cell differentiation in response to dietary antigens and commensal bacteria3. Nevertheless, vast numbers of potentially commensal-reactive effector and memory T cells populate intestinal mucosae 4. Recent evidence suggests that in mice, tolerance to commensal-derived antigens may be lost during pathogen-induced epithelial damage and subsequent transient exposure to commensals 5,6 . In humans, circulating memory T cells recognise peptides derived from gut bacteria and can cross-react to pathogens, which may confer immunological advantage during subsequent new infections 7,8 . While this process may be beneficial MANUSCRIPT during homeostasis, deranged responses to commensals may promote inflammatory conditions such as inflammatory bowel disease (IBD).
IBD (including Crohn’s disease (CD) and ulcerative colitis (UC)) results from a prolonged disturbance of gut homeostasis, the precise aetiology of which is uncertain. One hypothesis is that, in genetically susceptible individuals, IBD may be triggered by intestinal dysbiosis that promotes aberrant immune stimulation 9. Indeed, in mouse models of colitis, intestinal microbiota promote inflammation in part by stimulating microbiota-reactive CD4 + T cells 5,10 . Whether this drives IBD in humans, however, remains unknown. AlthoughACCEPTED CD4 + T cell responses to intestinal bacteria are known to occur in humans 11-13 , 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 individuals and IBD patients.
Using two independent assays, we observed that for almost all enteric bacteria examined, bacteria-reactive CD4 + T cells were present at a frequency of 40 to 500 per million CD4 + T cells in adult peripheral blood. Bacteria-reactive T cells were also prevalent in the gut mucosa, with prominent enrichment for proteobacteria-reactivity. Microbiota-responsive T cells showed a diverse
TCRV β repertoire and potently stimulated inflammatory responses by intestinal epithelial and stromal cells. Intriguingly, T cells from IBD patients displayed a normal spectrum of microbial responses, but expressed high amounts of IL-17A, consistent with increased amounts of
Th17-polarising cytokines in inflamed intestinal tissue. Collectively, these data demonstrate that microbiota-reactive CD4 + T cells are prevalent and normal constituents of the human immune system that are functionally altered during IBD pathogenesis. MANUSCRIPT
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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 obtained from IBD patients attending the John Radcliffe Hospital Gastroenterology unit or from healthy in-house volunteers. IBD patients (total, n=119; UC, n=59; CD, n=60) diagnosed by endoscopic, histological and radiological criteria were recruited for the study. Healthy volunteers
(n=30) without any known underlying acute or chronic pathological condition served as control donors. Demographic and clinical characteristics of IBD patients are summarized in Supplementary
Tables 5, 6, and 7. All donors provided informed, written consent. The NHS Research Ethics
System provided ethical approval (reference numbers 09/H0606/5 for IBD patients and 11/YH/0020 for controls; OCHRe ref 15/A237 for cord blood samples). For details regarding cell isolation, see
Supplemental Experimental Procedures.
CD154-based Detection of Antigen-Specific T Cells. CD154 detection was done as previously described 14,15 . Briefly, cells were plated at 5x10 6/cm MANUSCRIPT2 for 7-12h with heat-inactivated bacteria. 5 µg/ml brefeldin A (BFA, Sigma Aldrich) was added at 2 h. After 8-12 h, cells were harvested and treated as described in the Intracellular Cytokine, CD154 And Transcription Factor Staining section
(see below). For MACS enrichment of CD4 +CD154 + T cells, see Supplemental Experimental
Procedures.
Antigen-specific Recall Response (CFSE dilution assay) And T Cell Culture. Memory CD4 +
CD45RO + CD45RA - T cells were enriched from PBMC with untouched memory CD4 + T cell enrichment kit (Miltenyi Biotec), sorted to >97% purity on a FACS ARIA III (BD) using CD45RA and CD45RO ACCEPTED 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 were irradiated (45 Gy) and then pre-incubated for 3 h with bacterial lysates before T-cell co-culture.
For MHCII blockade, 10 µg/ml of a pan-HLA class-II blocking antibody (HLA-DR, DP, DQ;
(Tü39)) was added 30 minutes before T-cell co-culture. T cell lines were generated by sorting
CFSE low ICOS high CD4 + T cells after seven days of stimulation and expanding them with IL-2 (300
U/ml) and anti-CD3/CD28 beads (beads/T cell ratio, 1:4, Dynals) for 10-14 days. Supernatants were collected from 1x10 6 CD4 + T cells stimulated for 24h with PMA (5 ng/ml) and ionomycin (500 ng/ml; Sigma).
Flow Cytometry and Cell Sorting . PBMCs and LPMCs were stained according to standard protocols. For details, see Supplemental Experimental Procedures.
Intracellular Cytokine, CD154, And Transcription Factor Staining. For intracellular cytokine staining, cells were stained with fixable viability MANUSCRIPT dye eFluor® 780 (eBioscience) and surface markers, fixed with 2% formaldehyde (Merck), and stained for cytokines in MACS buffer containing 0.05% saponin (Sigma-Aldrich). Transcription factor expression was analysed using the
FoxP3 staining buffer set (eBioscience) according to manufacturer’s instructions.
Stimulation of Intestinal Cell Lines. CCD18Co (non-transformed human colon fibroblasts,
ATCC), and LIM1863 (human colon epithelial cells; a kind gift of Dr. Robert Whitehead, Ludwig
Institute for Cancer Research) cells were cultured in humidified incubators with 5% CO 2 at 37°C in
DMEM or RPMI media (Sigma) with 10% FCS (Sigma) and 100 U penicillin/0.1 mg/ml streptomycin. CellsACCEPTED 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-γ (B27), anti–TNF-α (Remicade), and anti–IL22 (IL22JOP).
Statistics . Statistical analyses were performed with GraphPad Prism v6.0 for Macintosh
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(GraphPad Software). Statistically significant p values were indicated as follows: ns, not significant;
* P≤0.05; ** P≤ 0.01; *** P≤0.001; **** P≤0.0001. Tests are specified in figure legends.
MANUSCRIPT
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Results
Healthy adults possess circulating memory CD4 + T cells that are reactive to intestinal microbiota
CD154 (also known as CD40 ligand) is rapidly upregulated by CD4 + T cells following antigen stimulation, irrespective of their differentiation phenotype, MHC alleles, or the precise nature of the antigenic epitope 14,15 . We therefore used CD154 to detect naïve and memory CD4 + T cell responses among peripheral blood mononuclear cells (PBMCs) after stimulation with heat-inactivated bacteria
(Figure 1A ). Seven aerobic and anaerobic intestinal bacterial species representing the four dominant gut phyla were chosen: Escherichia coli , Lactobacillus acidophilus , Bifidobacterium animalis subsp. lactis , Faecalibacterium prausnitzii , Bacteroides vulgatus , Roseburia intestinalis , and Ruminococcus obeum (Supplementary Figure 1A and Supplementary Table S1). These bacteria are common in the healthy intestine but are altered in relative abundance during inflammation 16,17 . Furthermore, we analysed responses to Salmonella typhimurium and Clostridium difficile due to their association with IBD 18,19 . T cell responsesMANUSCRIPT to the above bacteria were compared to those against well characterized barrier surface-related microbes that drive robust Th17 responses
(Staphylococcus aureus and Candida albicans ) or strong Th1 responses ( Mycobacterium tuberculosis )20,21 . The presence of a large pool of M. tuberculosis -reactive memory Th1 cells in non-exposed individuals has previously been documented. The responses in healthy controls are directed towards non-tuberculous mycobacteria (NTMs) rather than towards MTB 20,22,23 . The superantigen Staphylococcus enterotoxin B (SEB) was used as a positive stimulation control.
Stimulation with enteric bacteria reproducibly induced detectable numbers of CD4 +CD154 + T cells in the peripheralACCEPTED blood. CD4 + T cells reactive to S. aureus , C. albicans , and M. tuberculosis were generally more abundant (Figure 1B and Supplementary Figure 1B, C).
Activation markers CD69 and ICOS were upregulated on activated antigen-reactive
CD4 +CD154 + T cells (Supplementary Figure 1D). Responses were MHCII-dependent
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(Supplementary Figure 1E), and LPS failed to induce CD154 expression, confirming that CD154 upregulation was not a consequence of non-specific microbial responses (Figure 1B and
Supplementary Figure 1C). Based on CD154 + cell frequencies, we calculated that enteric bacteria-reactive CD4 + T cells were present at precursor frequencies of 40 to 500 cells per 10 6 circulating CD4 + T cells for almost all enteric bacteria surveyed (Figure 1C and Supplementary
Figure 1F ).
The newborn gut is primarily colonised with maternal vaginal and faecal bacteria after birth 24 . To understand whether T cell reactivity to microbes develops after birth, we compared
CD154 expression in umbilical cord blood with adult blood after enteric bacteria stimulation. As expected, appreciable responses to microbiota were observed only in adult blood. However,
CD4 +CD154 + T cell frequencies after SEB stimulation were similar between adult and cord blood
(Figure 1D and Supplementary Figure 1G).
Human memory T cells downregulate the naïve marker CD45RA and produce cytokines more efficiently than naïve T cells 25,26 . In healthy MANUSCRIPT individuals, the majority of bacteria-reactive CD4 + T cells had a memory phenotype (over 80% on average), indicating that they had been primed in vivo (Figure 1E, F and Supplementary Figure 2A, B). Microbiota-reactive
CD4 +CD154 +CD45RA - T cells expressed high amounts of TNF-α and IL-2 when compared to
CD4 +CD154 +CD45RA + T cells (Figure 1G and Supplementary Figure 2C ). Therefore, the circulating pool of memory CD4 + T cells contains numerous microbiota-reactive cells that arise after birth and produce cytokines including TNF-α and IL-2.
Circulating microbiota-reactiveACCEPTED CD4 + T cells express surface molecules that permit mucosal trafficking
Memory T cells express numerous adhesion molecules and chemokine receptors to access different tissues under steady-state and inflammatory conditions 4,27,28 . For example, α4β7 integrin
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and CCR9 regulate T cell migration to distinct parts of the gut. Blockade of α4β7 integrin has shown clinical efficacy for treating IBD, whereas CCR9 blockade yielded mixed results 29,30 . To identify homing receptors expressed by bacteria-reactive CD4 + T cells, we enriched CD4 +CD154 + T cells using magnetic beads to visualise rare enteric-bacteria-reactive T cells, and analysed them by flow cytometry (Figure 2A ). Microbiota-reactive T cells had a central memory phenotype, with over 60% expressing high levels of CCR7 ( Figure 2B ). Furthermore, 5–10% of CD4 +CD154 + T cells expressed the gut-homing surface markers integrin β7 and CCR9 ( Figure 2C, D). Relative to total memory CD4 + T cells and CD4 +CD154 - T, enteric bacteria-reactive T cells had high expression of the mucosa-homing receptors CCR4 and CCR6 (above 60%), low expression of
CCR10, and comparable expression of CXCR3 and CCR2 (Figure 2D and Supplementary Figure
2D ). Microbiota-reactive CD4 + T cells also expressed high amounts of CD161, a marker enriched on Th17 cells (Figure 2D, E). The majority of memory CD4 +CD154 + T cells co-expressed CCR7,
CCR4, CD161, and CCR6 in various combinations, some of which also expressed integrin β7 (Figure 2E, pie chart ). Therefore, circulating microbiota-reactive MANUSCRIPT CD4 + T cells are equipped with several homing receptors that promote mucosal access.
When comparing gut microbiota-reactive CD4 + T cells with those reactive to non-enteric organisms (including S. aureus -, M. tuberculosis -, and C. albicans ), enteric bacteria-reactive T cells were partially enriched only in CCR4 expression (Figure 2F ). Thus, the homing receptor phenotype of enteric bacteria-reactive T cells is consistent with that of T cells reactive to a broad diversity of mucosal microbes .
Microbiota-reactiveACCEPTED CD4 + T cells are enriched in gut tissue The gut harbours over 3x10 10 CD4 + T cells, but their specificity is unknown 4,5 . We therefore estimated the abundance of human microbiota-reactive CD4 + T cells in the gut by examining non-inflamed colon specimens using the CD154 assay (Figure 3A). Lamina propria CD4 + T cells
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showed a dominant EM and CM phenotype and expressed both tissue-resident and gut-related markers, with 80% of cells being CD69 + (Supplementary Figure 3A).
We next stimulated lamina propria mononuclear cells (LPMCs) with microbial lysates or
SEB. We combined intracellular CD154 detection with TNF-α staining to increase assay sensitivity, as lamina propria CD4 + T cells expressed low amounts of CD154 without stimulation
(Supplementary Figure 3B,C ). Compared with peripheral blood frequencies of unrelated donors, there were similar frequencies of S. aureus and SEB reactivity, and reduced M. tuberculosis -reactivity in the gut. However, gut CD4 + T cells were enriched in reactivity towards intestinal bacteria and C. albicans (Figure 3B and Supplementary Figure 3D ). Bacteria-reactive cells comprised 150–4000 cells per 10 6 gut-resident memory CD4 + T cells for all enteric bacteria tested. Given that peripheral blood contained 40–500 bacteria-reactive cells per 10 6 memory CD4 +
T cells (for each bacteria tested), this suggests that bacteria-reactive T cells are 3–8 fold more frequent in gut tissue, as compared to those in circulation. The strong enrichment of S. typhimurium and E. coli -reactivity in the gut was confirmed by assessing MANUSCRIPT CD154 and TNF-α expression in CD4 + T cells from donor-matched blood and intestinal tissue ( Figure 3C ). Since the gut harbours up to
3x10 10 memory T cells (versus 5–10x10 9 in blood) 4, many of which are bacteria-reactive, the absolute number of gut-resident microbiota-reactive CD4 + T cells is likely to be at least 10 times greater than that in peripheral blood.
Gut-resident bacteria-reactive (CD154 +TNF-α+) T cells produced high amounts of IFN-γ,
IL-17A, and IL-2, while production of IL-22, GM-CSF, and IL-4 was generally low ( Figure 3D and Supplementary Figure 3E,F ). Interestingly, lamina propria T cells showed increased IL-17A expression and reducedACCEPTED IFN-γ production relative to cells with similar reactivity in peripheral blood (Figure 3D and Supplementary Figure 3E).
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Enteric bacteria-reactive CD4 + T cells are clonally diverse
To assess the clonal diversity of circulating bacteria-reactive memory CD4 + T cells, we expanded
CFSE-labelled CD4 + T cells using whole bacteria and autologous irradiated monocytes as APCs
(Supplementary Figure 4A )21 . S. aureus-, M. tuberculosis-, and SEB-reactive T cells served as controls. Antigen-reactive T cells proliferated in an MHCII-dependent manner and were readily detectible after 3–6 days (Figure 4A and Supplementary Figure 4B –D). Proliferating cells expressed several activation markers including ICOS, CD25, and OX40 ( Figure 4A and
Supplementary Figure 4B,E,F ). Consistent with the CD154 assay, S. aureus , M. tuberculosis , and
SEB strongly induced T cell proliferation ( Figure 4A,B and Supplementary Figure 3B,C ).
Flow cytometry analysis revealed a diverse TCRV β repertoire in bacteria-reactive T cells, similar to polyclonal stimulation with phytohaemagglutinin (PHA) but different to stimulation with
SEB, which is known to activate a restricted V β repertoire (Figure 4C )31 . To directly assess the clonal diversity of bacteria-reactive CD4 + T cells, we isolated CFSE low bacteria-reactive memory T cells and assessed TCRV β clonotypes by multiplex PCRMANUSCRIPT and deep sequencing. 150–800 clonotypes were detected for each reactivity (Supplementary Figure 4G ). The largest clonal diversity was detected among E. coli - and S. typhimurium- reactive cells, consistent with frequencies observed in the CD154 assay (see Figure 1B,C and Supplementary Figure 1B,F ). While closely related species (e.g. E. coli versus S. typhimurium ) had 3–8% overlap in T cell clonotypes, little clonotype sharing was observed between T cells reactive to more distantly related bacteria (Supplementary
Figure 3H). Indeed, E. coli - and B. animalis-reactive CD4 + T cell lines were strongly restimulated when cultured with autologous monocytes loaded with E. coli or B. animalis lysates, respectively . In contrast, E. coliACCEPTED-reactive T cells responded weakly to the closely related S. typhimurium , while B. animalis -reactive T cells responded weakly to L. acidophilus , F. prausnitzii , and C. difficile
(Supplementary Figure 5A ). These data confirm the low degree of cross-reactivity predicted from
TCRV β sequencing.
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Microbiota-reactive memory CD4 + T cells are functionally heterogeneous and produce barrier-promoting cytokines
To functionally characterise circulating microbiota-reactive memory cells, we analysed CFSE low cells using flow cytometry after stimulation with enteric bacteria for 6 days. Enteric microbiota-reactive cells produced Th1- and Th17-related cytokines including IFN-γ, IL-17A, and
IL-22, but only low amounts of the Th2 cytokine IL-4, comparable to cells reactive towards S. aureus or C. albicans (Figure 4D and Supplementary Figure 5B,C ). In contrast, memory T cells reactive towards SEB, M. tuberculosis , influenza vaccine components, or tetanus toxoid showed a polarized Th1 profile with low expression of IL-17A (Figure 4D and Supplementary Figure
5B,C). Boolean gating revealed a high degree of functional heterogeneity in expanded microbiota-reactive memory T cells, with frequent co-expression of IL-17A, IL-22, and IFN-γ
(Figure 4E). Bacteria-reactive cells co-expressed the transcription factors ROR γt and T-bet, which are characteristic of Th17 and Th1 cells, respectively ( Figure 4F,G and Supplementary Figure 5D,E). Intriguingly, a subset of CD4 + T cells reactive MANUSCRIPT to F. prausnitzii , L. acidophilus , or B. animalis produced the immunoregulatory cytokine IL-10 in addition to IFN-γ and IL-17A
(Supplementary Figure 5F). Thus, unlike T cells that are reactive towards M. tuberculosis or vaccine antigens, enteric microbiota-reactive T cells are functionally distinct and produce barrier-promoting and immunoregulatory cytokines.
Microbiota-reactive memory T cells promote intestinal stromal and epithelial cell activation
During periods of epithelial damage and exposure to commensals, activation of microbiota-reactive memory T cellsACCEPTED could promote protective immune responses. To assess their tissue-modulating capabilities, cell-free supernatants of microbiota-reactive memory T cells were used to stimulate
CCD18Co intestinal myofibroblasts and LIM1863 colonic epithelial cells. CCD18Co and LIM1863 cells were then assessed for expression of various immune response genes that were selected a
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priori to represent responses to major T cell-derived cytokines. Both cell types responded by expressing several cytokine and chemokine genes known to be induced by IL-17A (including IL1B ,
CSF2 , IL6 , CXCL1 , and CXCL8 ), as well as IFN-γ-inducible genes including CXCL9 , CXCL10 , and
CXCL11 (Figure 5A,B)32 . Conversely, supernatants from SEB-stimulated memory T cells (which produce little IL-17A) mainly induced IFN-γ-dependent genes. Thus, stimulation of non-hematopoietic intestinal cells by microbiota-reactive T cells may promote recruitment and activation of myeloid cell populations to facilitate pathogen control and tissue repair.
We next assessed the effects of individual cytokines in E. coli -reactive T cell supernatants using combinations of neutralizing antibodies. This experiment revealed distinct IFN-γ- and IL-
17A/TNF-α–dependent groups of response genes in both intestinal epithelial cells and fibroblasts.
IFN-γ blockade strongly reduced expression of several chemokine genes including CXCL9 ,
CXCL10 , CXCL11 , CCL2 , and CCL7 (IFN-γ-dependent module; Figure 5C,D ). Intriguingly, single blockade of IL-17A, IL-22, or TNF-α did not affectMANUSCRIPT stromal or epithelial cell activation (Figure 5C,D ). However, combined blockade of IL-17A and TNF-α influenced a large number of genes including CSF2 , IL1B , TNF , CXCL1 , CXCL8 , CXCL5 , CXCL6 , and CCL20 (IL-
17A/TNF-α-dependent module). Triple blockade of IFN-γ, IL-17A, and TNF-α completely inhibited stromal and epithelial cell activation. IL-22 blockade did not affect cytokine or chemokine production, but attenuated induction of the anti-microbial peptide REG3G in LIM1863 cells. Given that the products of T cell-stimulated stromal and epithelial cells are highly expressed in the inflamed mucosa of IBD patients (Figure 5E and 7D ), this signature might reflect the activation of microbiota-reactive T cells following epithelial disruption, a key feature of IBD.
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Microbiota-reactive CD4 + T cells in inflamed intestinal tissue show a Th17-skewed phenotype in IBD patients
IBD is thought to arise in part from aberrant adaptive immune responses to microbiota 9. Human
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CD4 + T cells in IBD have been functionally characterised mainly by polyclonal stimulation 33-35 .
Therefore, we evaluated microbiota-reactive CD4 + T cell responses in IBD patients using the
CD154 detection approach. Circulating microbiota-reactive CD4 + T cell frequencies were decreased in IBD patients compared with healthy donors, which might reflect their selective recruitment to the inflamed gut (Figure 6A and Supplementary Figure 6A ). However, intestinal memory CD4 + T cells from IBD patients did not display reciprocally higher frequencies of microbial specificity
(Figure 6B and Supplementary Figure 6B ). We next calculated the frequency of memory CD4 +
T cells in inflamed mucosae using flow cytometry. Memory CD4 + T cells were present at higher frequencies in inflamed tissue from IBD patients compared to tissue from matched non-lesional sites of IBD patients and healthy controls (Figure 6C ). These findings were confirmed using a previously published bioinformatics approach known as CIBERSORT in an independent cohort 36
(Supplementary Figure 6C). Based on both approaches, memory CD4 + T cells are typically 2–4 fold more frequent in inflamed tissue from IBD patients compared to tissue from healthy controls. Thus, because inflamed tissue contains a higher abu MANUSCRIPTndance of memory CD4 + T cells than healthy mucosa, it can be inferred that gut-resident microbiota-reactive CD4 + T cells are similarly enriched in patients with active IBD (Figure 6C, Supplementary Figure 6C).
To evaluate functional alterations in microbiota-reactive CD4 + T cells in IBD, intracellular
CD154 detection was combined with cytokine analysis. Compared with healthy controls, circulating microbiota-reactive CD4 + T cells from IBD patients displayed increased IL-17A and IL-2 production, but decreased expression of IFN-γ (Figure 7A and Supplementary Figure 6D, E ).
Interestingly, increased IL-17A production was observed in all enteric bacteria-reactive responses, but not in S. aureus,ACCEPTED M. tuberculosis , or SEB responses ( Figure 7A and Supplementary Figure 6E,F ). These changes were observed in both CD and UC and were independent of disease activity or therapy ( Supplementary Figure 6G). However no difference in IL-10 production was observed between healthy donors and IBD patients (Supplementary Figure 6H). IFN-γ and IL-17A
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co-expression is thought to identify pathogenic CD4+ T cells in mouse colitis models 37 , so we assessed their co-expression in E. coli-reactive memory CD4 + T cells. Compared with controls, IBD patients displayed significantly increased frequencies of IL-17A +IFN-γ- cells and a marginal increase in IL-17A +IFN-γ+ cells, while the IL-17A -IFN-γ+ fraction was significantly reduced
(Figure 7B ). E. coli-reactive CD4 + T cells from inflamed intestinal tissue showed an increase in IL-
17A single producers similar to that seen in peripheral blood ( Figure 7C ).
Because the Th17-inducing cytokines IL1B , IL6 , and IL23A were highly enriched in the inflamed intestinal tissue of IBD patients (Figure 7D ), we reasoned that they might promote Th17 polarization of bacteria-reactive T cells. Indeed, treatment of microbiota-reactive CD4 + T cells from healthy donors and IBD patients with IL-1β, IL-6, or IL-23 for one week during stimulation with E. coli, S. typhimurium, L. acidophilus , or B. animalis (CFSE dilution assay) resulted in a 1.5–2-fold increase in IL-17A production ( Figure 7E and Supplementary Figure 6I).
Taken together, these experiments demonstrate that circulating and gut-resident microbiota-reactive CD4 + T cells express increased frequenciesMANUSCRIPT of IL-17A in IBD. Intestinal tissues from patients with active IBD express gene modules driven by Th1/Th17-derived cytokines, suggesting that bacteria-reactive memory cells could contribute to the tissue response.
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Discussion
The gastrointestinal tract harbours a large and diverse population of commensal bacteria, and how the immune system interacts with them is subject to intense investigation. Here we used two different methodologies to characterise microbiota-reactive CD4 + T cell frequencies and phenotypes in the blood and intestinal tissue of healthy individuals and those with IBD. For each bacterial strain tested, the healthy CD4 + T cell repertoire contains reactive cells at a frequency of 40–4000 per million, consistent with other antigen-reactive memory T cells 38 . Microbiota-reactive CD4 + T cells were mainly of a memory phenotype, present in both blood and gut tissue, had a diverse TCRVβ repertoire, and showed little clonotype sharing. Notably, microbiota-reactive CD4 + T cells were functionally heterogeneous in terms of homing receptor expression and effector functions and could stimulate intestinal cells via production of IL-17A, IFN-γ, IL-22, and TNF-α. Moreover, microbiota-reactive CD4 + T cells were recruited to sites of inflammation and showed increased IL- 17A production in patients with IBD. MANUSCRIPT Characterizing T cell responses to bacteria is technically challenging due to their complex antigenic make-up. We therefore used the CD154 and CFSE dilution assays, both of which exploit microbial complexity to provide large numbers of antigens. The combination of CFSE dilution and
TCRVβ sequencing allowed us to quantify clonotype heterogeneity and sharing between different bacteria-reactive T cells. Given the phylogenetic similarity of several bacteria used in this study, the paucity of clonotype sharing was surprising. Nevertheless, enteric bacteria-reactive T cells could be cross-reactive to other antigens not assessed in this study, and may have been primed during immune responses to other targets 39 . High interclonal and intraclonal functional heterogeneity in human CD4 + TACCEPTED cell responses to microbes and vaccines was recently observed 20 . However, clonotype sharing between different microbial stimuli has not previously been studied and requires further investigation.
Microbiota-reactive CD4 + T cells showed substantial phenotypic and functional
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heterogeneity. The majority of circulating enteric bacteria-reactive CD4 + T cells co-expressed chemokine receptors including CCR4, CCR6, and CCR7, while a smaller fraction expressed the gut-related homing receptors α4β7 and CCR9. These receptors promote access to secondary lymphoid organs and various mucosal tissues, including the intestine 4,28 . Furthermore, circulating and gut-resident microbiota-reactive T cells displayed both Th17 and Th1 characteristics, and in some cases produced IL-10 40 . Gut-resident cells showed a clear Th17 bias when compared to circulating populations, which was more pronounced in IBD.
Based on our observations, we can speculate that continuous sampling of luminal antigens by intestinal DCs causes low-level stimulation of gut-resident CD4 + T cells to produce cytokines that support epithelial integrity, barrier function, and intestinal homeostasis 41,42 . Indeed, cytokine production by commensal-reactive CD4 + T cells might play a more significant role in supporting gut homeostasis than previously thought ( Supplementary Figure 7). However, this homeostatic circuit might be disrupted in IBD due to dysbiotic changes and/or perturbed myeloid cell activity, causing inappropriate T cell activation and a pathogenic MANUSCRIPT imbalance of cytokine production 9. While IL-17A is frequently cited as a pathogenic cytokine, it is also critical for promoting mucosal barrier function and protection from pathogens 32 . Absence of IL-17A was recently shown to increase epithelial injury and compromise barrier function in mouse models of colitis 43,44 . Indeed,
IL-17A is a critical driver of neutrophil recruitment, and its absence could therefore exacerbate mucosal inflammation by facilitating bacterial invasion and dispersal 45 . Notably, blockade of IL-
17A in CD caused disease exacerbation despite being well tolerated and therapeutically effective in psoriasis 46 . Thus, IL-17A likely plays a key tissue-protective role in humans, suggesting that the increased Th17 ACCEPTEDpolarization of microbiota-reactive T cells in IBD patients could reflect an effort to bolster tissue integrity.
Host-microbial homeostasis depends on minimising contact between microorganisms and mucosal surfaces via the combined action of epithelial cells, mucus, IgA, antimicrobial peptides,
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and immune cells 1,2 . Active immune responses to gut flora have been linked to disease 9. However, this concept should be revisited in light of our current findings and the observation that healthy individuals generate antibody responses to commensals 47,48 . At least two plausible mechanisms could explain the genesis of these microbiota-reactive responses. First, mucosal dendritic cells constantly survey the luminal microenvironment and thereafter migrate to secondary lymphoid tissues to initiate B and T cell responses 49,50 . Second, during GI infections in mice, immune responses against commensals and pathogens are induced in parallel 51 . Thus, continuous luminal sampling of intestinal microbiota and periodic epithelial breaches during gastrointestinal infections might provide a plethora of memory T cells with potential reactivity towards newly encountered pathogens 7,8 . Therefore, contrary to the notion that they promote inflammatory pathology, acquired commensal-reactive T cell responses may be essential to promote barrier function and IL-10 mediated immune regulation, two cornerstones of intestinal homeostasis .
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Acknowledgments
We thank Helen Ferry, Jenny Middleton, Sam Bullers, Sebastian Rogatti Granados, Priya
Siddhanathi, James Chivenga, Ngonidzashe Charumbira, Jennifer Hollis, Linda Holden, Fiona
Goddard, Karen Doig, Nicole Stoesser, Nicole Gordon and Claire Pearson for FACS sorting, technical support, patient sample collection and lab management. We thank Harry Flint, Carolina V.
Arancibia-Cárcamo, Emily Thornton, Tobias Schwerd, David Danko, Arnold Han, Mark Davis, M.
Hussein Al-Mossawi, Paul Bowness, and all laboratory members for valuable support and discussions. We also thank Thomas Penz from the Biosequencing Facility at CeMM. We acknowledge the Oxford Radcliffe and GI Biobanks and the Oxford IBD cohort study supported by the NIHR Oxford Biomedical Research Centre (grant no. HBRWAE04 Task HB81.G). We thank all volunteers and patients who took part in this study.
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Figure Legends
Figure 1. Healthy adults possess circulating memory CD4 + T cells that are reactive to intestinal microbiota.
PBMCs were stimulated with heat-inactivated bacteria for 8-12h, and bacteria-reactive CD4 + T cells were detected by intracellular CD154 expression.
(A) Experimental setup.
(B) CD154 + frequencies among peripheral CD4 + T cells in adults after short-term stimulation with heat-inactivated bacteria (n=30 independent donors).
(C) Estimated microbiota-reactive cells per million CD4 + T cells in adult blood (n=30). The numbers of microbiota-reactive T cells were calculated based on the frequencies of CD4 +CD154 + T cells. Background (no microbe stimulation) was subtracted from bacterial stimulations. Significance calculated in relation to LPS stimulation.
(D) Frequencies (± SEM) of CD154 + cells among CD4 + T cells in adult (n=30) or cord blood (n=3) after short-term stimulation with heat-inactivated bacteria. MANUSCRIPT (E) Expression of CD45RA on CD4 +CD154 + T cells, showing four representative stimulations of the same donor.
(F) Mean (± SEM) frequencies of memory cells within CD4 +CD154 + T cells. Each symbol represents an antigen-reactive population from one individual (n=19-30).
(G) Expression of CD45RA, TNF-α, and IL-2 by CD4 +CD154 + T cells after short-term PBMC stimulation with bacterial lysates. Statistics: panels B, C, and F, one-way ANOVA with Sidak’s multiple comparison test; panel D, Mann-Whitney test. ACCEPTED Figure 2. Circulating microbiota-reactive CD4 + T cells express several surface molecules that promote mucosal trafficking.
CD4 +CD154 + T cells were analysed by flow cytometry after magnetic enrichment following
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short-term PBMC stimulation with heat-inactivated bacteria.
(A) Experimental setup and enrichment efficiency after stimulation with SEB.
(B) Representative CD45RA and CCR7 expression on enriched CD4 +CD154 + T cells. Central memory T cell frequencies are shown (right bar graph). Frequencies (± SEM) of 4 independent experiments are depicted with n=8 independent donors.
(C) Heat map depicting mean frequencies of surface marker and chemokine receptor expression on enriched CD4 +CD154 + T cells (n=5-8 independent donors).
(D) Surface marker and chemokine receptor expression frequencies among CD4 +CD154 + T cells after short-term stimulation with B. animalis . Data representative of 5-8 independent donors.
(E) Co-expression of surface molecules with CCR6 after short-term stimulation of CD4 +CD154 +
CD45RA - T cells with B. animalis (left panel). Boolean gating analysis shows each possible combination of CCR7, CCR4, CCR6, and CD161 expression (right panel).
(F) Surface marker and chemokine receptor expression frequencies among total memory CD4 + T cells and CD4 +CD154 + T cells after short-term stimulationMANUSCRIPT with B. animalis, S. aureus, M. tuberculosis, C. albicans, or SEB . Frequencies (± SEM) of 4 independent experiments are depicted with n=5-8 independent donors. Statistics: panel F, One-way ANOVA with Sidak’s multiple comparison test.
Figure 3. Microbiota-reactive CD4 + T cells are enriched in gut tissue
LPMCs were isolated from non-inflamed and tumour-free surgical specimens from colorectal cancer patients. (A) ExperimentalACCEPTED setup. LPMCs were stimulated with heat-inactivated bacteria for 8-12h and assessed for intracellular expression of cytokines and CD154.
(B) Estimated microbiota-reactive cells per million CD4 + T cells in adult blood and intestinal tissue from unrelated donors, based on CD154 staining (n=17 for control mucosa; n=25-31 for blood).
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(C) Matched LPMCs and PBMCs were stimulated with heat-inactivated E. coli (blue symbols) or S. typhimurium (red symbols), analysed for CD154 expression, and compared with respect to the estimated microbiota-reactive cells per million CD4+ T cells. Connected dots represent matched samples.
(D) Frequencies (±SEM) of IL-17A, IFN-γand IL-22 production by CD154 +TNF-α+ memory
CD4 + T cells isolated from LPMCs or PBMCs from unrelated donors after stimulation with heat-inactivated bacteria (n=10-23 donors). Significance calculated between the respective stimulations in control mucosa and peripheral blood. Statistics: panels B and D, Mann-Whitney test; panel C, paired t-test.
Figure 4. Microbiota-reactive memory CD4 + T cells are clonally diverse and functionally heterogeneous
Memory CD4 + T cells were labelled with CFSE or VPD (violet proliferation dye-450) and stimulated with heat-inactivated bacteria in the presence MANUSCRIPT of autologous monocytes. (A) CFSE profiles and ICOS expression on days 3 and 6 of stimulation in a representative donor.
(B) Percentage of CFSE low CD4 + T cells of each individual donor (n=18).
(C) Pie charts showing TCRVβ expression by proliferating VPD low cells measured by V β antibody staining on day 7 of stimulation. Average of three independent donors is depicted. TCRV β usage of the different reactivities of three independent donors is summarized in Supplementary Table 2.
(D) Mean (± SEM) cytokine production frequencies of proliferating CFSE low cells and non- activated CFSE high cells after PMA/ionomycin stimulation (n=12-13 independent donors). (E) Boolean gatingACCEPTED analysis showing each possible combination of IL-17A, IFN-γ,and IL-22 production by CFSE low proliferating cells. Data from 9 independent donors.
(F, G) ROR γt and T-bet expression in proliferating CFSE low cells measured by intracellular staining on day 7 of stimulation. (G) Boolean gating analysis showing each possible combination of ROR γt,
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T-bet, and GATA-3 production by CFSE low proliferating cells. Data from 3 independent donors.
Statistics: panel B, one-way ANOVA with Sidak’s multiple comparison test; panel D, one-way
ANOVA with Bonferroni’s multiple comparison test.
Figure 5. Microbiota-reactive memory T cells promote intestinal stromal and epithelial cell activation.
Healthy donor memory CD4 + T cells from peripheral blood were labelled with CFSE and stimulated with heat-inactivated bacteria in the presence of autologous monocytes.
CD4 +CFSE low ICOS high cells were FACS-sorted on day 7 and expanded for 10-14 days with anti-
CD3/CD28 beads. Expanded cells were stimulated at equal numbers with PMA/ionomycin for 24h to produce conditioned supernatants.
(A, B) Cell-free supernatants from different T cell specificities was used to stimulate CCD18Co intestinal myofibroblasts and LIM1863 colon epithelial cells. Gene expression in stimulated cells was measured by qPCR and normalised to control MANUSCRIPT treatment (media containing PMA/ionomycin alone). Results of independent stimulations were pooled together into the following categories:
Proteobacteria -reactive T cells (S. typhimurium- and E. coli-reactive); Actinobacteria -reactive T cells (B. animalis-reactive); Firmicutes -reactive T cells (F. prausnitzii- and L. acidophilus-reactive).
Data are from three independent T cell donors.
(C, D) Supernatants from E. coli -reactive CD4 + T cells were used to stimulate CCD18Co (C) or
LIM1863 (D) cells. Supernatants were pre-treated with one or more cytokine-neutralizing antibodies as indicated. Gene expression was median-normalized, log 2 transformed, and plotted as a heat map. Data representativeACCEPTED of 2–3 independent experiments. (E) Q-PCR analysis of mucosal biopsies from the Oxford IBD cohort, categorised by endoscopic assessment of disease activity. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 5. Statistics: panels A, B, and E, One-way ANOVA with
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Sidak’s multiple comparison test.
Figure 6. Abundance of circulating and gut-resident enteric bacteria-reactive CD4 + T cells in
IBD
(A) PBMCs from healthy donors or IBD patients were stimulated with the indicated heat-inactivated bacteria and analyzed for CD154 expression. Frequencies (± SEM) of CD154 + cells among CD4 + T cells are depicted (n=30-38). Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6.
(B) LPMCs were isolated from inflamed surgical specimens from IBD patients or non-inflamed and tumour-free surgical specimens from colorectal cancer patients. Isolated LPMCs were stimulated with the indicated heat-inactivated bacteria and analysed for CD154 expression. Frequencies (±
SEM) of CD154 + cells among CD4 + T cells are depicted (n=10-20).
(C) LPMCs from endoscopic intestinal biopsies were obtained from healthy controls or matched lesional and non-lesional sites of IBD patients from MANUSCRIPTan independent Oxford cohort (n=12 controls and 17 IBD). Dots represent frequencies of CD45RA −CD4 + memory T cells among total live
LPMCs from different donors; connected dots represent matched biopsies. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 7. Statistics: panels
A, B and C, Mann-Whitney test; panel C, one-way ANOVA with Sidak’s multiple comparison test.
Figure 7. Microbiota-reactive CD4 + T cells show a Th17-skewed phenotype in IBD patients.
(A-B) PBMCs isolated from healthy donors and IBD patients were stimulated with heat-inactivated bacteria or SEB ACCEPTEDand analysed for intracellular CD154 and cytokine expression. (A) Frequencies (± SEM) of IL-17A, IFN-γ, and IL-22 expression in CD154 +TNF-α+ memory
CD4 + T cells (n=23-33 independent donors). Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6.
32 ACCEPTED MANUSCRIPT
(B) Frequencies (± SEM) of IL-17A and IFN-γ co-expression in CD154 +TNF-α+ memory CD4 + T cells after short-term stimulation with heat-inactivated bacteria (n=23-34 independent donors).
(C) LPMCs from inflamed IBD surgical specimens or non-inflamed and tumour-free surgical specimens from colorectal cancer patients were stimulated with heat-inactivated E. coli. Boolean gating shows each possible combination of IL-17A, IFN-γ, and IL-22 production by
CD154 +TNF-α+ memory CD4 + T cells (n=6 and n=7 independent donors for IBD and controls, respectively).
(D) Q-PCR analysis of IL1B, IL6 , and IL23A in intestinal mucosal specimens categorised by endoscopic assessment of disease activity. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 5.
(E) CD4 +CD45RO +CD45RA -CD25 -CD8 - memory CD4 + T cells were isolated from healthy donor blood, labelled with CFSE, and stimulated with autologous monocytes pulsed with B. animalis in the presence or absence of the indicated cytokines. Data represent mean (± SEM) fold changes in IL-17A or IFN-γ expression frequencies relative toMANUSCRIPT cells expanded without cytokines. Statistics: panels A, B and C, Mann-Whitney test; panels D and E, one-way ANOVA with Sidak’s multiple comparison test.
ACCEPTED
33 ACCEPTED MANUSCRIPT
Firmicutes Bacteroidetes A Proteobacteria E E. coli B. animalis Actinobacteria 100 100
+ Bacterial lysate 80 Multicolor 80 6 12 Intracellular 60 8-16h flow cytometry 60 94 88 stain (Detection of +BFA surface molecules) 40 40 PBMCs 20 20
0 0 2 3 4 5 0 102 103 104 105 0 10 10 10 10 B **** *** ** * ** * ** ** ** * **** 10 F. prausnitzii SEB 100 100
1 80 80 7 42 % of maximum 60 93 60 58 cells (%)
+ 40 40 0.1 ns 20 20
0.01 0 0 0 102 103 104 105 0 102 103 104 105 of CD4 + 0.001 CD45RA CD154+ CD4+ T cells + 0.0001 Total CD4 T cells CD154 0.00001 F **** **** **** **** **** **** **** **** **** **** ns 100 LPS SEB E. coli 90 R. obeumS. aureus no microbe B. animalis B. vulgatus C. albicans 80 L. acidophilusF. prausnitziiR. intestinalis M. tuberculosis 70 **** 60 C 105 50 **** *** ** * ** * ** ** * * 40 104
Memory (%) 30 20 3 cells 10 + 10
2 0
CD4 10
6 MANUSCRIPT SEB
/10 E. coli 101 R. obeum S. aureus B. animalis B. vulgatus C. albicans L. acidophilusF. prausnitziiR. intestinalis Microbiota-reactive cells 0 M. tuberculosis 10 Total CD4 T cells gated on CD4+CD154+ T cells E. coli SEB G 5 R. obeumS. aureus 105 3.31 0.77 10 3.09 0.99
B. animalis B. vulgatus C. albicans E. coli 4 L. acidophilusF. prausnitziiR. intestinalis 104 10 M. tuberculosis 3 103 10
2 102 10 E. coli B. animalis L. acidophilus F. prausnitzii 0 0 D 0.08 0.03 0.03 0.015 30.9 65 57.7 38.2 *** ** *** ** 2 3 4 5 3 4 5 0 10 10 10 10 0 10 10 10 B. animalis
5 105 7.6 2.63 10 7.6 2.63 0.06 4 0.02 0.02 0.010 104 10
3 3 0.04 10 10
2 102 10 0.01 0.01 0.005 0 0 0.02 21.3 68.4 29.2 60.5 3 4 5 0 102 103 104 105 0 10 10 10 cells (%) F. prausnitzii + 5 0 0 0 0.000 105 4.46 1.79 10 6.25 0
CD45RA 4 SEB S. aureus M. tuberculosis C. albicans 104 10
8 ns 0.15 0.15 0.15 3 3 ***ACCEPTED*** ** 10 10 of CD4 2 2 + 10 10 6 0 0 0.10 0.10 0.10 23.2 70.5 43.8 50 3 4 5 0 102 103 104 105 0 10 10 10 4 5 105 36.5 3.93 10 32.1 8.31 CD154
0.05 0.05 0.05 4 104 SEB 2 10
3 103 10
2 0 0 0 0 102 10 0 0 Adult blood 26.7 32.9 25.1 34.5 3 4 5 Cord blood 0 102 103 104 105 0 10 10 10 TNF-α IL-2 Figure 1 ACCEPTED MANUSCRIPT
Bacterial lysate + + +αCD40 CD4 CD154 T cells +αCD28 + CD154 Total CD4 T cells A surface Multicolor B 5 8-16h stain CD154 10 Naive magnetic flow cytometry enrichment (Detection of 104 PBMCs 100 chemokine receptors CD4 -
and surface molecules) 3 + 10 80 SEB, gated on CD3 cells 0 CD45RA Original fraction Positive fraction negative fraction 60 EM CM 105 105 105 4.8 49 0.5 0 102 103 104 105 40
104 104 104 CCR7
5 cells (%) T
10 of CD45RA
+ 20 103 103 103 104 0
CD154 0 0 0 103 CCR7 0 E. coli 2 3 4 5 2 3 4 5 2 3 4 5 CD45RA 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 T cells EM CM B. animalis CD4 2 3 4 5 F. prausnitzii 0 10 10 10 10 L. acidophilusMemory CD4 FMO
CD4+ CD154+ T cells D Total CD4+ T cells 105 8.25 0.8 8.96 0.13 6.65 2.44 8.08 1.01
C 104
103 IntegrinCCR9 β7 CCR2CCR4CCR6CCR7CCR10CXCR3CXCR5CD161 % 0 80 E. coli 81.6 9.34 89.8 1.09 20.7 70.2 5.47 85.4 60 B. animalis Integrin β7 CCR2 CCR4 CCR6
CD45RA 5 40 L. acidophilus 10 7.45 0 7.61 0.04 8.08 0.97 8.67 0.38 104 20 F. prausnitzii 103
Memory 0 0 CD4 T cells 91.7 0.8 91.6 0.74 80.9 10.1 21.4 69.5 0 103 104 105 0 103 104 105 0 102 103 104 105 MANUSCRIPTCCR9 CCR10 CXCR3 CD161 Integrin β7 CCR9 CCR2 gated on F 20 20 80 + + ns ns ns ns ns ns ns ns ns ns ns ns ns ns ns + + - CD4 CD154 CD4 CD154 CD45RA T cells memory T cells 15 15 60 E Total CD4+ CD45RA- T cells 105 83.3 10.1 90.6 2.27 10 10 40
104 Integrin β7 5 5 20 CCR4 0 CCR6 CCR7 0 0 0
CD161 T cells 6.37 0.19 6.5 0.6 + CCR4 CCR6 CCR7 100 100 100 n=4 ns ns ns ns ** ns ns ns ns ns Integrin β7 CCR9 ns ** * * *** CD4
+ 80 80 80 CCR6 105 15 78.5 16.1 77.4 20.6 73 60 60 60
104 40 40 40
0 20 20 20
1.24 5.33 0.33 6.23 3.14 3.28 0 0 0 0 103 104 105 0 103 104 105 0 102 103 104 105 CCR10 CXCR3 CXCR5 CD161 20 50 20 80 CCR4 CCR7 CD161 ns ns ns ns *** ns ns ns ** ns ns ns ns ns ns ns ns ns * *** ACCEPTED 40 Percentage of CD154 15 15 60 30 10 10 40 20 5 20 5 10
0 0 0 0 s s s s SEB SEB SEB SEB T cells T cells T cells T cells S. aureus S. aureus S. aureus S. aureus B. animalis B. animalis B. animalis B. animalis C. albicanMemory CD4 C. albicanMemory CD4 C. albicanMemory CD4 C. albicanMemory CD4 M. tuberculosis M. tuberculosis M. tuberculosis M. tuberculosis Figure 2 ACCEPTED MANUSCRIPT
Firmicutes A Bacteroidetes D IL-17A Proteobacteria 60 Actinobacteria * *** *** ns ns ns ns **** **** ns ns + Bacterial lysate 50 Intracellular Multicolor 8-16h stain flow cytometry +BFA (Detection of intracellular 40 LPMCs cytokines and PBMCs surface molecules) 30
B E. coli B. animalis L. acidophilus 20 6000 400 1500 ns **** * (0.057) 10 5000 300 4000 1000 0 3000 200 IFN-γ cells (%) + 2000 500 100 100 90 **** * **** **** ** ** 1000 ns ** * ns ns CD4 cells + + 80 0 0 0 70
CD4 60
F. prausnitzii B. vulgatus R. intestinalis R. obeum 6
300 250 250 1600 CD154 50 ** ** **** ** + 1400 40 250 200 200 1200 30 200 150 150 1000 20 150 800 10 100 100 of TNF- α 600 + 100 0 400 50 50 50 200 IL-22 0 0 0 0 15 Control mucosa Cytokine SEB S. aureus M. tuberculosis C. albicans Peripheral blood
40000 2500 1000 2500 * * ns ns **** *** ns * ns ns ns ns ns ns ns
Microbiota-reactive cells/10 10 2000 800 2000 30000 MANUSCRIPT 1500 600 1500 20000 1000 400 1000 5 10000 500 200 500
0 0 0 0 0 Control mucosa (n=17) s Peripheral blood (n=25-31) SEB E. coli R. obeumS. aureus B. animalis B. vulgatus C. albican L. acidophilusF. prausnitziiR. intestinalis C M. tuberculosis Peripheral blood *
Mucosa
0 1000 2000 3000 4000 5000 Microbiota-reactiveACCEPTED cells /106 CD4+ cells
Figure 3 ACCEPTED MANUSCRIPT
A no microbe E. coli B. animalis L. acidophilus SEB B 5 105 0 10 0.56 105 0.17 105 0.27 105 39.6 * 4 100 * 104 10 104 104 104 ***
0.14 0.62 0.5 0.49 2.4 **** ****
3 Day 3 103 10 103 103 103 80 0 cells (%)
0 0 0 0 +
0.14 0.06 0.12 0.1 0.9 0 102 103 104 105 60 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CD3 3.27 1.31 0.7 + 1.45 5 5 5 5 10 105 10 10 10
ICOS 0.03 19.5 13.6 12.1 65.1
4 4 4 4 4 40 10 0.1 10 10 10 10
3 3 3 3 Day 6 10 10 10 103 10 of CD4 20 2 2 2 2
10 10 10 10 low 0 0 0 0 0 0.22 1.14 1.81 4.38 21 3 4 5 0 103 104 105 0 10 10 10 0 103 104 105 0 103 104 105 0 103 104 105 0 CFSE CFSE SEB E. coli
no microbe B. animalis C E. coli B. animalis PHA L. acidophilusF. prausnitzii
low D IL-17A IFN-γ * ns *** *** *** **** **** 100 **** **** **** 100
cells 80 80 + L. acidophilus F. prausnitzii SEB 60 60 CD4
40 40 cells (%) +
gated on proliferation dye 20 20
TRBV9 TRBV20-1 TRBV28 TRBV29-1 TRBV5-1 CD4 0 0
TRBV5-6 TRBV5-5 TRBV4-1,2,3 TRBV4-3 TRBV12-3,4 low TRBV3-1 TRBV25-1 TRBV10-3 TRBV6-5,6,9 TRBV6-2 TRBV6-6 TRBV27 TRBV14 TRBV19 TRBV18 IL-22 IL-4 TRBV30 TRBV11-2 TRBV2 TRBV13 Unkown TRBV 50 25 * * ** ns ns ns ns ns ns ns 20 of CFSE 40 MANUSCRIPT+ E 30 15 E. coli B. animalis SEB 20 10 Cytokine
10 5
0 0
high high L. acidophilus F. prausnitzii SEB SEB E. coli E. coli CFSE CFSE B. animalis B. animalis IL-17A+ + + + - - - - L. acidophilusF. prausnitzii L. acidophilusF. prausnitzii IFN-γ + + - - + + - - IL-22+ - + - + - + - G E. coli B. animalis SEB
F SEB E. coli B. animalis 105 1 0.1 105 17.1 0.37 105 5.8 0.7
104 104 104
103 103 103
102 102 102 RORγt + + + + - - - - ROR γ t ACCEPTED
0 0 0 T-bet + + - - + + - - 72.6 26.3 31.5 51 15.5 78 GATA-3 + - + - + - + - 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 105 46.3 0.8 105 18.6 0.35 105 9.35 0.5
104 104 104
103 103 103
2 2 2 T-bet 10 10 10
0 0 0 27.2 25.7 29.9 51.1 11.9 78.3 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 CFSE Figure 4 ACCEPTED MANUSCRIPT A IL1B CSF2 IL6 CXCL1 CXCL5 CXCL8
) 25 15 ** 15 15 15 20 2 ns **** ns *** ** **** ** **** ns** **** ** **** * ns* ** ns * 20 **** * * * 15 10 10 10 10 15 10 10 5 5 5 5 5 5 control treated (log
Expression relative to 0 0 0 0 0 0 CXCL9 CXCL10 CXCL11 ) 2 30 **** ns 15 **** ns 15 **** ns ns ns ns ns ns ns Control treated 20 10 10 SEB expanded T cells Proteobacteria-reactive T cells 10 5 5 Actinobacteria-reactive T cells control treated (log Expression relative to 0 0 0 Firmicutes-reactive T cells TNF CXCL1 CXCL5 CXCL8 CXCL9 CXCL10 CXCL11 ICAM1 B **** ns 25 **** ns 20 **** ns 25 ns ns 6 **** ns ) 15 * 10 ns ns** 15 ns ** 8 ns ns* ns 2 ns ns ns * * ns ns ns * 8 * * * 20 20 6 15 10 10 4 6 15 15 4 10 4 10 10 5 5 2 2 5 2 5 5
control treated (log 0 0 0 0 0 0 0 0 Expression relative to C D αIFN-γ + - - - + + + - - - + + - + + - αIFN-γ + - - - + + + - - - + + - + + - αIL-22 - + - - + - - + + - + - + + + - αIL-22 - + - - + - - + + - + - + + + - αIL-17A - - + - - + - + - + + + + - + - αIL-17A - - + - - + - + - + + + + - + -
control αTNF-α - - - + - - + - + + - + + + + - control αTNF-α - - - + - - + - + + - + + + + - CXCL2 TNF CSF2 CXCL5 CXCL3 MANUSCRIPT CXCL2 IL1B CXCL1 CXCL5 CXCL3 CXCL6 MUC1 CXCL8 CXCL8 CCL3 IL6 IL6 REG3G CXCL1 CXCL9 ICAM1 CXCL10 CXCL10 CXCL11 CXCL11 ICAM1 CXCL9 CCL2 CCL7 -3 -2 -1 0 1 2 3 LIM1863 CCL2 -3 -2 -1 0 1 2 3 CCD18co IL-17A-/TNF-α-dependent module IFN-γ-dependent module
CXCL2 CSF2 CXCL3 CXCL5 CXCL6 CXCL8 CXCL1 ns ns ns ns ns ns ns E 10 **** ns 10 **** ns 10 **** ns 20 **** ns 15 **** ns 15 **** * 10 **** ns ) 2 8 8 8 15 8 10 10 6 6 6 10 6 4 4 ACCEPTED4 5 5 5 4 2 2 2 0 2 0 0 0 0 0 -5 0 -2 -2 -2 -10 -5 -5 -2 CXCL10 CXCL11 CXCL9 ns ns ns 10 ** ns 10 ** ns 10 ** ns 8 8 8 Controls (n=9) Active IBD, lesional site (n=30) 6 6 6 Active IBD, uninvolved site (n=20) 4 4 4 IBD, remission (n=14) 2 2 2 0 0 0 Expression relative to control median (log Figure 5 -2 -2 -2 ACCEPTED MANUSCRIPT A PBMCs C E. coli B. animalis L. acidophilus ** 0.06 0.03 0.03 45 **** **** *** ** 40 0.04 0.02 0.02 35
0.02 0.01 0.01 30 25 ns 0.00 0.00 0.00 of total live cells (%)
- 20 F. prausnitzii B. vulgatus R. intestinalis R. obeum 0.015 *** 0.015 *** 0.008 **** 0.008 *** 15
0.006 0.006 10 cells (%)
+ 0.010 0.010 CD45RA + 0.004 0.004 5
0.005 0.005 CD4 0 0.002 0.002 of CD4 + Controls (n=13) 0.000 0.000 0.000 0.000 Active IBD, lesional site (n=15) Active IBD, uninvolved site (n=15)
CD154 SEB S. aureus M. tuberculosis C. albicans 8 ** 0.15 ns 0.10 * 0.08 *
0.08 6 0.06 0.10 0.06 4 0.04 0.04 0.05 2 0.02 0.02
0 0.00 0.00 0.00 Control (n=30) IBD (n=38)
LPMCs B E. coli B. animalis L. acidophilus MANUSCRIPT 0.5 ns 0.04 ns 0.03 ns 0.4 0.03 0.02 0.3 0.02 0.2 0.01 0.01 0.1
0.0 0.00 0.00 F. prausnitzii B. vulgatus R. intestinalis R. obeum 0.025 ns 0.04 ns 0.020 ns 0.025 ns cells (%) + 0.020 0.020 0.03 0.015 0.015 0.015 0.02 0.010 of CD4
+ 0.010 0.010 0.01 0.005 0.005 0.005 TNF- α
+ 0.000 0.00 0.000 0.000 SEB S. aureus M. tuberculosis C. albicans 4 ns 0.15 ACCEPTEDns 0.020 ns 0.25 ns CD154 0.20 3 0.015 0.10 0.15 2 0.010 0.10 0.05 1 0.005 0.05
0 0.00 0.000 0.00 Control mucosa (n=17-20) Inflamed mucosa (n=10-15) Figure 6 ACCEPTED MANUSCRIPT
A E. coli B E. coli 70 IL-17A 100 IFN-γ 40 IL-22 IL-17A+ IFN-γ- IL-17A+ IFN-γ+ IL-17A- IFN-γ+ ns 50 60 80 **** 90 ** ns 60 **** 70 ** 80 50 30 40 50 70 60 40 40 60 30 50 50 20 30 40
cells (%) 30
+ 40 cells (%) 20 + 30 30 20 20 10 20 20
CD4 10
+ 10 CD4 10
10 + 10 0 0 0 0 0 0 SEB SEB
CD154
+ CD154
+ + - + + - + 70 IL-17A 100 IFN-γ 40 IL-22 50 IL-17A IFN-γ 60 IL-17A IFN-γ 80 IL-17A IFN-γ ns ns ns ns ns ns 90 60 70 80 40 50 30 60 50 70
of TNF-α 40
+ 60 of TNF- α 50 40 + 30 50 20 30 40 30 40 20 30 30 20 20 10 Cytokine 20
20 Cytokine 10 10 10 10 10 0 0 0 0 0 0 Controls (n=23) Controls (n=23) IBD (n=33) IBD (n=34)
C E. coli D IL1B IL6 IL23A 10 10 10 ns ns ns **** ns **** ns **** ns 8 8 8 ) 2 * 6 MANUSCRIPT6 6 4 4 4 2 2 2 Control mucosa Inflamed mucosa median (log 0 0 0 α + + + + + + + + TNF- -2 -2 -2 + + + + - - - - IL-17A Expression relative to control Controls (n=9) IFN-γ + + - - + + - - Active IBD, lesional site (n=30) IL-22 + - + - + - + - Active IBD, uninvolved site (n=20) IBD, remission (n=14)
E IL-17A IFN-γ 2.5 2.5 ** ns ns * ns ns ns ns 2.0 2.0
+ 1.5 1.5
1 1
Cytokine ACCEPTED
(relative to control) 0.5 0.5
0 0 IL-1β − + − − + − + − − + IL-6 − − + − + − − + − + IL-23 − − − + + − − − + +
Figure 7 ACCEPTED MANUSCRIPT
Supplemental Information
Induction of Local and Systemic CD4 + T-cell Responses to Intestinal Microbes in
Healthy Individuals and Alterations in Patients With Inflammatory Bowel Diseases
Ahmed N. Hegazy*, Nathaniel R. West*, Michael J. T. Stubbington, Emily Wendt, Kim
Suijker, Angeliki Datsi, Sebastien This, Camille Danne, Suzanne Campion, Sylvia Duncan,
Benjamin M. J. Owens, Holm H. Uhlig, Andrew McMichael, Oxford IBD Cohort
Investigators, Andreas Bergthaler, Sarah A. Teichmann, Satish Keshav, Fiona Powrie #
*Co-first authors
#Corresponding author
MANUSCRIPT
ACCEPTED
1 ACCEPTED MANUSCRIPT
Supplemental Figure Legends
Supplementary Figure 1. Bacterial strains used in the study and detection of enteric bacteria-specific memory CD4 + T cells (related to Figure 1)
(A) Bacterial species are listed according to their family and phylum classification. Coloured circles relate the bacteria to their gaseous requirement.
(B) Frequencies of CD154 + cells among peripheral CD4 + T cells in adults after short-term stimulation with heat-inactivated bacteria. Dots represent different donors (n=30).
(C) Representative flow cytometric plots of different stimulations showing CD154 expression against CD4 within CD3 + cells after short-term stimulation with heat-inactivated bacteria.
Frequencies of CD154 within CD4 + T cells are depicted.
(D) Representative plot of CD69 and ICOS expression on CD4 +CD154 + T cells after short-term stimulation with heat-inactivated S. typhimurium.
(E) PBMCs were stimulated with the indicated heat-inactivated bacteria in presence or absence of MHC-II blocking antibodies and analysed MANUSCRIPT for CD154 expression. Percentage of reduction in CD154 + frequencies after MHCII blockade compared to control treated is depicted. Frequencies (± SEM) of 3 independent experiments is depicted (n=5-6 independent donors).
(F) Estimated microbiota-reactive cells per million CD4 + T cells in adult blood (n=30). The numbers of microbiota-reactive T cells were calculated based on the frequencies of
CD4 +CD154 + T cells. Background (no microbe stimulation) was subtracted from bacterial stimulations. Significance calculated in relation to LPS stimulation. (G) FrequenciesACCEPTED ± SEM of CD154 + cells among CD4 + T cells in adult (n=30) or cord blood (n=3) after short-term stimulation with heat-inactivated bacteria.
2 ACCEPTED MANUSCRIPT
Statistics: panels E and G, Mann-Whitney test; C, and F, panel F, one-way ANOVA with
Sidak’s multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001;
**** P≤0.0001.
Supplementary Figure 2. Detection and characterisation of microbiota-specific memory
CD4 + T cells (related to Figure 1 and 2)
(A) Frequencies of CD154 + cells among naïve and memory peripheral CD4 + T cells in adults after short-term stimulation with heat-inactivated bacteria. Dots represent different donors
(n=30).
(B) Percentage of memory cells within CD4 +CD154 + T cells. Each symbol represents an antigen-reactive population from one individual; the line indicates the mean of experiments performed independently with blood obtained at different times (n=19-30). Significance calculated in relation to total CD4 + T cells. (C) Percentages of cytokine-expressing cells among MANUSCRIPT memory CD4 +CD45RA -CD154 + T cells are shown. Frequencies (± SEM) of 27 independent donors are depicted.
(D) Surface marker and chemokine receptor expression frequencies among CD4 +
CD45RA - CD154 + and CD4 + CD45RA - CD154 - T cells after short-term stimulation with B. animalis . Frequencies within CD4 + CD45RA - CD154 + or CD4 + CD45RA - CD154 - T cells are depicted.
Statistics: panel A, Mann-Whitney tests; panels B, One-way ANOVA with Sidak’s multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001. ACCEPTED Supplementary Figure 3. Detection and characterisation of microbiota-specific CD4 + T cells within gut-resident memory T cells (related to Figure 3)
(A) Representative flow cytometry plots showing CD45RA, Integrin β7, CD69, CCR7, and
CD127 expression in gut-resident CD3 +CD4 + T cells. Data representative of ten independent
3 ACCEPTED MANUSCRIPT
donors.
(B, C) Gating strategy for identification of enteric bacteria-specific memory CD4 + T cells in
LPMCs and PBMCs after stimulation with heat-inactivated E. coli in a representative donor.
(D) LPMCs were stimulated with heat-inactivated bacteria and analysed for CD154 expression. Estimated gut resident microbiota-reactive cells per million CD4 + T cells compared with adult blood is depicted (n=17 for control mucosa; n=25-31 for blood).
(E, F) Cytokine production by CD4 +CD154 +TNF-α+ T cells after short-term stimulation with the indicated heat-inactivated bacteria. Frequencies (± SEM) of 10-23 independent donors.
Significance calculated between the respective stimulations in control mucosa and peripheral blood.
Statistics: panel D, E and F, Mann-Whitney test; ns, not significant; * P≤0.05; ** P≤0.01; ***
P≤0.001; **** P≤0.0001.
Supplementary Figure 4. Expansion and detection of microbiota-specific memory CD4 + T cells (related to Figure 4) MANUSCRIPT Total memory CD4 + T cells were isolated, labelled with CFSE and cultured with autologous irradiated monocytes in the presence or absence of the indicated heat-inactivated bacteria and blocking antibodies to MHCII.
(A) Experimental setup.
(B) Shown are the CFSE profiles and ICOS expression on days 3 and 6 of stimulation in a representative donor.
(C) Frequencies (± SEM) of CFSE low proliferating cells after stimulation with S . aureus and M. tuberculosisACCEPTED (n=5-21). Data representative of 5-10 independent experiments. Each dot represents an independent donor.
(D) Frequencies (± SEM) of CFSE low proliferating CD4 + T cells in presence or absence of anti–MHC-II. Data representative of two independent experiments.
(E, F) CFSE profiles and expression of CD25 and OX40 on day 6 of stimulation in a
4 ACCEPTED MANUSCRIPT
representative donor. (F) Geometric mean and frequencies (± SEM) of ICOS, CD25, and
OX40 expression on CFSE low CD4 + T cells on day 6.
(G) Number of unique TCR β clonotypes detected in bacteria-reactive CD4 + T cells. TCRV β sequencing was performed from 3 independent donors. Each circle represents an independent donor. TCRV β usage of the different reactivities of three independent donors is summarized in Supplementary Table 3.
(H) Heat map showing the frequency of shared clonotypes between different bacteria-reactive
CD4 + T cell responses. SEB and PHA expanded memory T cells were used as controls. Data from 3 independent donors. The CDR3 sequences are summarized in Supplementary Table 4.
Statistics: panels C, D and F, One-way ANOVA with Sidak’s multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; **** P≤0.0001.
Supplementary Figure 5. Cross-reactivity, cytokine production and transcription factor expression by antigen-reactive memory CD4 + T cells (related to Figure 5) (A) CD154 + memory CD4 T cells from PBMCs wereMANUSCRIPT sorted after short-term stimulation with E. coli and B. animalis lysates and expanded them for 10-14 days with CD3/CD28 and IL-2.
The E. coli Nissle - and B. animalis-reactive T cell lines were CFSE-labelled and then co- incubated with autologous monocytes loaded with the various bacterial lysates. Frequencies
(± SEM) of CFSE low proliferating cells after 5 days of stimulation with the indicated bacteria are shown. Data from 3-6 independent donors.
(B-F) Total memory CD4 + T cells were isolated, labelled with CFSE and cultured with autologous irradiated monocytes in the presence of the indicated heat-inactivated bacteria or antigens. ACCEPTED (B, C) Production of cytokines by proliferating CFSE low cells measured by intracellular staining after PMA/Ionomycin stimulation on day 7 of primary stimulation with monocytes and heat-inactivated bacteria. Frequencies (± SEM) of 3-13 independent donors are depicted.
Each dot represents an independent donor.
5 ACCEPTED MANUSCRIPT
(D, E) ROR γt, T-bet and GATA-3 expression on day 7 of primary stimulation with monocytes and influenza seasonal vaccination or heat-inactivated S. aureus in a representative donor.
(E) Boolean gating analysis showing each possible combination of ROR γt T-bet and
GATA-3 production by CFSE low proliferating cells. Data generated from 3 independent donors.
(F) Production of IL-10 by expanded CFSE low CD4 + cells after PMA/Ionomycin stimulation.
Frequencies (± SEM) and IFN-γ / IL-17A co-expression within IL-10 + and IL-10 - cells are depicted from 5-8 independent donors.
Statistics: panels A, C, One-way ANOVA with Sidak’s multiple comparison test; panel F,
Kruskal-Wallis test with Dunn’s multiple comparison; ns, not significant; *** P≤0.001; ****
P≤0.0001.
Supplementary Figure 6. Functional characteristics of enteric bacteria-reactive CD4 + T cells in IBD (related to Figure 7) (A) PBMCs isolated from healthy donors or IBD MANUSCRIPTpatients were stimulated with the indicated heat-inactivated bacteria and analysed for CD154 expression. Frequencies (± SEM) of reactive CD154 + cells among CD4 + T cells in peripheral blood are depicted (n=30-38).
Demographic and clinical characteristics of IBD patients are summarized in Supplementary
Table 6.
(B) LPMCs were isolated from inflamed surgical specimens from IBD patients or non-inflamed and tumour-free surgical specimens from colorectal cancer patients. Isolated
LPMCs were stimulated with the indicated heat-inactivated bacteria and analysed for CD154 expression. FrequenciesACCEPTED (± SEM) of reactive CD154+ TNF-α+ cells among CD4 + T cells in peripheral blood are depicted (n=10-20).
(C) Microarray analysis of intestinal biopsies obtained at endoscopy (n=6 controls, 24 UC and
37 CD; Gene Expression Omnibus entry GSE16879). The relative abundance of memory
CD4 + T cells in control and inflamed IBD tissue was estimated in silico using CIBERSORT
6 ACCEPTED MANUSCRIPT
(see Supplementary Methods for details).
(D-F) PBMCs isolated from healthy and IBD patients were stimulated with the indicated heat-inactivated bacteria or SEB and analysed for CD154 expression and intracellular cytokine expression. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6.
(D) IL-2 expression in CD154 + TNF-α+ memory CD4 + T cells after stimulation with the indicated heat-inactivated bacteria or SEB. Frequencies (± SEM) of 23-33 independent donors. Each dot represent an independent donor.
(E, F) IL-17A, IFN-γ, IL-22, and IL-2 expression in CD154 + TNF-α + memory CD4 + T cells after stimulation with the indicated heat-inactivated bacteria. Each dot represents an independent donor. Demographic and clinical characteristics of IBD patients are summarized in Supplementary Table 6.
(G) IL-17A expression in CD154 + TNF-α + memory CD4 + T cells after stimulation with S. typhimurium . IBD patients were categorised by disease MANUSCRIPT phenotype (UC, CD), disease activity according to clinical notes, and current treatment.
(H) Total memory CD4 + T cells were isolated from healthy controls or IBD patients blood, labelled with CFSE and cultured with autologous irradiated monocytes in the presence of the indicated heat-inactivated bacteria or antigens. Production of IL-10 by expanded CFSE low
CD4 + cells after PMA/Ionomycin stimulation on day 7 of stimulation. Frequencies (± SEM) are depicted from 9-11 independent donors.
(I) CD4 +CD45RO +CD45RA -CD25 -CD8 - memory CD4 + T cells were isolated from healthy controls or ACCEPTED IBD patients blood, labelled with CFSE, and stimulated with autologous monocytes pulsed with B. animalis in the presence or absence of the indicated cytokines. Data represent mean (± SEM) fold changes in IL-17A or IFN-γ expression frequencies relative to cells expanded without cytokines.
Statistics: panels A-E and G-H, Mann-Whitney test; panel F, One-way ANOVA with Sidak’s
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multiple comparison test; ns, not significant; * P≤0.05; ** P≤0.01; *** P≤0.001; ****
P≤0.0001.
Supplementary Figure 7. Microbiota-specific memory T cell responses in intestinal homeostasis and inflammation
Microbiota-specific memory CD4 + T cells are abundant in peripheral blood and mucosa.
Luminal sampling of intestinal microbiota by dendritic cells and repetitive epithelial breaches after birth might induce CD4 + T cell responses targeted towards intestinal bacteria. Enteric bacteria-specific CD4 + T cells produce barrier-protective cytokines such as IL-17A, IFN-γ and IL-22. Furthermore, certain commensal-specific CD4 + T cells also produce IL-10. We speculate that the cytokine production by commensal-specific CD4 + T cells might play a significant role in supporting gut homeostasis and the mutualistic relationship with intestinal microbiota. In inflammation, epithelial break down and leakage leads to increased availability of luminal antigens and bacterial stimuli, which acMANUSCRIPTtivate the innate system and the expression of Th17 inducing cytokines (IL-1β, IL-6 and IL-23). Recruited microbiota-specific memory
CD4 + T cells accumulate in inflamed mucosa and are polarised towards more IL-17A production. Activated memory cells express IL-17A, TNF-α, and IFN-γ, which stimulate intestinal cells such as stromal and epithelial cells to express various chemokine ligands and cytokines. This may promote recruitment and activation of myeloid cell populations.
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Supplemental Tables
Supplementary Table 1. Table S1. Enteric bacterial species and control species used in the study (related to Figure 1).
Supplementary Table 2. T cell receptor (TCR) V β usage in expanded microbiota-reactive T cells measured by V β antibody panel from Beckman Coulter (related to Figure 3).
Supplementary Table 3. T cell receptor (TCR) V β usage in expanded microbiota-reactive T cells using multiplex PCR assay and deep sequencing (related to Figure 3).
Supplementary Table 4. CDR3 sequences of microbiota-reactive memory CD4 + T cells
(related to Figure 3).
Supplementary Table 5. Clinical characteristics of Oxford cohort patients assessed in this study for gene expression analysis. (related to Figure 5E and 7D).
Supplementary Table 6. Clinical characteristics of Oxford cohort patients assessed in this study for the analysis of microbiota-specific CD4 MANUSCRIPT T cells. (related to Figure 7A, B, and Supplementary 6A-H).
Supplementary Table 7. Clinical characteristics of Oxford cohort patients assessed in this study for cell accumulation in the mucosa. (related to Figure 3B, C and Figure 7C).
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Supplemental Experimental Procedures
Human Samples and Cell Isolation. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (GE Healthcare Life Sciences) density gradient centrifugation, resuspended in PBS with 2mM EDTA and 0.02% BSA, and further processed. Gut specimens were obtained from patients with IBD undergoing surgery for severe, chronically active, or complicated disease. Control gut specimens from macroscopically healthy areas were collected from colorectal cancer patients as non-inflammatory controls. One intestinal pinch biopsy was obtained from healthy donors (Colorectal cancer screening, or other non-IBD related conditions) or IBD patients during routine endoscopy, from lesional, and non-lesional sites, attending the John Radcliffe Hospital (Oxford, UK). Inflammation status of biopsies was binarized into either inflamed or uninflamed categories based on endoscopic assessment.
Further information about the analysed IBD patients can be found in Supplementary Table
5, Supplementary Table 6, and Supplementary Table 7 . LPMCs were isolated as described 1. In brief, mucosa MANUSCRIPT was dissected and washed in 1 mM DTT at room temperature for 15 min to remove mucus. Specimens were washed three times with
0.75 mM EDTA at 37°C for 45 min to detach epithelial crypts and digested overnight with
0.1 mg/ml collagenase D (Roche). Cells were centrifuged for 30 min in a Percoll gradient and collected at the 40–60% interface. All solutions were supplemented with antibiotics
(penicillin/streptomycin, 40 g/ml gentamicin, and 0.025 g/ml amphotericin B).
Flow Cytometry . Cells were stained with the following monoclonal antibodies as described 2:
CD4-BV510 (OKT4), CD4 PE-Dazzle (RPA-T4), CD3-PE-CF594 or –BV510 (UCHT1), CD45RA-BV711ACCEPTED (HI100), CD45RO-BV570 (UCHL1), CD161-eF450 (HP3G10), CCR7-PE (G034H7), CCR2-APC (K036C2), CCR6-BV605 (G034E3), CCR9-PE-Cy7 (L053E8),
CXCR3-PE/Cy5 (1C6/CXCR3), CCR4-PE-Cy7 (1G1), CCR10-PE (314305), Integrin
7-FITC/APC (FIB504), CD69-PE-Cy7 (FN50), IL-2-BV650 (MQ1-17H12), TNF- -PB
(MAb11), CD154-FITC/PE-Cy5 (24-31), IL-17A BV711 (eBio64DEC17), IL-22-PE-Cy7
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(22URTI), IFN-γ-AF700 (B27), GM-CSF-APC (BVD2-21C11), IL-4-PE (8D4-8)
(eBioscience, Biolegend and Becton Dickinson). FACS panels contained up to 12 fluorochromes. Compensation beads were used for compensations (BDBioscience). TCRV β usage was assessed using the IOTest Beta Mark TCR Vbeta Repertoire Kit (Beckman
Coulter). Samples were acquired on FACS LSRFortessa and FACSLSRII (Becton
Dickinson); ≥2x10 5 memory CD4 + T cells were acquired. Data were analysed with FlowJo
(Tree Star) and SPICE. For analysis of cytokine expression by microbiota-specific T cells, a minimum of 20 CD4 + CD154 + TNF-α + T cells was used; donors with lower events were excluded.
CD154 Enrichment of Antigen-specific T Cells. For MACS enrichment of CD4 +CD154 + T cells, CD154 Enrichment and Detection Kit was used (Miltenyi Biotec). Briefly, cells were plated at 5x10 6/cm 2 for 7-12 h with heat-inactivated bacteria in the presence of anti-CD40 blocking antibody (HB14) and anti-CD28 stimulation antibody (CD28.6). Anti-CD40 blockade prevents down-regulation of CD154, MANUSCRIPT while CD28 co-stimulation optimizes induction of CD154 expression. For MHCII blockade, 10 µg/ml of a pan-HLA class-II blocking antibody (HLA-DR, DP, DQ; (Tü39)) was added 30 minutes before bacterial stimulation. Cord and adult blood analysis was performed identically. Antigen-presenting cell
(APCs) abundance is similar in cord and adult blood3,4 .
Cross Reactivity Assay. CD154 + CD4 + T cells were isolated using MACS enrichment and
FACS sorting from total PBMCs after stimulation with E. coli Nissle and B. animalis lysates as described above . CD154 + CD4 + T cells were expanded for 10-14 days with IL-2 and anti- CD3/CD28 beadsACCEPTED (beads/T cell ratio, 1:4, Dynals). The expanded T cell lines were washed in IL-2 free medium and incubated for 12h without IL-2 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). The expanded T cell lines were labelled with CFSE, and then
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were co-incubated with autologous monocytes loaded with various bacterial lysates. T cells were co-cultured with the irradiated autologous monocytes at a ratio of 2:1 for 5-7 days. The
CFSE dilution was measured at the end of the culture. Autologous CD14 + monocytes were isolated from PBMC using anti-CD14 microbeads (Miltenyi Biotec) and frozen until the T cell co-culture. Monocytes were thawed down, irradiated (45 Gy) and then pre-incubated for
3 h with bacterial lysates before T-cell co-culture.
Intracellular Cytokine Analysis. For intracellular cytokine staining, cells were restimulated with PMA (5 ng/ml) and ionomycin (500 ng/ml; Sigma) or bacterial lysates. 5 µg/ml brefeldin
A (Sigma-Aldrich) was added at 2 h. After 4-12 h, cells were stained with fixable viability dye eFluor® 780 (eBioscience) and surface markers, fixed with 2% formaldehyde (Merck), and stained for cytokines in buffer containing 0.05% saponin (Sigma-Aldrich). In some assays, cytokine analysis was combined with intracellular CD154 detection as described 5,6 .
Transcription Factors Staining. Transcription factor expression was analysed using the FoxP3 staining buffer set (eBioscience) according MANUSCRIPT to manufacturer’s instructions. Briefly, cells were stained with fixable viability dye eFluor® 780 (eBioscience) and surface markers, fixed with 1x Fixation/Permeabilization buffer, followed by antibody staining and washing in
1x permeabilization buffer. Tbet-Pacific Blue (4B10) and GATA-3-Alexa-647 (TWAJ) were from eBioscience. ROR γt-PE (Q21-559) was from BDBioscience.
Preparation of Bacterial Lysates. Different bacteria were cultured in their respective optimal media for 16h at 37 °C, washed in sterile PBS and heat-inactivated at 65 °C for 1 hour, followed by three freeze-thaw cycles. Extremely oxygen sensitive bacteria were provided by ACCEPTED Sylvia Duncan, University of Abeerden, after heat-inactivation. Suspensions were centrifuged at max speed for 15 min and supernatants collected. Protein concentration was quantified using Nanodrop (Thermo Fisher Scientific). The following bacterial strains were used: Bacteroides vulgatus (Bv 1447), Bifidobacterium animalis subsp. lactis Bi-07,
Clostridium difficile (OXF1003, Toxin AB -), E. coli (Nissle 1917 ), Faecalibacterium
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prauznitzii (A2-165), Lactobacillus acidophilus (NCFM), Roseburia intestinalis (M50/1),
Ruminococcus obeum (A2-162), Salmonella enterica serovar typhimurium (NCTC 12023),
Staphylococcus aureus (NCTC 6571). Further information regarding the bacterial stains is in
Supplementary Table 1. Bacterial lysates were titrated in CFSE dilution assay and an optimal concentration was used (5-15 g/ml). Tetanus Toxoid (Calbiochem) and influenza seasonal vaccine (OPTAFLU, Novartis) were used at 5 g/ml. Heat-killed Mycobacterium tuberculosis (H37Ra) and Candida albicans were from InvivoGen. Ultrapure LPS-EB from
E. coli 0111:B4 (InvivoGen) was used as a stimulation control. SEB was used at 1 µg/ml
(Sigma).
RNA Extraction, cDNA Synthesis and qPCR. Tissue was homogenized using a motor with sterile RNase/DNase-free disposable pestles (both VWR) in RLT buffer (QIAGEN). Cells were lysed directly in RLT buffer. RNA was isolated using RNeasy Mini kit (QIAGEN) or
Quick-RNA TM MiniPrep kit (Zymo Research, Irvine, USA) followed by cDNA preparation using High-Capacity cDNA Reverse Transcription MANUSCRIPT Kit (Applied Biosystems) with random hexamers. Q-PCR was performed using a CFX96 (Bio-Rad) or ViiA7 384-well real-time PCR system (Applied Biosystems) with TaqMan assays (Life Technologies) and PrecisionPLUS
Mastermix (Primerdesign). Expression levels were normalized to a house-keeping (hk) gene
(RPLP0 ) and expressed as 2^-(CTgene-CThk). Heat maps were made using Cluster 3.0 and
Java TreeView (Michael Eisen, Stanford University).
CIBERSORT Analysis. To calculate enrichment of cell populations using CIBERSORT 7, we analyzed the GSE16879 dataset on default settings. For each sample, relative expression of PTPRC (CD45,ACCEPTED representing relative leukocyte content) was calculated separately using median-normalized microarray data. This value was then multiplied by CIBERSORT cell type scores (e.g. proportion of memory CD4 + T cells in the total leukocyte fraction) to estimate cell type enrichment levels. Finally, for cell types of interest, fold differences between IBD and control specimens were calculated to estimate relative cell type abundance
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in active IBD lesions versus healthy tissue.
TCRV βββ Sequencing and Analysis. Memory T cells (3.6x10 6) were cultured with autologous monocytes pulsed with heat-inactivated bacteria from three healthy donors. Expanded
CFSE low ICOS +CD4 + T cells were sorted into DNase/RNase free water with BSA (10 mg/ml) in 96 well plates (100 cells/well) and stored at -80°C. TCRV β sequence analysis was obtained by a series of three nested PCR reactions as described previously 8. Reverse transcription and preamplification were performed with a One-Step RT-PCR kit (Qiagen) using multiplex PCR with multiple V β region primers and a C β region primer. After the first reaction, an aliquot was used for the second PCR using a set of multiple internally nested TCRVβ primers and internally nested C β region primer with HotStarTaq DNA polymerase kit (Qiagen). In the final PCR reaction, an aliquot of the second PCR was used and amplification was performed using barcoding primers containing the common 23 base sequence (incorporated into the
β β second set of V primers) and a third internallyMANUSCRIPT nested C primer and Illumina Pair-End primers. After the third PCR reaction, each PCR product should have a unique set of barcodes incorporated that specifies plate, row and column and have Illumina Paired-End sequences that enabled sequencing on the Illumina MiSeq platform. The PCR products were combined at equal proportion by volume, run on a 1.2% agarose gel, and a band around 350 to 380 bp was excised and gel purified using a Qiaquick gel extraction kit (Qiagen). Barcoded products were sequenced on the Illumina MiSeq platform.
Sequence reads were trimmed using 'Trim Galore' software
(http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) with default settings.
Reads were demultiplexedACCEPTED using a custom Python script according to the presence of index sequences. A further custom script then split reads from each well according to the presence of primer sequences used in the PCR amplification. These reads were then analysed by
MICXR v1.6 (ref to http://www.nature.com/nmeth/journal/v12/n5/full/nmeth.3364.html)
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using settings appropriate for the anticipated TCR locus of origin (A or B). MIXCR provided
CDR3 sequences along with counts of the number of times each sequence was observed.
Detected CDR3 sequences were filtered to exclude those with ≤10 counts per well. There were 48 wells per donor of SEB-stimulated or PHA-stimulated cells whilst there were
96-wells of bacterially-stimulated cells. Furthermore, some wells did not contain any valid
TCR sequences after filtering for low read counts suggesting that amplification or sequencing had failed for those wells. To permit comparisons of the number of TCR sequences between plates with differing numbers of successful wells, counts were scaled to the theoretical maximum of 96 wells.
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Supplemental References
1. Geremia A, Arancibia-Cárcamo CV, Fleming MPP, et al. IL-23-responsive innate
lymphoid cells are increased in inflammatory bowel disease. J Exp Med 2011;208:1127–
1133.
2. Brodie T, Brenna E, Sallusto F. OMIP-018: Chemokine receptor expression on human T
helper cells. Cytometry A 2013.
3. Marodi L, Leijh PCJ, Furth RV. Characteristics and Functional Capacities of Human
Cord Blood Granulocytes and Monocytes. Pediatr Res 1984;18:1127–1131.
4. Ebba Sohlberg SS-HKBES-E. Cord blood monocyte subsets are similar to adult and
show potent peptidoglycan-stimulated cytokine responses. Immunology 2011;133:41.
5. Frentsch M, Arbach O, Kirchhoff D, et al. Direct access to CD4+ T cells specific for
defined antigens according to CD154 expression. Nat Med 2005;11:1118–1124.
6. Bacher P, Schink C, Teutschbein J, et al. Antigen-Reactive T Cell Enrichment for Direct, High-Resolution Analysis of the Human Naive MANUSCRIPT and Memory Th Cell Repertoire. The Journal of Immunology 2013;190:3967–3976.
7. Newman AM, Liu CL, Green MR, et al. Robust enumeration of cell subsets from tissue
expression profiles. Nat. Methods 2015;12:453–457.
8. Han A, Glanville J, Hansmann L, et al. Linking T-cell receptor sequence to functional
phenotype at the single-cell level. Nat Biotechnol 2014;32:684–692.
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Supplementary Table 1. Enteric bacterial species and control species used in the study Bacterial species Strain Family Phylum Kingdom Phylum Gram Growth Changes Refere Designation abundance stain characteristics during IBD on nce (of the species) Phylum level Bacteroides vulgatus Bv 1447 Bacteroidaceae Bacteroidetes Bacteria ~20-40% Negative Obligate ê 1, 2, 3, anaerobe 4 Bifidobacterium animalis subsp. Bi-07 Bifidobacteriaceae Actinobacteria Bacteria ~1-10% Positive Obligate ê (at family 1, 2, 3, lactis anaerobe level) 4 Clostridium difficile OXF1003, Clostridiaceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3, Toxin AB- anaerobe 4 Escherichia coli Nissle 1917 Enterobacteriaceae Proteobacteria Bacteria ~5-15% Negative Facultative é 1, 2, 3, anaerobe 4 Faecalibacterium prauznitzii A2-165) Ruminococcaceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3, anaerobe 4 Lactobacillus acidophilus NCFM Lactobacillaceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3, anaerobe 4 Roseburia intestinalis M50/1 Lachnospiraceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3, anaerobe 4 Ruminococcus obeum A2-162 Lachnospiraceae Firmicutes Bacteria ~50-70% Positive Obligate ê 1, 2, 3, MANUSCRIPT anaerobe 4 Salmonella enterica serovar NCTC 12023 Enterobacteriaceae Proteobacteria Bacteria ~5-15% Negative Facultative é 1, 2, 3, typhimurium anaerobe 4 Staphylococcus aureus NCTC 6571 Staphylococcaceae Firmicutes Bacteria ~50-70% Positive Facultative not invovled 1, 2, 3, anaerobe (at family 4 level) Mycobacterium tuberculosis H37Ra Mycobacteriaceae Actinobacteria Bacteria ~1-10% Positive Obligate not invovled 1, 2, 3, aerobe (at family 4 level) Candida albicans Debaryomycetaceae Saccharomycetes Fungi Facultative é 1, 2, 3, (Class) anaerobe 4
References 1. Morgan XC, Tickle TL, Sokol H, et al. DysfunctionACCEPTED of the intestinal microbiome in inflammatory bowel disease and treatment. Genome Biol. 2012;13:R79. 2. Kostic AD, Xavier RJ, Gevers D. The microbiome in inflammatory bowel disease: current status and the future ahead. Gastroenterology 2014;146:1489–1499. 3. Sokol H, Leducq V, Aschard H, et al. Fungal microbiota dysbiosis in IBD. Gut 2016. 4. Gevers D, Kugathasan S, Denson LA, et al. The Treatment-Naive Microbiome in New-Onset Crohn’s Disease. Cell Host Microbe 2014;15:382–392. ACCEPTED MANUSCRIPT
Supplementary Table 2. T cell receptor (TCR) V β usage in expanded microbiota-specific T cells measured by V β antibody panel from Beckman Coulter (related to Figure 3) Donors Microbe Vβ1β1β1 Vβ2β2β2 Vβ3β3β3 Vβ4β4β4 Vβ5.1β5.1β5.1 Vβ5.2β5.2β5.2 Vβ5.3β5.3β5.3 Vβ7.1β7.1β7.1 Vβ7.2β7.2β7.2 Vβ8β8β8 Vβ9β9β9 Vβ11β11β11 Vβ12β12β12 TRBV9 TRBV21-1 TRBV28 TRBV29-1 TRBV5-1 TRBV5-6 TRBV5-5 TRBV4-1,2,3 TRBV4-3 TRBV12-3,4 TRBV3-1 TRBV25-1 TRBV10-3 Donor A S. typhimurium 1.98 8.34 3.47 1.61 4.97 0.463 0.451 5.69 0.485 7.15 3.55 0.482 0.525 Donor A E. coli 5.32 9.6 3.8 1.2 7.78 1.32 0.393 0.892 1.25 4.25 1.86 0.968 3.85 Donor A B. animalis 1.86 11.1 3.87 0.504 8.59 0.301 0.705 0.888 0.135 1.9 3.03 0.272 0.904 Donor A L. acidophilus 2.06 11.7 4.68 0.744 7.18 0.513 0.239 0.647 0.216 2.4 1.94 0.275 1 Donor A F. prausnitzii 1.39 8.16 2.38 0.682 7.13 0.321 0.368 1.12 0.581 2.2 2.82 0.374 0.333 Donor A C. difficile 0.821 9.46 4.61 1.52 9.56 0.219 0.592 1.43 0 1.54 3.27 0 1.73 Donor A SEB 0.0745 0.0962 25.9 0.418 0.00517 0.446 0.0707 0.0943 0.07 0.165 0.027 0.0528 8.07 Donor A PHA 2.22 13.5 5.99 2.08 1.07 6.21 0.838 0.932 0.673 2.5 3.11 0.44 2.07 Donor B S. typhimurium 3.39 9.76 5.28 2.27 7.64 0.672 0.721 0.569 0.696 5.5 4.63 0.331 0.88 Donor B E. coli 3.52 11.9 4.32 1.96 7.97 0.714 0.921 1.02 1.56 5 4.22 0.184 0.74 Donor B B. animalis 2.36 4.07 7.63 4.29 7.72 0.461 3.41 1.12 1.48 3.23 1.81 0.575 1.13 Donor B L. acidophilus 8.58 11 3.49 4.26 9.74 0.762 1.81 0.499 2.15 4.19 0.955 0.416 0.949 Donor B F. prausnitzii 3.06 5.41 6.07 2.15 7.49 0.535 0.748 0.748 1.77 3.85 1.03 0.416 2.57 Donor B C. difficile 1.95 9.02 2.8 1.46 4.5 0.185 0.781 0.588 0.308 1.44 1.43 0.238 13.3 Donor B SEB 0.226 0.175 29.4 0.939 0.0283 0.434 0.107 0.0555 0.0598 0.274 0.077 0.0443 10 Donor B PHA 0.808 10.1 4.83 0.908 0.698 3.32 0.531 0.363 0.147 1.93 2.64 0.227 1.42 Donor C S. typhimurium 1.52 1.21 4.72 32.9 0.998 0.31 0.0963 0.84 0 9.8 0.357 0.0977 0.13 Donor C E. coli 1.34 1.22 4.53 26.3 1.28 0.181 0.167 0.642 0 9.4 0.337 0.136 0.256 Donor C B. animalis 1.36 1.61 1.58 1.8 1.92 0.378 0.188 1.2 0.0113 1.08 0.286 0.21 0.216 Donor C L. acidophilus 0.816 5.71 1.26 3.29 1.32 0.333MANUSCRIPT 1.18 1.22 0.00834 2.58 0.943 0.261 0.683 Donor C F. prausnitzii 0.498 1.51 0.6 1.73 0.585 0.234 0.109 0.269 0.0247 1.07 0.271 0.382 0.222 Donor C C. difficile 0.471 1.09 0.536 0.778 0.615 0.218 0.145 0.563 0.0046 4.63 0.165 0.208 0.258 Donor C SEB 0.191 0.155 19.9 1.43 0.0354 0.482 0.114 0.0692 0.0188 0.234 0.0615 0.0577 8.98 Donor C PHA 2.01 10.2 5.36 2.43 0.963 6.44 0.958 0.626 0.00905 4.24 2.61 0.732 1.87
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Supplementary Table 2. T cell receptor (TCR) V β usage in expandedACCEPTED microbiota-specific T cells measured by V β antibody panel from Beckman Coulter
Donors Microbe Vβ13.1β13.1β13.1 Vβ13.2β13.2β13.2 Vβ13.6β13.6β13.6 Vβ14β14β14 Vβ16β16β16 Vβ17β17β17 Vβ18β18β18 Vβ20β20β20 Vβ21.3β21.3β21.3 Vβ22β22β22 Vβ23β23β23 Unkown V βββ TRBV6-5,6,9 TRBV6-2 TRBV6-6 TRBV27 TRBV14 TRBV19 TRBV18 TRBV30 TRBV11-2 TRBV2 TRBV13 Donor A S. typhimurium 1.75 1.24 1.45 2.72 1.78 4.92 0.398 0.862 1.09 2.28 0.079 42.265 Donor A E. coli 2.07 1.73 2.09 3.2 0.731 5.7 0.515 4.25 0.901 5.48 0.267 30.583 Donor A B. animalis 0.727 6.54 0.998 1.89 1.09 7.64 0.197 1.98 1.41 15.5 0.955 27.014 ACCEPTED MANUSCRIPT
Donor A L. acidophilus 1.07 1.78 0.726 0.627 0.746 14.5 1.29 0.79 1.09 13 0.212 30.575 Donor A F. prausnitzii 1.09 0.556 1.35 1.27 0.896 3.21 0.921 1.39 1.91 19.2 0.463 39.885 Donor A C. difficile 1.09 1.35 1.39 2.07 0.369 2.48 1.13 3.5 1.86 11.9 0.0548 38.0542 Donor A SEB 0.11 5.22 0.333 2.02 0.333 14.6 0.0234 2.73 0.0444 0.354 0.177 38.56553 Donor A PHA 3.16 2.82 1.9 2.95 1.14 4.6 1.55 2.42 2.85 2.26 0.37 32.347 Donor B S. typhimurium 0.912 3.07 3.71 2.06 0.43 5.21 0.335 0.167 3.12 6.83 0.706 31.111 Donor B E. coli 2.47 3.98 2.96 2.71 0.792 5.14 1.41 0.2 3.11 4.82 0.432 27.947 Donor B B. animalis 1.49 4.37 4.08 1.37 0.421 6.43 0.812 0.559 0.677 4.04 0.118 36.347 Donor B L. acidophilus 2.67 2.82 3.02 2.7 0.148 6.01 0.409 0.36 1.2 6.69 0.076 25.096 Donor B F. prausnitzii 1.07 1.17 4.92 6.59 0.972 8.73 0.237 0.917 0.764 14.1 0.115 24.568 Donor B C. difficile 2.93 0.638 8.27 1.82 0.2 3.29 0.548 0.298 2.81 7.49 1.94 31.766 Donor B SEB 0.104 7.76 0.724 2.03 0.424 23 0.0317 0.052 0.0674 0.542 0.788 22.657 Donor B PHA 1.94 2 4.05 1.8 0.716 3.37 1.5 0.0536 2.16 0.869 0.223 53.3964 Donor C S. typhimurium 1.05 0.133 0.81 4.43 0.084 0.942 0.0269 0.228 0.796 4.51 0.0442 33.9669 Donor C E. coli 0.785 0.176 0.878 2.55 0.0657 1.26 0.0797 0.347 0.791 4.57 0.0959 42.6127 Donor C B. animalis 1.05 0.317 0.803 0.52 0.068 2.27 0.222 0.979 0.339 1.3 0.133 80.1597 Donor C L. acidophilus 1.41 0.233 0.415 1.33 0.0705 3 0.193 2.37 0.898 1.06 0.139 69.27716 Donor C F. prausnitzii 0.543 0.52 0.291 1.43 0.0855 3.92 0.0425 1.03 0.33 0.645 0.102 83.5563 Donor C C. difficile 0.327 0.092 0.138 0.482 0.0694 1.61 0.109 0.735 0.161 0.411 0.106 86.078 Donor C SEB 0.206 3.48 0.345 3.34 0.381 19.9 0.0309 4.82 0.0836 1.04 0.257 34.3879 Donor C PHA 3.55 1.7 2.16 3.4 1.09 4.67 1.09 3.14 3.11 1.79 0.487 35.36495
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Supplementary Table 3. T cell receptor (TCR) V β usage in expanded microbiota specific T cells using multiplex PCR assay and deep sequencing (related to Figure 3) S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vβββ TRBV9 1.617783985 1.462695991 1.669805399 1.107458038 0.883673765 0.521920668 0.397526502 0.700678783 ['1'] TRBV20-1 10.59124767 11.1543723 9.780288763 10.24398685 8.083441982 9.251043841 0.309187279 8.430041603 ['2'] TRBV28 1.0358473 1.346943071 2.209667294 3.910711196 2.013617268 0.417536534 21.37809187 0.569301511 ['3'] TRBV29-1 5.52839851 5.713985057 5.599497803 6.29866759 5.606258149 7.215553236 0.618374558 4.072695424 ['4'] TRBV5-1 8.216945996 5.819214985 4.959196485 3.4781104 2.491670288 6.3282881 0.530035336 4.510619663 ['5.1'] TRBV5-6 0.442271881 0.904977376 0.69052103 0.19034435 0.8402144 0.339248434 0.044169611 0.131377272 ['5.2'] TRBV5-5 0.453910615 0.904977376 0.69052103 0.19034435 0.8402144 0.339248434 0.044169611 0.131377272 ['5.3'] TRBV4-1 1.0358473 1.062822267 0.715630885 0.103824191 1.115457048 1.957202505 0.088339223 1.401357565 ['7.1'] TRBV4-2 3.852420857 3.335788698 3.992467043 2.993597508 6.156743445 5.414926931 0.706713781 4.050799212 ['7.1'] TRBV4-3 3.86405959 3.388403662 4.017576899 2.993597508 6.156743445 5.401878914 0.706713781 4.13838406 ['7.1', '7.2'] TRBV4-3 3.86405959 3.388403662 4.017576899 2.993597508 6.156743445 5.401878914 0.706713781 4.13838406 ['7.1', '7.2'] TRBV12-3 2.420856611 2.462380301 1.820464532 2.422564458 0.86918731 0.978601253 0.132508834 1.22618787['8'] TRBV12-4 2.420856611 2.462380301 1.820464532 2.422564458 0.86918731 0.978601253 0.132508834 1.22618787['8'] TRBV3-1 0.186219739 0.410396717 0.615191463 0.519120955 0.376647834 0.130480167 0 0['9'] TRBV25-1 0.034916201 0.021045985 0.037664783 0 0.014486455 0 0.088339223 0['11'] TRBV10-3 0 0.021045985 0 0 0 0 0.088339223 0['12'] TRBV6-1 0.477188082 0.820793434 1.858129316 1.69579512 1.839779806 0.247912317 0.618374558 0.262754543 ['13.1'] TRBV6-5 3.526536313 3.893507313 4.268675455 3.928015228 4.389395915 2.518267223 1.192579505 5.517845413 ['13.1'] TRBV6-6 3.479981378 3.809323372 4.18079096 3.8587991 4.244531363 2.466075157 1.192579505 5.386468141 ['13.1', '13.6'] TRBV6-9 0 0 0.012554928 0 0 0.013048017 0 0['13.1'] TRBV6-2 0.53538175 0.894454383 1.305712492 1.055545942 0.782268579 0.874217119 7.155477032 0.634990147 ['13.2'] TRBV6-6 3.479981378 3.809323372 4.18079096 3.8587991 4.244531363 2.466075157 1.192579505 5.386468141 ['13.1', '13.6'] TRBV27 0.151303538 0.168367884 0.640301318 0.46720886 0.970592496 0.013048017 4.06360424 0.021896212 ['14'] TRBV14 1.163873371 0.936546354 0.376647834 2.145699948 0.608431117 0.365344468 0.132508834 1.248084081 ['16'] TRBV19 0.034916201 0.031568978 0.062774639 0.034608064 0.173837462 0.013048017 0.574204947 0['17'] TRBV18 0.104748603 0.242028833 0.138104206 0.346080637 0.217296827 0.130480167 0 0.065688636['18'] TRBV30 4.67877095 4.198674103 4.105461394 6.765876449 4.679125018 6.537056367 29.99116608 6.481278739 ['20'] TRBV11-2 1.175512104 1.11543723 2.071563089 3.374286209 0.796755034 0.495824635 0.265017668 0.372235603 ['21.3'] TRBV2 0.698324022 1.410081027 0.263653484 0.46720886 0.217296827 0.287056367 0.044169611 0['22'] TRBV13 0.058193669 0.073660949 0.025109856 0 0.014486455 0.052192067 0 0.021896212['23'] TRBV1 0 0 0 0MANUSCRIPT 0 0 0 0 UNK TRBV7-1 0 0 0 0 0 0 0 0 UNK TRBV7-2 12.13919926 11.38587814 10.80979284 5.917978889 9.054034478 16.03601253 1.899293286 16.70680972 UNK TRBV8-1 0 0 0 0 0 0 0 0 UNK TRBV5-2 0 0 0 0 0 0 0 0 UNK TRBV6-4 0.034916201 0.168367884 0.025109856 0 0 0 1.01590106 0 UNK TRBV6-3 0.53538175 0.894454383 1.305712492 1.055545942 0.782268579 0.874217119 7.155477032 0.634990147 UNK TRBV7-3 4.213221601 3.683047459 4.143126177 4.031839419 4.621179197 2.583507307 1.899293286 3.656667397 UNK TRBV8-2 0 0 0 0 0 0 0 0 UNK TRBV5-3 0.244413408 0.199936862 0.050219711 0.017304032 0.014486455 0.03914405 0.309187279 0.043792424 UNK TRBV10-1 0 0 0 0 0 0 0 0 UNK TRBV11-1 1.047486034 1.125960223 1.644695543 3.270462018 0.753295669 0.456680585 0.220848057 0.394131815 UNK TRBV12-1 0 0 0 0 0 0 0 0 UNK TRBV10-2 0.081471136 0.052614964 0.037664783 0 0.173837462 0 5.344522968 0.153273484UNK TRBV12-2 0 0 0 0 0 0 0 0 UNK TRBV7-4 0.046554935 0.11575292 0.025109856 0.051912096 0.02897291 0.052192067 0.750883392 0.10948106 UNK TRBV5-4 0.675046555 0.926023361 0.502197112 1.401626579 1.434159061 1.696242171 0.176678445 0.131377272 UNK TRBV7-5 0 0 0 0 0 0 0 0 UNK TRBV6-7 0 0 0 0 0 0 0 0 UNK TRBV7-6 1.745810056 2.230874461 1.694915254 1.12476207 1.941184992 3.444676409 1.369257951 3.590978761 UNK TRBV5-8 0.675046555 0.947069347 0.527306968 1.540058834 1.463131972 1.761482255 0.176678445 0.131377272 UNK TRBV6-8 0ACCEPTED 0 0 0 0 0 0 0 UNK TRBV7-8 4.166666667 3.314742713 4.369114878 2.595604776 6.634796465 3.405532359 1.766784452 4.92664769 UNK TRBV7-7 1.641061453 2.051983584 1.556811048 1.12476207 1.839779806 3.314196242 1.369257951 3.284431793 UNK TRBV5-7 0.40735568 0.820793434 0.665411174 0.19034435 0.724322758 0.326200418 0.044169611 0.10948106 UNK TRBV7-9 3.514897579 4.082921183 3.892027621 4.499048278 3.215993047 3.001043841 0.35335689 3.70045982 UNK TRBV11-3 1.198789572 1.199621172 2.084118016 3.374286209 0.796755034 0.495824635 0.265017668 0.416028027 UNK TRBV12-5 0.162942272 0.242028833 0.11299435 0.103824191 0 0.03914405 0 0.10948106UNK TRBV15 2.118249534 1.125960223 0.351537979 0.692161274 0.622917572 1.200417537 0.35335689 1.707904533 UNK TRBV16 0.046554935 0.021045985 0 0.899809656 0 0 0 0.043792424 UNK TRBV17 0 0 0 0 0 0 0 0 UNK TRBV21-1 0 0 0 0 0 0 0 0 UNK ACCEPTED MANUSCRIPT
TRBV22-1 0 0 0 0 0 0 0 0 UNK TRBV23-1 0 0 0 0 0 0 0 0 UNK TRBV24-1 0.174581006 0.147321898 0.075329567 0.242256446 0.246269738 0.11743215 3.136042403 0.021896212 UNK TRBV26 0 0 0 0 0 0 0 0 UNK
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Table S3. T cell receptor (TCR) V β usage in expanded microbiota specific T cells using multiplex PCR assays and deep sequencing
Donor 1 S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vβββ TRBV9 0.730868444 0.879443019 0.414421881 0 1.560348784 0.058309038 0.484848485 0.736196319 ['1'] TRBV20-1 7.09372313 9.967020887 14.46332366 12.77573529 10.9683341 9.329446064 0.242424242 7.668711656 ['2'] TRBV28 0.816852966 0.586295346 3.315375052 11.67279412 2.248737953 0.349854227 26.42424242 0.306748466 ['3'] TRBV29-1 9.200343938 7.108831074 9.241607957 8.180147059 11.70261588 11.31195335 0.242424242 4.846625767 ['4'] TRBV5-1 7.308684437 4.067423965 3.895565686 3.125 1.147315282 6.472303207 0.242424242 3.312883436 ['5.1'] TRBV5-6 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0.061349693['5.2'] TRBV5-5 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0.061349693['5.3'] TRBV4-1 2.708512468 1.026016856 0.994612516 0 0.045892611 1.224489796 0.121212121 0.736196319 ['7.1'] TRBV4-2 2.235597592 4.873580066 4.517198508 0.275735294 7.847636531 5.131195335 0.848484848 5.153374233 ['7.1'] TRBV4-3 2.235597592 4.910223525 4.558640696 0.275735294 7.847636531 5.131195335 0.848484848 5.214723926 ['7.1', '7.2'] TRBV4-3 2.235597592 4.910223525 4.558640696 0.275735294 7.847636531 5.131195335 0.848484848 5.214723926 ['7.1', '7.2'] TRBV12-3 5.54600172 2.161964089 0.414421881 0.091911765 1.147315282 0.699708455 0.121212121 0.552147239 ['8'] TRBV12-4 5.54600172 2.161964089 0.414421881 0.091911765 1.147315282 0.699708455 0.121212121 0.552147239 ['8'] TRBV3-1 0.042992261 0 0 0.091911765 0 0 0 0['9'] TRBV25-1 0 0 0 0 0.045892611 0 0.242424242 0['11'] TRBV10-3 0 0 0 0 0 0 0 0['12'] TRBV6-1 0.171969046 0.036643459 0.165768753 0.091911765 0.321248279 0.233236152 0.363636364 0['13.1'] TRBV6-5 3.740326741 5.313301576 1.367592209 6.433823529 0.413033502 1.982507289 0.484848485 3.926380368 ['13.1'] TRBV6-6 3.740326741 5.240014657 1.367592209 6.617647059 0.36714089 1.982507289 0.484848485 3.803680982 ['13.1', '13.6'] TRBV6-9 0 0 0 0 0 0 0 0 ['13.1'] TRBV6-2 0.085984523 0.916086479 0.704517199 1.654411765 0.183570445 1.10787172 8 0.920245399['13.2'] TRBV6-6 3.740326741 5.240014657 1.367592209 6.617647059 0.36714089 1.982507289 0.484848485 3.803680982 ['13.1', '13.6'] TRBV27 0.386930353 0.293147673 1.036054704 0.183823529 2.8453419 0 4 0['14'] TRBV14 2.622527945 0.256504214 0.331537505 0.459558824 0.413033502 0 0 0.797546012['16'] TRBV19 0 0 0 0 0.045892611 0 0.121212121 0['17'] TRBV18 0.085984523 0.109930377 0 0.091911765 0.183570445 0 0 0 ['18'] TRBV30 2.708512468 5.386588494 6.092001658 5.974264706 6.05782469 5.189504373 27.15151515 7.791411043 ['20'] TRBV11-2 0.859845228 0.513008428 1.243265644 0.183823529 1.193207894 0 0.121212121 0.306748466['21.3'] TRBV2 0.085984523 0.43972151 0.041442188 0 0.091785223 0 0 0 ['22'] TRBV13 0 0 0 0 0 0 0 0 ['23'] TRBV1 0 0 0 0 0 0 0 0 UNK TRBV7-1 0 0 0 0MANUSCRIPT 0 0 0 0 UNK TRBV7-2 14.144454 17.0392085 11.1893908 3.952205882 12.80403855 15.62682216 1.454545455 19.57055215 UNK TRBV8-1 0 0 0 0 0 0 0 0 UNK TRBV5-2 0 0 0 0 0 0 0 0 UNK TRBV6-4 0.085984523 0 0.041442188 0 0 0 2.181818182 0UNK TRBV6-3 0.085984523 0.916086479 0.704517199 1.654411765 0.183570445 1.10787172 8 0.920245399UNK TRBV7-3 5.331040413 3.187980945 6.506423539 0 4.726938963 0.758017493 0.96969697 3.190184049 UNK TRBV8-2 0 0 0 0 0 0 0 0 UNK TRBV5-3 0.085984523 0.43972151 0 0 0 0.116618076 0.242424242 0 UNK TRBV10-1 0 0 0 0 0 0 0 0 UNK TRBV11-1 0.816852966 0.513008428 1.160381268 0.183823529 1.147315282 0 0 0.36809816UNK TRBV12-1 0 0 0 0 0 0 0 0 UNK TRBV10-2 0.042992261 0.036643459 0.041442188 0 0.550711335 0 7.636363636 0.306748466UNK TRBV12-2 0 0 0 0 0 0 0 0 UNK TRBV7-4 0.042992261 0 0 0 0 0 1.212121212 0.18404908UNK TRBV5-4 0.128976784 0.329791132 0.621632822 0 0.183570445 5.422740525 0.121212121 0UNK TRBV7-5 0 0 0 0 0 0 0 0 UNK TRBV6-7 0 0 0 0 0 0 0 0 UNK TRBV7-6 1.977644024 0.989373397 3.688354745 4.136029412 1.78981184 3.731778426 0.848484848 4.662576687 UNK TRBV5-8 0.128976784 0.329791132 0.621632822 0 0.183570445 5.422740525 0.121212121 0UNK TRBV6-8 0 0 0 0 0 0 0 0 UNK TRBV7-8 4.170249355 3.151337486 9.117281392 5.055147059 5.32354291 3.206997085 1.454545455 5.214723926 UNK TRBV7-7 1.891659501ACCEPTED 0.952729938 3.398259428 4.136029412 1.743919229 3.381924198 0.848484848 4.478527607 UNK TRBV5-7 0.343938091 0.146573837 0.041442188 0.091911765 0.091785223 0 0 0UNK TRBV7-9 3.611349957 3.261267864 0.994612516 10.47794118 3.717301514 2.857142857 0.96969697 2.944785276 UNK TRBV11-3 0.859845228 0.513008428 1.243265644 0.183823529 1.193207894 0 0.121212121 0.36809816UNK TRBV12-5 0.042992261 0 0 0 0 0.174927114 0 0.122699387UNK TRBV15 1.504729149 1.392451447 0.041442188 0 0 0.174927114 0.121212121 1.840490798 UNK TRBV16 0 0 04.779411765 0 0 0 0UNK TRBV17 0 0 0 0 0 0 0 0 UNK TRBV21-1 0 0 0 0 0 0 0 0 UNK TRBV22-1 0 0 0 0 0 0 0 0 UNK TRBV23-1 0 0 0 0 0 0 0 0 UNK TRBV24-1 0.085984523 0.109930377 0 0 0.137677834 0 1.696969697 0.061349693 UNK ACCEPTED MANUSCRIPT
TRBV26 0 0 0 0 0 0 0 0 UNK
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Table S3. T cell receptor (TCR) V β usage in expanded microbiota specific T cells using multiplex PCR assays and deep sequencing
Donor 2 S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vβββ TRBV9 2.232142857 2.699144174 1.598495534 3.249630724 0.166574125 0.30159414 0.295420975 1.194659171 ['1'] TRBV20-1 10.75487013 11.32323897 9.685002351 13.22008863 8.995002776 7.755277897 0.738552437 7.589599438 ['2'] TRBV28 2.353896104 3.324555629 4.37235543 7.311669129 4.664075514 0.258509263 26.44017725 1.475755446 ['3'] TRBV29-1 3.652597403 7.142857143 4.607428303 2.511078287 1.499167129 0.646273158 1.624815362 3.021784961 ['4'] TRBV5-1 11.28246753 5.99078341 5.500705219 5.022156573 7.273736813 4.308487721 0.59084195 5.762473647 ['5.1'] TRBV5-6 0.689935065 1.382488479 2.256699577 0.295420975 2.165463631 0.646273158 0.147710487 0.281096275 ['5.2'] TRBV5-5 0.730519481 1.382488479 2.256699577 0.295420975 2.165463631 0.646273158 0.147710487 0.281096275 ['5.3'] TRBV4-1 0.649350649 0.954575379 1.081335214 0.295420975 2.498611882 1.981904352 0 1.405481377['7.1'] TRBV4-2 1.988636364 1.283739302 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 1.967673928 ['7.1'] TRBV4-3 2.029220779 1.316655695 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 2.037947997 ['7.1', '7.2'] TRBV4-3 2.029220779 1.316655695 0.188058298 0.812407681 3.498056635 8.358466178 0.59084195 2.037947997 ['7.1', '7.2'] TRBV12-3 0.730519481 1.81040158 2.3977433 4.135893648 1.887840089 2.585092632 0.147710487 3.09205903 ['8'] TRBV12-4 0.730519481 1.81040158 2.3977433 4.135893648 1.887840089 2.585092632 0.147710487 3.09205903 ['8'] TRBV3-1 0.487012987 1.152073733 2.209685002 1.772525849 1.388117712 0.129254632 0 0['9'] TRBV25-1 0.081168831 0.032916392 0 0 0 0 0 0['11'] TRBV10-3 0 0 0 0 0 00.147710487 0['12'] TRBV6-1 0.852272727 1.184990125 1.269393512 1.772525849 0.72182121 0.473933649 0.886262925 0.421644413 ['13.1'] TRBV6-5 4.748376623 2.995391705 5.547719793 5.391432792 2.554136591 3.274450668 1.1816839 8.854532677 ['13.1'] TRBV6-6 4.586038961 2.92955892 5.500705219 5.317577548 2.554136591 3.274450668 1.1816839 8.643710471 ['13.1', '13.6'] TRBV6-9 0 0 0 0 0 0 0 0 ['13.1'] TRBV6-2 0.527597403 0.658327847 0.329102022 0.664697194 1.054969461 0.043084877 2.954209749 0.281096275 ['13.2'] TRBV6-6 4.586038961 2.92955892 5.500705219 5.317577548 2.554136591 3.274450668 1.1816839 8.643710471 ['13.1', '13.6'] TRBV27 0.081168831 0.032916392 1.034320639 0.960118168 0.166574125 0 3.397341211 0.070274069['14'] TRBV14 0.487012987 0.855826201 0.94029149 2.215657312 1.83231538 0.215424386 0.443131462 1.405481377 ['16'] TRBV19 0 0 0 0.073855244 0 0.043084877 0.295420975 0['17'] TRBV18 0.040584416 0.230414747 0.235072873 0.369276219 0.499722376 0.344679018 0 0.210822207['18'] TRBV30 4.788961039 4.377880184 2.3977433 5.539143279 2.94280955 8.746230073 30.42836041 3.72452565 ['20'] TRBV11-2 1.866883117 1.217906517 3.667136812 1.550960118 1.166018878 0.430848772 0.147710487 0.562192551 ['21.3'] TRBV2 1.948051948 2.567478604 0.799247767 1.624815362 0.610771793 0.732442913 0 0['22'] TRBV13 0.202922078 0.098749177 0 0 0 0 0 0.070274069['23'] TRBV1 0 0 0 0 0 0 0 0 UNK TRBV7-1 0 0 0 0MANUSCRIPT 0 0 0 0 UNK TRBV7-2 9.699675325 6.978275181 7.663375646 3.249630724 10.77179345 9.392503231 1.920236337 10.11946592 UNK TRBV8-1 0 0 0 0 0 0 0 0 UNK TRBV5-2 0 0 0 0 0 0 0 0 UNK TRBV6-4 0 0 0.047014575 0 0 0 0.443131462 0UNK TRBV6-3 0.527597403 0.658327847 0.329102022 0.664697194 1.054969461 0.043084877 2.954209749 0.281096275 UNK TRBV7-3 3.125 3.982883476 1.645510108 5.612998523 2.387562465 3.619129685 2.658788774 3.935347857 UNK TRBV8-2 0 0 0 0 0 0 0 0 UNK TRBV5-3 0.162337662 0.131665569 0.094029149 0.073855244 0.055524708 0 0.59084195 0UNK TRBV10-1 0 0 0 0 0 0 0 0 UNK TRBV11-1 1.542207792 1.11915734 2.162670428 1.1816839 1.054969461 0.344679018 0.147710487 0.562192551 UNK TRBV12-1 0 0 0 0 0 0 0 0 UNK TRBV10-2 0.162337662 0.065832785 0 0 0 0 5.169867061 0.070274069 UNK TRBV12-2 0 0 0 0 0 0 0 0 UNK TRBV7-4 0.040584416 0.164581962 0.047014575 0.073855244 0 0.129254632 0.59084195 0.070274069 UNK TRBV5-4 1.176948052 1.481237656 0.470145745 1.698670606 1.665741255 1.206376562 0 0.421644413UNK TRBV7-5 0 0 0 0 0 0 0 0 UNK TRBV6-7 0 0 0 0 0 0 0 0 UNK TRBV7-6 1.866883117 3.324555629 1.833568406 0.664697194 1.665741255 4.825506247 2.215657312 2.951510892 UNK TRBV5-8 1.176948052 1.547070441 0.564174894 2.363367799 1.776790672 1.292546316 0 0.421644413UNK TRBV6-8 0 0 0 0 0 0 0 0 UNK TRBV7-8 5.438311688 4.805793285 3.94922426 3.249630724 7.828983898 3.920723826 2.067946824 4.427266339 UNK TRBV7-7 1.785714286ACCEPTED 3.159973667 1.692524683 0.664697194 1.554691838 4.739336493 2.215657312 2.670414617 UNK TRBV5-7 0.568181818 1.11915734 2.209685002 0.295420975 1.998889506 0.603188281 0.147710487 0.281096275 UNK TRBV7-9 3.571428571 5.892034233 5.970850964 1.846381093 4.664075514 1.206376562 0 4.567814476UNK TRBV11-3 1.948051948 1.316655695 3.667136812 1.550960118 1.166018878 0.430848772 0.147710487 0.562192551 UNK TRBV12-5 0.324675325 0.032916392 0.047014575 0.443131462 0 0 0 0.210822207UNK TRBV15 1.339285714 1.11915734 1.175364363 0.443131462 0.610771793 0.086169754 1.033973412 2.178496135 UNK TRBV16 0.081168831 0 0 0 0 0 0 0.140548138UNK TRBV17 0 0 0 0 0 0 0 0 UNK TRBV21-1 0 0 0 0 0 0 0 0 UNK TRBV22-1 0 0 0 0 0 0 0 0 UNK TRBV23-1 0 0 0 0 0 0 0 0 UNK TRBV24-1 0.324675325 0.098749177 0.282087447 0.443131462 0 0.387763895 3.397341211 0UNK ACCEPTED MANUSCRIPT
TRBV26 0 0 0 0 0 0 0 0 UNK
MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT
Table S3. T cell receptor (TCR) V β usage in expanded microbiota-specific T cells using multiplex PCR assay and deep sequencing
Donor 3 S. typhimurium E. coli B. animalis L. acidophilus F. prausnitzii C. difficile SEB PHA Vβββ TRBV9 1.762230405 0.883297645 2.598540146 0.599340725 0.821074239 0.882028666 0.393700787 0.198150594 ['1'] TRBV20-1 12.62493425 11.88436831 6.540145985 8.210967935 5.371193979 10.17089305 0 10.03963012['2'] TRBV28 0.315623356 0.294432548 0.087591241 0 0.20526856 0.551267916 11.41732283 0['3'] TRBV29-1 4.497632825 3.533190578 3.649635037 7.222055739 3.592199795 9.481808159 0.131233596 4.227212682 ['4'] TRBV5-1 6.785902157 6.959314775 5.372262774 2.96673659 0.547382826 7.552370452 0.787401575 4.623513871 ['5.1'] TRBV5-6 0.341925302 1.070663812 0.175182482 0.179802218 0.581594252 0.303197354 0 0.066050198['5.2'] TRBV5-5 0.341925302 1.070663812 0.175182482 0.179802218 0.581594252 0.303197354 0 0.066050198['5.3'] TRBV4-1 0.263019463 1.177730193 0.291970803 0.059934073 1.060554225 2.287761852 0.131233596 2.113606341 ['7.1'] TRBV4-2 6.049447659 3.881156317 5.98540146 4.764758765 6.534382484 3.665931643 0.656167979 4.821664465 ['7.1'] TRBV4-3 6.049447659 3.961456103 6.01459854 4.764758765 6.534382484 3.638368247 0.656167979 4.953764861 ['7.1', '7.2'] TRBV4-3 6.049447659 3.961456103 6.01459854 4.764758765 6.534382484 3.638368247 0.656167979 4.953764861 ['7.1', '7.2'] TRBV12-3 1.604418727 3.211991435 2.452554745 2.48726401 0.034211427 0.082690187 0.131233596 0.198150594 ['8'] TRBV12-4 1.604418727 3.211991435 2.452554745 2.48726401 0.034211427 0.082690187 0.131233596 0.198150594 ['8'] TRBV3-1 0.078905839 0.107066381 0.058394161 0.149835181 0.034211427 0.192943771 0 0['9'] TRBV25-1 0.026301946 0.026766595 0.087591241 0 0 0 0 0['11'] TRBV10-3 0 0.053533191 0 0 0 0 0.131233596 0['12'] TRBV6-1 0.420831142 1.097430407 3.416058394 2.187593647 3.660622648MANUSCRIPT 0.110253583 0.656167979 0.396301189 ['13.1'] TRBV6-5 2.603892688 3.586723769 5.518248175 2.517231046 8.484 433801 2.287761852 1.968503937 4.095112285 ['13.1'] TRBV6-6 2.603892688 3.479657388 5.343065693 2.367395865 8.176530961 2.177508269 1.968503937 4.029062087 ['13.1', '13.6'] TRBV6-9 0 0 0.02919708 0 0 0.027563396 0 0['13.1'] TRBV6-2 0.815360337 1.070663812 2.335766423 1.018879233 1.060554225 1.295479603 9.973753281 0.660501982 ['13.2'] TRBV6-6 2.603892688 3.479657388 5.343065693 2.367395865 8.176530961 2.177508269 1.968503937 4.029062087 ['13.1', '13.6'] TRBV27 0.052603893 0.187366167 0.116788321 0.359604435 0.068422853 0.027563396 4.724409449 0['14'] TRBV14 0.710152551 1.498929336 0.058394161 2.667066227 0 0.633958104 0 1.585204756['16'] TRBV19 0.078905839 0.080299786 0.145985401 0.029967036 0.376325693 0 1.312335958 0['17'] TRBV18 0.157811678 0.347965739 0.175182482 0.419538508 0.068422853 0.055126792 0 0['18'] TRBV30 5.812730142 3.185224839 3.766423358 7.521726101 4.721176873 5.760749724 32.67716535 7.661822985 ['20'] TRBV11-2 0.920568122 1.472162741 1.664233577 5.154330237 0.273691413 0.771775083 0.524934383 0.264200793 ['21.3'] TRBV2 0.263019463 1.177730193 0.087591241 0.149835181 0.068422853 0.137816979 0.131233596 0['22'] TRBV13 0 0.107066381 0.058394161 0 0.034211427 0.110253583 0 0['23'] TRBV1 0 0ACCEPTED 0 0 0 0 0 0UNK TRBV7-1 0 0 0 0 0 0 0 0UNK TRBV7-2 12.49342451 10.84047109 12.49635037 7.641594246 5.200136846 20.47960309 2.362204724 19.81505945 UNK TRBV8-1 0 0 0 0 0 0 0 0UNK TRBV5-2 0 0 0 0 0 0 0 0UNK TRBV6-4 0.026301946 0.428265525 0 0 0 0 0.262467192 0UNK ACCEPTED MANUSCRIPT
TRBV6-3 0.815360337 1.070663812 2.335766423 1.018879233 1.060554225 1.295479603 9.973753281 0.660501982 UNK TRBV7-3 4.234613361 3.800856531 4.02919708 4.704824693 5.918576805 2.783902977 2.230971129 3.896961691 UNK TRBV8-2 0 0 0 0 0 0 0 0UNK TRBV5-3 0.394529195 0.080299786 0.058394161 0 0 0.027563396 0.131233596 0.132100396 UNK TRBV10-1 0 0 0 0 0 0 0 0UNK TRBV11-1 0.867964229 1.579229122 1.664233577 5.1243632 0.273691413 0.744211687 0.524934383 0.264200793 UNK TRBV12-1 0 0 0 0 0 0 0 0UNK TRBV10-2 0.052603893 0.053533191 0.058394161 0 0 0 3.018372703 0.066050198 UNK TRBV12-2 0 0 0 0 0 0 0 0UNK TRBV7-4 0.052603893 0.160599572 0.02919708 0.059934073 0.068422853 0.027563396 0.393700787 0.066050198 UNK TRBV5-4 0.683850605 0.91006424 0.437956204 1.738088103 2.22374273 0.248070562 0.393700787 0UNK TRBV7-5 0 0 0 0 0 0 0 0UNK TRBV6-7 0 0 0 0 0 0 0 0UNK TRBV7-6 1.525512888 2.248394004 0.204379562 0.329637399 2.22374273 2.425578831 1.181102362 3.038309115 UNK TRBV5-8 0.683850605 0.91006424 0.437956204 1.708121067 2.22374273 0.33076075 0.393700787 0UNK TRBV6-8 0 0 0 0 0 0 0 0UNK TRBV7-8 3.340347186 2.221627409 1.284671533 1.528318849 6.87649675 3.169790518 1.837270341 5.085865258 UNK TRBV7-7 1.394003156 1.953961456 0.175182482 0.329637399 2.086897024 2.37045204 1.181102362 2.575957728 UNK TRBV5-7 0.341925302 1.070663812 0.145985401 0.179802218 0.410537119 0.303197354 0 0.066050198UNK TRBV7-9 3.419253025 3.211991435 4.642335766 3.626011387 1.950051317 4.217199559 0 3.698811096UNK TRBV11-3 0.920568122 1.605995717 1.693430657 5.154330237 0.273691413 0.771775083 0.524934383 0.330250991 UNK TRBV12-5 0.131509732 0.588865096 0.233576642 0MANUSCRIPT 0 0 0 0UNK TRBV15 2.998421883 0.936830835 0.058394161 1.018879233 1.0947 65652 2.398015436 0 1.122853369UNK TRBV16 0.052603893 0.053533191 0 0 0 0 0 0UNK TRBV17 0 0 0 0 0 0 0 0UNK TRBV21-1 0 0 0 0 0 0 0 0UNK TRBV22-1 0 0 0 0 0 0 0 0UNK TRBV23-1 0 0 0 0 0 0 0 0UNK TRBV24-1 0.131509732 0.214132762 0 0.23973629 0.478959973 0 4.461942257 0UNK TRBV26 0 0 0 0 0 0 0 0UNK
ACCEPTED ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAGACCAAGGGCCGCGGATGGGATGCCGCAGTATTTT 22554 CAWRPRAADGMPQYF TGTGCCTGGGAGCCAGTGGAGGCACTCACAGATACGCAGTATTTT 7027 CAWEPVEALTDTQYF TGTGCCAGCAGCTTCGGGGACAGTATCCAAGAGACCCAGTACTTC 5842 CASSFGDSIQETQYF TGTGCCTGGGACACTAGCGGGCAGGGCAATGAGCAGTTCTTC 5596 CAWDTSGQGNEQFF TGTGCCTGGGGGGACGGAAGCAATCAGCCCCAGCATTTT 5367 CAWGDGSNQPQHF TGTGCCAGCAGCTTCCTTCGGGGGAACACTGAAGCTTTCTTT 5210 CASSFLRGNTEAFF TGCGCCAGCAGCCAAGAGACGGGCCACGAGAGGAACATTCAGTACTTC 4524 CASSQETGHERNIQYF TGTGCCAGCAGCTTTGAGGCAGGGCGGATCACTGAAGCTTTCTTT 4454 CASSFEAGRITEAFF TGCAGTGCTAGCGCTGGCCTGGGGTACAATGAGCAGTTCTTC 4435 CSASAGLGYNEQFF TGTGCCAGCAGCTTGGATAGCGGCTACAATGAGCAGTTCTTC 4025 CASSLDSGYNEQFF TGCGCCAGCAGCCAAGAGGTCGGGCCGTATGGCTACACCTTC 4016 CASSQEVGPYGYTF TGTGCCACCTCTTCACCGGGCTGGGCAGATACGCAGTATTTT 3751 CATSSPGWADTQYF TGTGCCTGGGACCCAGCGATCTCAGATACGCAGTATTTT 3581 CAWDPAISDTQYF TGTGCCGGCCAGACTAGCGGAGGTGTGGGTGAGCAGTTCTTC 3399 CAGQTSGGVGEQFF TGTGCCAGCAGCCAAGCCACTATCCCTCCCAATGAGCAGTTCTTC 3276 CASSQATIPPNEQFF TGTGCCACCAGCAGCTGGACAGGGTCTTCGGATGAGCAGTTCTTC 3081 CATSSWTGSSDEQFF TGTGCCAGCAGCCCCTTTACGGACGAGCAGTACTTC 3001 CASSPFTDEQYF TGCAGCGTTACGGGACTAGCGGGAGGCTCCTACAATGAGCAGTTCTTC 2750 CSVTGLAGGSYNEQFF TGTGCCAGCAGTCCGGGACTAGCGGTACAAGAGACCCAGTACTTC 2596 CASSPGLAVQETQYF TGCGCCAGCAGCCAAGATACAGGGGACCGCGGGGGCTACACCTTC 2425 CASSQDTGDRGGYTF TGTGCCAGCAGCTACTCTAGCGGGAGCGGTGGAGAGACCCAGTACTTC 2416 CASSYSSGSGGETQYF TGTGCCAGCAGCCATACGACTAGTAGCGGGTTCTACGAGCAGTACTTC 2271 CASSHTTSSGFYEQYF TGTGCCAGCTCCGGGGAGGCGCAGTATTTT 2263 CASSGEAQYF TGTGCCAGCAGCTTAGCATCCCCCACTGAAGCTTTCTTT 2239 CASSLASPTEAFF TGTGCCAGCAGCCCGTTACGGGGTGGAGATACGCAGTATTTT MANUSCRIPT2162 CASSPLRGGDTQYF TGTGCCAGCAGCTTAAAGGCAGCGACCGACCTGGCTGAAGCTTTCTTT 2014 CASSLKAATDLAEAFF TGTGCCTGGAGTGTGGGGGACTATGGCTACACCTTC 1868 CAWSVGDYGYTF TGTGCCAGCAGTCCTTGGGGGGGAGGGGGAGATACGCAGTATTTT 1822 CASSPWGGGGDTQYF TGTGCCAGCAGCTTGGCTAGCCAAGAGACCCAGTACTTC 1757 CASSLASQETQYF TGTGCCAGCAGCTTAAGCCTCATCCTCGCGGGCAATGAGCAGTTCTTC 1724 CASSLSLILAGNEQFF TGTGCCAGCAGCTTATATGTCCGAGAGACCCAGTACTTC 1716 CASSLYVRETQYF TGTGCCAGCAGCGCAGACTATGGCAATCAGCCCCAGCATTTT 1679 CASSADYGNQPQHF TGTGCCTGGAGTATCGGGACAGCTTACGAGCAGTACTTC 1636 CAWSIGTAYEQYF TGTGCCAGCAGCTTAGGCGGGGCGAGGACTGAAGCTTTCTTT 1573 CASSLGGARTEAFF TGTGCCTGGAGTGCGGGTCTACTGAGCTACGAGCAGTACTTC 1539 CAWSAGLLSYEQYF TGCAGCGTTGGAACAGCGCCCACTGAAGCTTTCTTT 1526 CSVGTAPTEAFF TGTGCCAGCAGCGGGACTAGCGGGAGTGAGCAGTTCTTC 1524 CASSGTSGSEQFF TGTGCCAGCTCGGGAGATACGCAGTATTTT 1433 CASSGDTQYF TGTGCCAGCAGCATGGGGTGGAGGACTGAAGCTTTCTTT 1430 CASSMGWRTEAFF TGTGCCAGCAGCCCGACCGGGACGTCGAGCTCCTACGAGCAGTACTTC 1410 CASSPTGTSSSYEQYF TGTGCCAGCGCTACAGGGGGGGATCAGCCCCAGCATTTTACCEPTED 1402 CASATGGDQPQHF TGTGCCAGCAGCTTATTGACAGGGGGGGCTTTCTTT 1402 CASSLLTGGAFF TGTGCCAGCAGCTTAGATTGGCAGACCGCGGATGAGCAGTTCTTC 1396 CASSLDWQTADEQFF TGTGCTAGCAGCCCATCCGGAGGTGCCGGCACAGATACGCAGTATTTT 1360 CASSPSGGAGTDTQYF TGCAGTGCTAGATCCGGGACAGGGGTCGGGAGCAATCAGCCCCAGCATTTT 1337 CSARSGTGVGSNQPQHF TGTGCCAGCAGCCCTTCGCGGTACAATGAGCAGTTCTTC 1312 CASSPSRYNEQFF TGCGCCAGCAGCCTTTATTTAGCGGGAGCGTCGACCGGGGAGCTGTTTTTT 1296 CASSLYLAGASTGELFF TGTGCCAGCAGCTTAGGGCTTAATCCCTCCTACAATGAGCAGTTCTTC 1204 CASSLGLNPSYNEQFF TGTGCCTGGAGCCTACAGGGATCCTATGGCTACACCTTC 1199 CAWSLQGSYGYTF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCTTCGGGGACAGTATCCAAGAGACCCAGTACTTC 16040 CASSFGDSIQETQYF TGTGCCAGCAGCCCCCAGACTGAAGCTTTCTTT 7638 CASSPQTEAFF TGTGCCAGCGGGGTCAATGAGCAGTTCTTC 6655 CASGVNEQFF TGTGCCTGGAGTGGGACAGGGGTTCGACAGCCCCAGCATTTT 6051 CAWSGTGVRQPQHF TGTGCCAGCAGCCCGGGGGGGCTGAACACTGAAGCTTTCTTT 5779 CASSPGGLNTEAFF TGCGCCAGCAGCCTCCAGGGCACTGAAGCTTTCTTT 5616 CASSLQGTEAFF TGTGCCAGCAGCTTTCTACAGGGGGCTCGCACTGAAGCTTTCTTT 4841 CASSFLQGARTEAFF TGTGCCAGCAGCCCGGGACTAGCATCGTACATTCAGTACTTC 4396 CASSPGLASYIQYF TGTGCCTGGAACACGGGCCGCCATGAGCAGTTCTTC 4380 CAWNTGRHEQFF TGTGCCACCAGCAGAGAAGGCGGCAGGGTCATGAACACTGAAGCTTTCTTT 4044 CATSREGGRVMNTEAFF TGTGCCAGTCGACAGGGGGCGGATGGCTACACCTTC 3608 CASRQGADGYTF TGTGCCTGGAGGGCGGAATACACTGAAGCTTTCTTT 3490 CAWRAEYTEAFF TGTGCCAGCAGCCCGGGGGGACTAGCGGGCGTCAATGAGCAGTTCTTC 3353 CASSPGGLAGVNEQFF TGTGCCAGCAGCTGGGGGACAGGGGACACAGATACGCAGTATTTT 3226 CASSWGTGDTDTQYF TGTGCCAGCAGCCCAGCAAGCGGGTCCTACGAGCAGTACTTC 3169 CASSPASGSYEQYF TGTGCCAGCAGCCAACACCGGACAGCTGGGGCCAACGTCCTGACTTTC 2850 CASSQHRTAGANVLTF TGCGCCAGCAGGAGAGGGGGGGTGGATTCACCCCTCCACTTT 2580 CASRRGGVDSPLHF TGTGCCTGGACTAGCGCCGCCCGAGAGACCCAGTACTTC 2563 CAWTSAARETQYF TGTGCCTGGAGTCGGGGGGGCCGAGCTTTCTTT 2498 CAWSRGGRAFF TGTGCCAGCAGCTCGACACCGACAGGGGGAGGCTACACCTTC 2476 CASSSTPTGGGYTF TGTGCCACCAGCAGGTACGGAGAACACTCTGAAGCTTTCTTT 2453 CATSRYGEHSEAFF TGTGCCAGCATGGCGTCGGCAGGCTACGAGCAGTACTTC 2369 CASMASAGYEQYF TGTGCCTGGACCCGGACTATCCAAGAGACCCAGTACTTC 2333 CAWTRTIQETQYF TGCAGTGCTAGATCCGGGACAGGGGTCGGGAGCAATCAGCCCCAGCATTTTMANUSCRIPT2271 CSARSGTGVGSNQPQHF TGTGCCTGGAACGAACAGGATTTAGCCGGGGAGCTGTTTTTT 2232 CAWNEQDLAGELFF TGTGCCAGCAGCCCGGGGGGCGGGAGCTCCTACAATGAGCAGTTCTTC 2209 CASSPGGGSSYNEQFF TGCGCCAGCAGCCAAGACTGGCTAGCGGGAGTCGAGCAGTACTTC 2173 CASSQDWLAGVEQYF TGTGCCAGCAGCTTAGGGAGGGGCACTGAAGCTTTCTTT 2172 CASSLGRGTEAFF TGTGCCAGCAGCCGTTGGGACGGACAAGAGACCCAGTACTTC 2161 CASSRWDGQETQYF TGTGCCAGCAGCGAACAGGGAGGGCAGCCCCAGCATTTT 2107 CASSEQGGQPQHF TGTGCCAGCAGCTTTTACATCGAAGAGACCCAGTACTTC 2053 CASSFYIEETQYF TGTGCCAGCAGTCCGGGACTAGCGGTACAAGAGACCCAGTACTTC 2032 CASSPGLAVQETQYF TGCGCCAGCAGCCGGGACCTAGCAACAGATACGCAGTATTTT 2025 CASSRDLATDTQYF TGTGCCAGCAGCTTGGATAGCGGCTACAATGAGCAGTTCTTC 1975 CASSLDSGYNEQFF TGTGCCAGCAGCTTCGTGGGGGGCACTGAAGCTTTCTTT 1941 CASSFVGGTEAFF TGTGCCAGCAGCCGCATGAGGGGGGAGGAGACCCAGTACTTC 1930 CASSRMRGEETQYF TGTGCCTGGACTGGCGACAGGTCGTACTACGAGCAGTACTTC 1892 CAWTGDRSYYEQYF TGTGCCTGGAGTGTCGGGTCGGATGGCTACACCTTC 1879 CAWSVGSDGYTF TGTGCCAGCAGCCTTCCCGTGAACACTGAAGCTTTCTTT 1871 CASSLPVNTEAFF TGTGCCAGCAGCGCAGAATGGAATCAGCCCCAGCATTTTACCEPTED 1801 CASSAEWNQPQHF TGTGCCAGCAGCCCGACGGGGGTAGGTGAAGCTTTCTTT 1765 CASSPTGVGEAFF TGTGCCTGGAACCGACTGGGAATGGATACGCAGTATTTT 1742 CAWNRLGMDTQYF TGTGCCAGCAGCTTAACGCACGCTAGCGGGGAGCAGTACTTC 1739 CASSLTHASGEQYF TGTGCCTGGAGAGATAACCTTGCGGAAAAACTGTTTTTT 1733 CAWRDNLAEKLFF TGTGCCAGCAGCTTGGTCTCTAGCGGGAGCGATACGCAGTATTTT 1675 CASSLVSSGSDTQYF TGTGCCTGGAGTGTACTCGGGCCGAGTCCCCAGTACGTC 1605 CAWSVLGPSPQYV TGCAGTGTTAATAGTCCGGCTTTAACGGGACAGCCCCAGCATTTT 1563 CSVNSPALTGQPQHF TGCGCCAGCAGCTCCCGGGACGGGACGAATTCACCCCTCCACTTT 1556 CASSSRDGTNSPLHF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCGCTTCCGGGACAGGGTCCTACGAGCAGTACTTC 33028 CASSASGTGSYEQYF TGCGCCAGCAGCCAAGCCCCCTCTGGGGTTAGGCCCGGGGAGCTGTTTTTT 32838 CASSQAPSGVRPGELFF TGTGCCTGGACGGGTGAAGGGGCCGCGGGGTTGCGCAATCAGCCCCAGCATTTT 22766 CAWTGEGAAGLRNQPQHF TGTGCCAGCAGCCAAGTCGGAAGTCTAATTGAAAAACTGTTTTTT 21458 CASSQVGSLIEKLFF TGTGCCTGGAACCTCTACCCGGGACTAGCGGGGACAGATACGCAGTATTTT 19128 CAWNLYPGLAGTDTQYF TGCAGCGTTCCAGGGGTCTGGTCCTACAATGAGCAGTTCTTC 16761 CSVPGVWSYNEQFF TGTGCCAGCAGCTTGGGGGGGACCAGACTGAAAAATGAAAAACTGTTTTTT 15048 CASSLGGTRLKNEKLFF TGTGCCAGCAGCTTAGTCGCGGGAGAGGGGGAGTTCTTC 11894 CASSLVAGEGEFF TGTGCCAGCAGCGTAGACGTCGGGAGGTCCTACGAGCAGTACTTC 9692 CASSVDVGRSYEQYF TGCGCCAGCAGCTTGGGCTTGGATGTTGAGACCCAGTACTTC 8359 CASSLGLDVETQYF TGTGCCAGCAGCTTGTGGGGGCAACAGACCCAGTACTTC 6894 CASSLWGQQTQYF TGTGCCACCTCGTTTCCAGCGCGCCACAATGAGCAGTTCTTC 6173 CATSFPARHNEQFF TGTGCCAGCAGCTTTGATTCGGGACAAGGACGGGAAAAGTACTTC 5746 CASSFDSGQGREKYF TGTGCCAGCAGCCCAAACTCACTAGCGGGAGTCAATGAGCAGTTCTTC 5624 CASSPNSLAGVNEQFF TGTGCCAGCAGCTTGGCGAGCGGGACCGCCGGCGAGCAGTTCTTC 3550 CASSLASGTAGEQFF TGTGCCAGCAGCTTCCTCCTCAACCCCCGGGATACGCAGTATTTT 3501 CASSFLLNPRDTQYF TGTGCCAGCAGCCACACCGGGACTAGCGGGACCCCGCAGTACTTC 3410 CASSHTGTSGTPQYF TGCAGTGCTAGTTTTGATTCTGGAAACACCATATATTTT 3320 CSASFDSGNTIYF TGTGCCAGCACCCATCTTAGCGGGACCTACAATGAGCAGTTCTTC 3173 CASTHLSGTYNEQFF TGTGCCAGCAGCAGGACCGTCGGGAATGGCTACACCTTC 2899 CASSRTVGNGYTF TGTGCCAGCAGTTACACCGGACAGGGGGTCTATGGCTACACCTTC 2664 CASSYTGQGVYGYTF TGTGCCAGCAGCTTCGGAGCCACCATTAACTATGGCTACACCTTC 2417 CASSFGATINYGYTF TGTGCCTGACAGGGTGGAATGGCTACACCTTC MANUSCRIPT2278 CA*QG_NGYTF TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 2235 CASSLVGNSGNTIYF TGTGCCAGCAGCTTAGAGATCGGGAATGAGCAGTTCTTC 2208 CASSLEIGNEQFF TGTGCCAGCAGCTTAAGACACACCACCGGGGAGCTGTTTTTT 2169 CASSLRHTTGELFF TGTGCCAGCAGCCTAGGACTAGCGGGAAACGTCCGGCATTACAATGAGCAGTTCTTC 2146 CASSLGLAGNVRHYNEQFF TGTGCCAGCAGCTCAGCGGGAGAGGTCAAGGAGCAGTTCTTC 2135 CASSSAGEVKEQFF TGTGCCAGCAGCTTCGACAGGGGGAATTCACCCCTCCACTTT 1987 CASSFDRGNSPLHF TGCAGCGTTGTCCCGGCCAGCCGGAACACCGGGGAGCTGTTTTTT 1796 CSVVPASRNTGELFF TGTGCCAGCAGTATTTCGAGCTCCTACAATGAGCAGTTCTTC 1749 CASSISSSYNEQFF TGCAGTGCCGGGGGACTAGCGATTAGCACAGATACGCAGTATTTT 1690 CSAGGLAISTDTQYF TGTGCCAGCGCATCAGGAGGGGCCAACGTCCTGACTTTC 1588 CASASGGANVLTF TGTGCCAGCAGCATCGGGAACTACAATGAGCAGTTCTTC 1500 CASSIGNYNEQFF TGTGCCTGGAGTGCTCTGGGGATGGGGGAGACCCAGTACTTC 1463 CAWSALGMGETQYF TGTGCCTGGAGTGGGGGGGTCGGCTATAATTCACCCCTCCACTTT 1455 CAWSGGVGYNSPLHF TGTGCCAGCAGCCCGCGCTCGGACAGGGGAGATTCACCCCTCCACTTT 1455 CASSPRSDRGDSPLHF TGTGCCTGGAGAGGGGCTAGCACAGATACGCAGTATTTTACCEPTED 1444 CAWRGASTDTQYF TGTGCCAGCAGCTTAAGGACTAGCGGGACGGAGACCCAGTACTTC 1442 CASSLRTSGTETQYF TGTGCCAGCAGCTTAGCCCGATGGGCAACTAATGAAAAACTGTTTTTT 1438 CASSLARWATNEKLFF TGCAGTGCTGGGGTAGGGAGGGACCACGGGGAGCTGTTTTTT 1436 CSAGVGRDHGELFF TGCGCCAGCAGCGGGGGACTTAGCTCCTACGAGCAGTACTTC 1415 CASSGGLSSYEQYF TGCAGTGCTGGGGGGGGAAGAGATACGCAGTATTTT 1372 CSAGGGRDTQYF TGTGCCAGCAGCTTGCCCAGGGGGCATCCCGGGGAGCTGTTTTTT 1237 CASSLPRGHPGELFF TGTGCCAGCCGACAGGGAACGAACACCGGGGAGCTGTTTTTT 1205 CASRQGTNTGELFF TGTGCCAGCAGTTACTTCGGGAACACTGAAGCTTTCTTT 1075 CASSYFGNTEAFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGTCGACTAGCGGAGGGGAGGTACGAGCAGTACGTC 30769 CASSRLAEGRYEQYV TGCAGTGCTTATTACACCTACCAAGAGACCCAGTACTTC 25592 CSAYYTYQETQYF TGTGCCTGGAGTTTGGACACCCAGTGGAGCGGCTACACCTTC 25497 CAWSLDTQWSGYTF TGCAGCGTTGCCCGAGACCCAAATGCCGGGGAGCTGTTTTTT 22391 CSVARDPNAGELFF TGTGCCAGCAGCTTGTACGTTGGGAAAGCTTTCTTT 17185 CASSLYVGKAFF TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 16483 CASSLVGNSGNTIYF TGTGCCTGGAGTGTCTTTCCAGCGACACAAGAGACCCAGTACTTC 16483 CAWSVFPATQETQYF TGTGCCTGGAGCAGGGGTGAGACCCAGTACTTC 13440 CAWSRGETQYF TGTGCCAGCTCCCGGGGTACCTACGAGCAGTACGTC 11904 CASSRGTYEQYV TGTGCCAGCAGCCAAGATTGGCGGAACACTGAAGCTTTCTTT 11250 CASSQDWRNTEAFF TGCAGCGTTGAAGGGACTAGCGGGAGTTACTCCTACAATGAGCAGTTCTTC 10468 CSVEGTSGSYSYNEQFF TGTGCCAGCAGCCCTCTAGCCCAAGACAGACGACCTTCCCTCCACTTT 9927 CASSPLAQDRRPSLHF TGTGCCAGCAGCTTAGGGTGGCAGGGGAGAGAGACCCAGTACTTC 9559 CASSLGWQGRETQYF TGTGCCAGCACTCCTGTCAGGGTCGGCACTGAAGCTTTCTTT 9529 CASTPVRVGTEAFF TGTGCCTGGAGCCAGCCGGGGTCGGAGACCCAGTACTTC 8373 CAWSQPGSETQYF TGTGCCAGCAGCTCCCTAGTGGTCTATGAGCAGTTCTTC 7806 CASSSLVVYEQFF TGTGCCTGGAGGGATGAGACCGATTATGGCTACACCTTC 7682 CAWRDETDYGYTF TGTGCCAGCAGCACAGACCGGGACGCCGTGAATTTCTTT 7380 CASSTDRDAVNFF TGTGCCTGGGGTGACAGGGGTGACACTGAAGCTTTCTTT 7085 CAWGDRGDTEAFF TGTGCCACCAGCAGAGTTAAGGGACGGGGAACTGAAGCTTTCTTT 6503 CATSRVKGRGTEAFF TGTGCCAGCAGTTACGTGGGAAATCTTAACTATGGCTACACCTTC 5636 CASSYVGNLNYGYTF TGTGCCAGCAGCTGGAGGGGGGCAGATACGCAGTATTTT 5460 CASSWRGADTQYF TGCGCCAGCAGGTCCAGTCCTGATGAGACCCAGTACTTC 5291 CASRSSPDETQYF TGTGCCAGCAGCGGAATTCCGGGCGCGAAGCAGTTCTTC 4367 CASSGIPGAKQFF TGCGCCAGCAGCCAAGGGACTAGCAGCGAGCAGTACTTC 4216 CASSQGTSSEQYF TGTGCCAGCAGCTTAAGCAACACCGGGGAGCTGTTTTTT 4008 CASSLSNTGELFF TGTGCCAGCAGCTTATCTTTAGGGGCTGGGGCCAACGTCCTGACTTTC MANUSCRIPT3770 CASSLSLGAGANVLTF TGCGCCAGCAGCCAAAGGGGGAGCTCCTACAATGAGCAGTTCTTC 3631 CASSQRGSSYNEQFF TGTGCCAGCAGCTGGGTTGGGGATACGCAGTATTTT 2651 CASSWVGDTQYF TGTGCCAGCAGTTCAGGACAGGGTTCCTACAATGAGCAGTTCTTC 2391 CASSSGQGSYNEQFF TGTGCCAGCAGCACAGGGGTTGAAGCTTTCTTT 2099 CASSTGVEAFF TGTGCCAGCAGCCATCGACAGGGGGCTGCAACTAATGAAAAACTGTTTTTT 2025 CASSHRQGAATNEKLFF TGTGCCTGGAGTCCATCTAATCAGCCCCAGCATTTT 1964 CAWSPSNQPQHF TGTGCCAGCAGCTTTAGGGGCAGGGATACTGAAGCTTTCTTT 1955 CASSFRGRDTEAFF TGTGCCAGCAGGGGTGGACTAGCGGGAGTTGTTGAGCAGTTCTTC 1858 CASRGGLAGVVEQFF TGTGCCAGCAGTGAAGGACAGGGGACGCAGTATTTT 1731 CASSEGQGTQYF TGTGCTAGCAGCGCACGGACTAGCGGGAACTACAATGAGCAGTTCTTC 1713 CASSARTSGNYNEQFF TGCAGCGTTGACGGTTTAGGGCCCTACGAGCAGTACTTC 1429 CSVDGLGPYEQYF TGTGCCAGCAGCTTAAACTCGGGAGATACGCAGTATTTT 1423 CASSLNSGDTQYF TGTGCCAGCAGCCAGTGGGACACCGGGGAGCTGTTTTTT 1329 CASSQWDTGELFF TGTGCCTGCCCGGAGCTGGGACAGGGGTGTTGAGGGCTACACCTTC 1319 CACPELGQ_GVEGYTF TGCAGTGGGACGTGGAATGAGCAGTTCTTC 1083 CSGTWNEQFF TGCAGTGCTAGAGATCATCAACGTGGACAGTTCGGTGAGCAGTTCTTC 1078 CSARDHQRGQFGEQFF TGCGCCAGCAGCTTCGAGGGGTTCGAATATGGCTACACCTTCACCEPTED 1059 CASSFEGFEYGYTF TGTGCCAGCAGCTCGCGAGTAATACGACAGGGAACCAATGGCTACACCTTC 1017 CASSSRVIRQGTNGYTF TGTGCCAGCAGCTTGGTAGAGAACACTGAAGCTTTCTTT 973 CASSLVENTEAFF TGTGCCAGCAGCTTTGGGGGAGGCTATGGCTACACCTTC 961 CASSFGGGYGYTF TGTGCCAGCAGCTTAAATGCAGCGGGAGTGGGCGAGACCCAGTACTTC 961 CASSLNAAGVGETQYF TGTGCCAGCAGCCTAACACCGGGGCTCACAGATACGCAGTATTTT 904 CASSLTPGLTDTQYF TGTGCCAGCAGCCAGGGTCGTGGCTACACCTTC 796 CASSQGRGYTF TGTGCCACCAGTGAAGCAACGGGACTTTACAGTGGGCAGTTCTTC 774 CATSEATGLYSGQFF TGTGCCAGCAGCTTGGAACGAACAGATACGCAGTATTTT 763 CASSLERTDTQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAAGGATCTAGCAAGGCCGGCAGATACGCAGTATTTT 50115 CAWKDLARPADTQYF TGTGCCAGCAGCTTCGGGGTAACTAGCACAGATACGCAGTATTTT 37834 CASSFGVTSTDTQYF TGCGCCAGCAGCCAAGAGGGGGGCCGGGGGAGCACAGATACGCAGTATTTT 36188 CASSQEGGRGSTDTQYF TGTGCCAGCAGTTACGGACCGACAGGGGGTTATGGCTACACCTTC 30465 CASSYGPTGGYGYTF TGCAGCGTTGATCCACGAGGAGCGGGAGCGGGGTACAATGAGCAGTTCTTC 26893 CSVDPRGAGAGYNEQFF TGTGCCAGCAGCGCGGACAGGGCGGCTAACTATGGCTACACCTTC 24874 CASSADRAANYGYTF TGTGCCAGCAGTTGGGGGTTTCGGACTGGCAATGAGCAGTTCTTC 11409 CASSWGFRTGNEQFF TGTGCCGGCGAGGGACCTAGAGCCGGGACAGGGTTCGATGAGCAGTTCTTC 9488 CAGEGPRAGTGFDEQFF TGTGCCAGCAGCCATACCGGGACTAGCTACACAGATACGCAGTATTTT 8664 CASSHTGTSYTDTQYF TGCGCCAGCAGCCTGGGGGAGGGAGGGCCCTGGGAGACCCAGTACTTC 8244 CASSLGEGGPWETQYF TGTGCCAGCAGCTCGTCTAGCGGGTCTTCCTACAATGAGCAGTTCTTC 6509 CASSSSSGSSYNEQFF TGTGCCAGCAGTGCCCGGGACAACTCTGGAAACACCATATATTTT 5638 CASSARDNSGNTIYF TGTGCCAGCAGTTCCTTTAGCGGGGGGTCCTACAATGAGCAGTTCTTC 5384 CASSSFSGGSYNEQFF TGTGCCAGCAGCCCCAGGAGGACAGGGGGGATCAACTATGGCTACACCTTC 4594 CASSPRRTGGINYGYTF TGTGCCAGCAGCGCGACAGGGATGGTGGCAGATACGCAGTATTTT 4318 CASSATGMVADTQYF TGTGCCAGCAGCTTATTAGGCGGAGAGACCCAGTACTTC 3500 CASSLLGGETQYF TGTGCCAGCAGCTTAGTGCCAGGGGTGGTTGAAAAACTGTTTTTT 3310 CASSLVPGVVEKLFF TGCAGTGCTAGAGATCGCAGGGGATGGTCCTACACCTTC 3123 CSARDRRGWSYTF TGCAGTGCTGGGGGGGGAAGAGATACGCAGTATTTT 3015 CSAGGGRDTQYF TGCGCCAGCAGCCAAGATGGCGCAGATACGCAGTATTTT 2748 CASSQDGADTQYF TGTGCCACCAGCTCCGGGACAGGGTTTTGGGAGCAGTACTTC 2518 CATSSGTGFWEQYF TGTGCCTGGAGTCTCCGCGGACTAGCGGGATATGGGGCCAACGTCCTGACTTTC 2396 CAWSLRGLAGYGANVLTF TGCAGCAGTTCAGTCCAGGGGGAGCAGTACTTC MANUSCRIPT1797 CSSSVQGEQYF TGTGCCAGCAGCTTCTATGGACAGGGGGCGTCCAGTGGAGAGACCCAGTACTTC 1794 CASSFYGQGASSGETQYF TGCAGTGCTAGATCGGGGGACAGGGGTGAAAAACTGTTTTTT 1556 CSARSGDRGEKLFF TGTGCCAGCATTCCGGGGGCGAGAAACACCATATATTTT 1538 CASIPGARNTIYF TGTGCCTGGAGTGTCGCCAGGGAGATCGGCTACACCTTC 1536 CAWSVAREIGYTF TGTGCCAGCAGCCAGGGACAGACTGAAGAGACCCAGTACTTC 1386 CASSQGQTEETQYF TGCAGTGCGCGAGAGCAAGGTAGCGGGACCTACGAGCAGTACGTC 1255 CSAREQGSGTYEQYV TGTGCCACCAGTGATTTAACGAAGCGGGCCCGACGGGAGACCCAGTACTTC 1165 CATSDLTKRARRETQYF TGTGCCAGCAGCGGCGGACAGGGGGTAGGAGGCTACACCTTC 1117 CASSGGQGVGGYTF TGTGCCAGCAGTTACTCTGGGGACACCATATATTTT 1097 CASSYSGDTIYF TGTGCCAGCAGACGGACACTGGGGCCCAATGAGCAGTTCTTC 1095 CASRRTLGPNEQFF TGTGCCAGCAGCTTAGTTGGAAACTCTGGAAACACCATATATTTT 1013 CASSLVGNSGNTIYF TGTGCCAGCAGCCATCGACAGGGGGCTGCAACTAATGAAAAACTGTTTTTT 925 CASSHRQGAATNEKLFF TGTGCCAGCAGCTTAATGGGCAGTAAAGAGACCCAGTACTTC 794 CASSLMGSKETQYF TGTGCCAGCAGTTACAGGAATCACAATGAGCAGTTCTTC 766 CASSYRNHNEQFF TGTGCCTGGAGTGTAGACCGGTTTCAGCCCCAGCATTTTACCEPTED 745 CAWSVDRFQPQHF TGCAGTGCTAGAGGAGGGGGGGATTCACCCCTCCACTTT 695 CSARGGGDSPLHF TGTGCCAGCGGGCCCCCCCGTAGCGATACGCAGTATTTT 661 CASGPPRSDTQYF TGTGCCAGCAGCACGGACGGGAATCAGCCCCAGCATTTT 650 CASSTDGNQPQHF TGTGCCAGCAGCTTAGGGAAGTTTGCAGTGGACCAGCCCCAGCATTTT 591 CASSLGKFAVDQPQHF TGCAGTGCTGCCAGTGACGAAAAACTGTTTTTT 554 CSAASDEKLFF TGCAGTGGAGGGGTAGGCTACACCTTC 507 CSGGVGYTF TGTGCCAGCAGTATAGGGCAGGGGCACACTGAAGCTTTCTTT 504 CASSIGQGHTEAFF TGTGCCAGCAGCTTAGCGCCCCTGGTCAATTCACCCCTCCACTTT 502 CASSLAPLVNSPLHF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAGTGTACTGTGGCTTCACCAAGAAGAGACCCAGTACTTC 36495 CAWSVLWLHQEETQYF TGTGCCCAGGAGAAGGGAAGGGGCGGCACAGATACGCAGTATTTT 33746 CAQEKGRGGTDTQYF TGTGCTAGCAGCCCCGGGCCCAGGGGTGAGCAGTTCTTC 29258 CASSPGPRGEQFF TGTGCCAGCAGCTCCCCGCGAAGGCAAGAGACCCAGTACTTC 25715 CASSSPRRQETQYF TGTGCCACCAGCAGAGATCCAGGGAGCTCCTACGAGCAGTACTTC 17695 CATSRDPGSSYEQYF TGTGCCAGCAGCGGAGGGACAGGGTCCAACTATGGCTACACCTTC 15472 CASSGGTGSNYGYTF TGCAGCGTTGGGACAGCCAATCAGCCCCAGCATTTT 15266 CSVGTANQPQHF TGCAGTGCTAGACAACAGCTTAATCAGCCCCAGCATTTT 11870 CSARQQLNQPQHF TGTGCCAGCAGCGATCACCCGGGACAGGGAATTTCCTACGAGCAGTACGTC 11755 CASSDHPGQGISYEQYV TGTGCCAGCAGCGCCGGGACAGGGGCGTTTCAGTACTTC 9062 CASSAGTGAFQYF TGTGCCAGCAGCTTAACTGGGCGGGACACTGAAGCTTTCTTT 8965 CASSLTGRDTEAFF TGTGCCAGCAGTCTAGGGGTAGGGAACTACGAGCAGTACTTC 8587 CASSLGVGNYEQYF TGCGCCAGCAGCCAGAGGCCGGGACCCTGGGGTCTCACCTTC 7695 CASSQRPGPWGLTF TGTGCCAGCAGCCAAGTGGGCGATCAGCCCCAGCATTTT 7323 CASSQVGDQPQHF TGTGCCTGGAGTAAGCGGCCGCACACAGATACGCAGTATTTT 7103 CAWSKRPHTDTQYF TGTGCCAGCAGCCTCGCCCCCGGGACAGGGAATGAAAAACTGTTTTTT 6747 CASSLAPGTGNEKLFF TGTGCCAGCAGCCGTGCTGGGAGGGGAAACACTGAAGCTTTCTTT 5783 CASSRAGRGNTEAFF TGCAGCGTTGGCCAGGGGAGGACTGAAGCTTTCTTT 5265 CSVGQGRTEAFF TGCAGTGCTAGAGATATAGGGGACAGCGGAATTTATAGCAATCAGCCCCAGCATTTT 5121 CSARDIGDSGIYSNQPQHF TGCGCCAGCAGCCAAGGGACTAGCGGGAGTTTAACCGGGGAGCTGTTTTTT 4853 CASSQGTSGSLTGELFF TGTGCCAGCACCTTGGAGGGGGCGCAGGAGACCCAGTACTTC 4464 CASTLEGAQETQYF TGTGCCAGCAGCTTAGGATCAGGGAGCTCTGGGGCCAACGTCCTGACTTTC 4456 CASSLGSGSSGANVLTF TGTGCCAGCAGCTTAGTCGCCGTAGGTAGCACAGATACGCAGTATTTT 4346 CASSLVAVGSTDTQYF TGCAGCGTTGTACTCTCGGGGAATGAAAAACTGTTTTTT MANUSCRIPT3906 CSVVLSGNEKLFF TGTGCCAGCAGTGAGGGGAACACTGAAGCTTTCTTT 3867 CASSEGNTEAFF TGTGCCAGCAGCTTCGGGGGGGACTATGGCTACACCTTC 3534 CASSFGGDYGYTF TGTGCCAGCAGCATCCTGGCGGATACGCAGTATTTT 3377 CASSILADTQYF TGTGCCAGCAGCTATGCGGGACTAGCGAATGAGCAGTTCTTC 3312 CASSYAGLANEQFF TGTGCCTGGAGTCGAGGGTTCCAAGAGACCCAGTACTTC 3103 CAWSRGFQETQYF TGCGCCAGCAGCTTTGAGGGAGGCACAGATACGCAGTATTTT 3050 CASSFEGGTDTQYF TGCAGTGCTAGGGGACAGGCCCCGTACCAAGAGACCCAGTACTTC 2614 CSARGQAPYQETQYF TGTGCCAGCGGGGATTTTGGAGGGCCAGATACGCAGTATTTT 2501 CASGDFGGPDTQYF TGTGCCAGCAGCATCGGACTAGCGGACAATGAGCAGTTCTTC 2491 CASSIGLADNEQFF TGCAGCGTTGTAGGGCTAGCCGGCGAGCAGTTCTTC 2376 CSVVGLAGEQFF TGCGCCAGCAGCCAAGATCCCGGGAGCCCTCTCACAGATACGCAGTATTTT 2168 CASSQDPGSPLTDTQYF TGTGCCAGCAGTTCCCATTGGGACCGCGGGCAGTTCTTC 2135 CASSSHWDRGQFF TGTGCCAGCAGCCCCGGGACTAGCGGGATCGGCAATGAGCAGTTCTTC 2129 CASSPGTSGIGNEQFF TGTGCCAGCAGCTCCGTGGGACTAGCGGGAGCCGAGCAGTTCTTC 1986 CASSSVGLAGAEQFF TGTGCCAGCAGCTTAAGTTCTAGCCGGCAGTATTTT 1835 CASSLSSSRQYF TGTGCCAGCAGCGACATCGGCACTGAAGCTTTCTTTACCEPTED 1547 CASSDIGTEAFF TGTGCCAGCAGCTTGAGATCCGGTTGGGGCCCCCTTAATTCACCCCTCCACTTT 1501 CASSLRSGWGPLNSPLHF TGTGCTAGCAGCTTAGATATCCAGGGGGTAGGGGGTGAGCAGTTCTTC 1486 CASSLDIQGVGGEQFF TGTGCCAGCAGCCAAGATCCCAGGGAGACCCAGTACTTC 1465 CASSQDPRETQYF TGTGCCAGCAGCGAAGTAGCGGGAGAGGGCGAGACCCAGTACTTC 1449 CASSEVAGEGETQYF TGTGCCAGCAGCTTAATCCCGGGACTAAGTGAGCAGTTCTTC 1449 CASSLIPGLSEQFF TGTGCCAGCAGCCCCCAGCGGGACCTTGAGCCCCAGCATTTT 1443 CASSPQRDLEPQHF TGTGCCTGGAGTGCCGGGACAGCTAATGAAAAACTGTTTTTT 1408 CAWSAGTANEKLFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAGTGGCCAAAACATTCAGTACTTC 5713 CAWSGQNIQYF TGTGCCGGCAGAGAGACCCAGTACTTC 2426 CAGRETQYF TGTGCCTGGAGTGTTCAGGGGGCGTATGGCTACACCTTC 2229 CAWSVQGAYGYTF TGTGCCAGCAGCTTAATGGTATGGGGATATCTTCCCTCGTTGCTCTTC 2138 CASSLMVWGYLPSLLF TGTGCCTGGAGTCGATCCGGGACTAGCGGCAATGAGCAGTTCTTC 1858 CAWSRSGTSGNEQFF TGTGCCTGGGAGTCTGGAGGGAACACTGAAGCTTTCTTT 1827 CAWESGGNTEAFF TGTGCCTGGAGCGAGGGACTCCAGCTGAGCACAGATACGCAGTATTTT 1769 CAWSEGLQLSTDTQYF TGTGCCAGCAGCCCAAATCGCCCCTACGAGCAGTACGTC 1666 CASSPNRPYEQYV TGTGCCTGGAGTGTAGCCGTTAACTATGGCTACACCTTC 1636 CAWSVAVNYGYTF TGTGCCTGGAGTATCGGACAGGGAAACACTGAAGCTTTCTTT 1520 CAWSIGQGNTEAFF TGTGCCTGGACGGCCCCCCAGGGTGGGGTGATCAATCAGCCCCAGCATTTT 1441 CAWTAPQGGVINQPQHF TGTGCCTGGAGTGAAAGGGTTACTCAGCCCCAGCATTTT 1381 CAWSERVTQPQHF TGTGCCTGGAGTGTTCAACTGGACTACGAGCAGTACTTC 1316 CAWSVQLDYEQYF TGTGCCTGGAGCCCCTCCAATACGCAGTATTTT 1255 CAWSPSNTQYF TGTGCCTGGGCGAAGGGCGGACGCAGCACAGATACGCAGTATTTT 1229 CAWAKGGRSTDTQYF TGTGCCGGGGGGCGGATGAACACTGAAGCTTTCTTT 1202 CAGGRMNTEAFF TGTGCCAGCAGCCCCGGACTAGCGGGAGGGATATACGAGCAGTACGTC 1164 CASSPGLAGGIYEQYV TGTGCCTGTATGACAGGGGGCTTGGCCACTGAAGCTTTCTTT 1146 CACMTGGLATEAFF TGTGCCAGCAGCTTAGTGTCCTGGTCCGAGCAGTACTTC 1120 CASSLVSWSEQYF TGTGCCAGCAGCTTGCCGGATTAGCAATCAGCCCCAGCATTTT 1069 CASSLPD_SNQPQHF TGTGCCTGGAGTACGGGAGGGGGGCTTGGCTACACCTTC 1020 CAWSTGGGLGYTF TGTGCCTGGAGTGTACTAGCGGGATCGAATGAGCAGTTCTTC 1011 CAWSVLAGSNEQFF TGTGCCTGGAGTGAGGGGCAGGGCAGCGGCTACACCTTC 992 CAWSEGQGSGYTF TGTGCCTGGCAGTCCGGGGTAGCGGGAGCAAACTCCTACAATGAGCAGTTCTTCMANUSCRIPT917 CAWQSGVAGANSYNEQFF TGTGCCGGCGGGAGGGGTAATCAGCCCCAGCATTTT 900 CAGGRGNQPQHF TGTGCCTGGAACAGGGGCGGGGATGGGGGCACAGATACGCAGTATTTT 856 CAWNRGGDGGTDTQYF TGCGCCAGCAGCCAAGATTGGACTAGCGGGCCGTACTCCTACAATGAGCAGTTCTTC 851 CASSQDWTSGPYSYNEQFF TGTGCCTGGAGACCGACAGGGGGCCGAGATCAGCCCCAGCATTTT 807 CAWRPTGGRDQPQHF TGTGCCTGGCGGACAGGGACCAACTATGGCTACACCTTC 790 CAWRTGTNYGYTF TGTGCCTGGAGTCCCCGAAGCGAGTCCCAGTACTTC 776 CAWSPRSESQYF TGTGCCAGCCGGGGACAAACGAACACTGAAGCTTTCTTT 767 CASRGQTNTEAFF TGTGCCTGGGGCAGTGCTTATGGCTACACCTTC 745 CAWGSAYGYTF TGCAGCGTACGGGACAGGCGGAATTCACCCCTCCACTTT 721 CSVRDRRNSPLHF TGTGCCTGGATGCGACAGACGAACACTGAAGCTTTCTTT 694 CAWMRQTNTEAFF TGTGCCTGGAGTTCGGGACAGCACAATCAGCCCCAGCATTTT 686 CAWSSGQHNQPQHF TGTGCCAGCAATCCGGGACTTGCCCCCTACGAGCAGTACTTC 662 CASNPGLAPYEQYF TGTGCCTGGACCACCCTGATGGAAAACACCATATATTTT 654 CAWTTLMENTIYF TGTGCCACCAGGGAGGAAGTCGAAAAACTGTTTTTT 651 CATREEVEKLFF TGTGCCTGGAGCAGCGGGAGGGAGAAACTGTTTTTT 611 CAWSSGREKLFF TGTGCCAGCAGACGGGTGACAGGGAGGAGCAATGAGCAGTTCTTCACCEPTED 606 CASRRVTGRSNEQFF TGTGCCTGGAACCGGGGCCGGCAAAACATTCAGTACTTC 604 CAWNRGRQNIQYF TGTGCCTGGAGTGTGGGGGGCGTGGTGAACACTGAAGCTTTCTTT 560 CAWSVGGVVNTEAFF TGTGCCTGGAGATCGGATAATAGCAATCAGCCCCAGCATTTT 549 CAWRSDNSNQPQHF TGTGCCTGGAGTGTTGGACTGTCAGGGGAGCTGTTTTTT 542 CAWSVGLSGELFF TGTGCCTGGAGTGCGGGGACTAGCACAGATACGCAGTATTTT 537 CAWSAGTSTDTQYF TGTGCCTGGAGTGTACAGGGGGGTAATACCGGGGAGCTGTTTTTT 531 CAWSVQGGNTGELFF TGTGCCAGCAGCTTAGCGGTGGCAGGACGGCCGCTACACCTTC 519 CASSLAV_QDGRYTF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGAGAGGGGTCTGGCACAGATACGCAGTATTTT 1975 CASREGSGTDTQYF TGTGCCAGCAGCAAGGGGACAGCAAATCAGCCCCAGCATTTT 1670 CASSKGTANQPQHF TGTGCCTGTGTGGGACAGATTTACAATGAGCAGTTCTTC 1380 CACVGQIYNEQFF TGTGCCTGGAGTGCGGGGGCGAACACTGAAGCTTTCTTT 1157 CAWSAGANTEAFF TGTGCCTGGAGTGCGGGTGCCAGAAGCACAGATACGCAGTATTTT 1027 CAWSAGARSTDTQYF TGTGCCTGGGCGAAGGGCGGACGCAGCACAGATACGCAGTATTTT 982 CAWAKGGRSTDTQYF TGTGCCCGGACAGGGGGCCCGGGGTCAACGGGCTACACCTTC 954 CARTGGPGSTGYTF TGTGCCTGGAGTGGAGACAGGGTGGAGACCCAGTACTTC 942 CAWSGDRVETQYF TGTGCCTGGAGTGGCCAAAACATTCAGTACTTC 930 CAWSGQNIQYF TGTGCCTGGAACGAGGCGGCAGGCAATCAGCCCCAGCATTTT 843 CAWNEAAGNQPQHF TGTGCCAGCAGCTTAGCGACCGACAGCGCTGAAGCTTTCTTT 822 CASSLATDSAEAFF TGTGCCAGCAGCCTCGGTCCTAGCGGGAGTGTTGGGGAGCAGTTCTTC 818 CASSLGPSGSVGEQFF TGTGCCGGGGTAGGGGGCACTGAAGCTTTCTTT 813 CAGVGGTEAFF TGTGCCAGCAGCCAAGGGGGGGGCCGCGAGCCCCAGCATTTT 809 CASSQGGGREPQHF TGTGCCAGCAGCCCAGGAAGGGGGTCCGGCACAGATACGCAGTATTTT 795 CASSPGRGSGTDTQYF TGTGCCTGTGATAGCGGGATCGCCTACAATGAGCAGTTCTTC 768 CACDSGIAYNEQFF TGTGCCTGGAGTGTACGGTCCCCAGATACGCAGTATTTT 737 CAWSVRSPDTQYF TGTGCCTGGAGTGTACAGCAGAACACTGAAGCTTTCTTT 713 CAWSVQQNTEAFF TGTGCCAGCAGCTTCAGGACAGTCGCATCCTACGAGCAGTACTTC 643 CASSFRTVASYEQYF TGTGCCTGGAAACTCCAGGGCTCCTACAATGAGCAGTTCTTC 638 CAWKLQGSYNEQFF TGTGCCAGCAGCCGCCCGGGACAGATGAACTATGGCTACACCTTC 634 CASSRPGQMNYGYTF TGTGCCAGCAGCTTAGGGGGCAGGGAATCTGTCTACGAGCAGTACTTC 598 CASSLGGRESVYEQYF TGTGCCTGGGACAAACCAGGGGGAAACTACAATGAGCAGTTCTTC 569 CAWDKPGGNYNEQFF TGTGCGGCCGGGACTTACACCTACGAGCAGTACTTC 561 CAAGTYTYEQYF TGTGCCAGCAGCCGGGACCTTTCCGGGGAGACCCAGTACTTC 561 CASSRDLSGETQYF TGTGCCTGGACGGGTTTCGGTAACAATGAGCAGTTCTTC MANUSCRIPT544 CAWTGFGNNEQFF TGTGCCTGGGACAGCGGCAGGTGGGAGCAGTACTTC 541 CAWDSGRWEQYF TGTGCCAGCAGCTTGAGCGGAGGCCAAGAGACCCAGTACTTC 535 CASSLSGGQETQYF TGTGCCTGGAGGGATCAGGGGGCTAACTATGGCTACACCTTC 519 CAWRDQGANYGYTF TGTGCCAGCAGCTTAGGGGCAGGGACTGAAGCTTTCTTT 516 CASSLGAGTEAFF TGTGCCAGCAGCTTAAACGGGCAGCGGGAGACCCAGTACTTC 509 CASSLNGQRETQYF TGTGCCTGGAGTGTAGGGACAGCGAACACTGAAGCTTTCTTT 505 CAWSVGTANTEAFF TGTGCCAGCAGCCAAAAAGTGATCCTAGCTACTAGCACAGATACGCAGTATTTT 501 CASSQKVILATSTDTQYF TGCGCCAGCAGCTATTGGGGGGGCGAGACCCAGTACTTC 486 CASSYWGGETQYF TGCGCCAGCAGCCAAGATTATGTGGAGACCCAGTACTTC 473 CASSQDYVETQYF TGTGCCTGGGGGAGTGGGGATACGCAGTATTTT 466 CAWGSGDTQYF TGTGCCTGGTCATGGGGTGGCTATGGCTACACCTTC 451 CAWSWGGYGYTF TGTGCCAGCAGCTTCGCGGCAACGGCTACTGAAGCTTTCTTT 445 CASSFAATATEAFF TGTGCCAGCAGCTTAGGGGCAGACACTGAAGCTTTCTTT 440 CASSLGADTEAFF TGTGCCAGCAGCTCACCGGGACAGGGCGACGAGCAGTACTTC 432 CASSSPGQGDEQYF TGCGCCAGCAGCCCTTTTTTTGTGGGACTAGAGATGGAGACCCAGTACTTC 429 CASSPFFVGLEMETQYF TGTGCCTGGAGTTCCTATCTGAATACGCAGTATTTT 413 CAWSSYLNTQYF TGTGCCTGGAGTACCCGTGGGACTCTCCAAGAGACCCAGTACTTCACCEPTED 408 CAWSTRGTLQETQYF TGTGCCAGCAGCCCGGTGACTTGGACCAATTCACCCCTCCACTTT 404 CASSPVTWTNSPLHF TGTGCCAGCAGCCAACGCGCGGGAGCCGCCTACGAGCAGTACGTC 397 CASSQRAGAAYEQYV TGTGCCTGGACCGAGGGGGGGCCCTCAGGAGCTTTCTTT 394 CAWTEGGPSGAFF TGTGCCAGCAGGCCGGGACTAGCGTCCTGGGCCGGGGAGCTGTTTTTT 393 CASRPGLASWAGELFF TGTGCCAGCAGCCAACTGGGAAGGGACACTGAAGCTTTCTTT 393 CASSQLGRDTEAFF TGTGGGGGGGACACAGGAGTAGAGAACACTGAAGCTTTCTTT 392 CGGDTGVENTEAFF TGTGCCTGGAGTACCGGGACAGTTTCCTATGGCTACACCTTC 385 CAWSTGTVSYGYTF TGTGCTAGCAGCAAAGGGGCCGGGGGCACCGGGGAGCTGTTTTTT 385 CASSKGAGGTGELFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGTATAGGGGGAGGGCAGTCCGAGCAGTACTTC 19802 CASSIGGGQSEQYF TGTGCCAGCAGCCCGGGCTCCTATAATTCACCCCTCCACTTT 6613 CASSPGSYNSPLHF TGCAGTGCTGGCCCTCAGGCACTAGCGGGTAATGAGCAGTTCTTC 4686 CSAGPQALAGNEQFF TGCGCCAGCAGCTCCTCCATAGCGGGAGATGGTGCGAATGAGCAGTTCTTC 4057 CASSSSIAGDGANEQFF TGTGCCAGCAGCACAATGCACGGACTGCAAGAGACCCAGTACTTC 3238 CASSTMHGLQETQYF TGTGCCTGGAGCGGGGATGATCAGCCCCAGCATTTT 2883 CAWSGDDQPQHF TGTGCCAGCAGCCCCCCTAGCGGGAACTACGAGCAGTACTTC 2455 CASSPPSGNYEQYF TGTGCCAGCAGCCTAGCGGAGCCCGGCACAGATACGCAGTATTTT 2429 CASSLAEPGTDTQYF TGTGCCTGGAGTCGGGAGTTCGAGCAGTACTTC 2383 CAWSREFEQYF TGTGCCAGCAGCTTAGTTGGCGGTTCTAGCACAGATACGCAGTATTTT 2363 CASSLVGGSSTDTQYF TGTGCCTGGAGTGTGGGGGCCTCAGAGACCCAGTACTTC 1823 CAWSVGASETQYF TGTGCCAGCACGCAAGCCGGGACTAGCGGGTCCCAATGGTACTTC 1801 CASTQAGTSGSQWYF TGTGCCAGCAGCCAAGATCGGGGGACAGGGGCCAAGGCTTGGGGCTACACCTTC 1722 CASSQDRGTGAKAWGYTF TGCGCCAGCAGCTTGGATGGGTATGGCTACACCTTC 1680 CASSLDGYGYTF TGTGCCACCAGCAGAGGCCGGGAAAACACTGAAGCTTTCTTT 1639 CATSRGRENTEAFF TGTGCCAGCAGCCCGGGACTCAGCACCGGGGAGCTGTTTTTT 1591 CASSPGLSTGELFF TGTGCCAGCAGCTTGGACGTCTACACTGAAGCTTTCTTT 1562 CASSLDVYTEAFF TGTGCCTGGGGGATGGGGTTCTACGAGCAGTACTTC 1544 CAWGMGFYEQYF TGTGCCAGCAGCAGGGGGCTGAGCTCCTACGAGCAGTACTTC 1494 CASSRGLSSYEQYF TGTGCCAGCAGCTTAGCAGGCTCCTATAATTCACCCCTCCACTTT 1492 CASSLAGSYNSPLHF TGTGCCAGCAGCTTCGAGGGGCGATGGGGCGAGCAGTACTTC 1453 CASSFEGRWGEQYF TGTGCCTGGAGTCTTGCTCTTAGCAATCAGCCCCAGCATTTT 1441 CAWSLALSNQPQHF TGTGCCAGCAGCAATTTCCCGGGACCGGACAATGAGCAGTTCTTC 1386 CASSNFPGPDNEQFF TGTGCCAGCAGTACTAGCGGAGCTTTCACCGGGGAGCTGTTTTTT 1339 CASSTSGAFTGELFF TGTGCCAGCAGCCCGGGGGGGGCGCTGGAGACCCAGTACTTC 1335 CASSPGGALETQYF TGTGCCTGGAGTGTCGGACTAGCCACGGACGAGCAGTACTTC 1293 CAWSVGLATDEQYF TGTGCCAGCAGCAAATATGAGGTGCAGTACTTC MANUSCRIPT1274 CASSKYEVQYF TGTGCCAGCAGCTCAGGACAGGTGACCTACGAGCAGTACTTC 1265 CASSSGQVTYEQYF TGCAGCGCAGAACGACAGAACACTGAAGCTTTCTTT 1230 CSAERQNTEAFF TGTGCCAGCAGCGAGGGACAGGCGTCCTGGGAGCAGTACTTC 1211 CASSEGQASWEQYF TGTGCCAGCACCATCCCAAACAGTCCCCAGTACTTC 1109 CASTIPNSPQYF TGTGCCAGCAGCCAATCTACGGGACTCATCACAGATACGCAGTATTTT 1100 CASSQSTGLITDTQYF TGTGCCTGGCCGTGGGGGGAAAGGTACGAGCAGTACTTC 1070 CAWPWGERYEQYF TGTGCCTGGAGCGGGAGCTCCCACGAGCAGTACTTC 1023 CAWSGSSHEQYF TGTGCCAGCAGCACCGGGGCGTCAGATACGCAGTATTTT 1023 CASSTGASDTQYF TGTGCCAGCAGTTCCCAGGGACGCTACAATGAGCAGTTCTTC 983 CASSSQGRYNEQFF TGTGCCAGCAGCTTAGGGGATACGCAGTATTTT 973 CASSLGDTQYF TGTGCCTGGAATACAGGGGCGAGGGGCTACACCTTC 954 CAWNTGARGYTF TGCGCCAGCAGCCAAGATGGGGGAGCTTCAGATACGCAGTATTTT 945 CASSQDGGASDTQYF TGTGCCAGCAGCCGGGGGGGGGCATTGGAGACCCAGTACTTC 914 CASSRGGALETQYF TGTGCCAGCAGCTTAGAGGGACTACCCCAGCATTTT 905 CASSLEGLPQHF TGTGCCTGGAGCGGAAACAGGGAATCAGCCCCAGCATTTT 898 CAWSGNR_NQPQHF TGTGCCAGCAGCCCACCAGCTAGAAACGCCTACGAGCAGTACTTC 870 CASSPPARNAYEQYF TGTGCCAGCAGTTACCATGGACTAGCCCAAGAGACCCAGTACTTC 853 CASSYHGLAQETQYF TGTGCCTGGCGCCCCGACAGGGCCAGTTCACCCCTCCACTTTACCEPTED 847 CAWRPDRASSPLHF TGCGCCAGCAGCTTGGACAGGGGGACTGAAGCTTTCTTT 832 CASSLDRGTEAFF TGTGCCTGGGACAGGGGCCTGGAGACCCAGTACTTC 830 CAWDRGLETQYF TGTGCCTGGAGTCCGGGACTAGCGGGACTCACAGATACGCAGTATTTT 823 CAWSPGLAGLTDTQYF TGTGCCAGCAGCGAGACGACGGTGAACACTGAAGCTTTCTTT 817 CASSETTVNTEAFF TGTGCCAGCAGCTCCCCCACTAGCTTACAAGAGACCCAGTACTTC 805 CASSSPTSLQETQYF TGTGCCTGGAGTGTACCCCCCGGGACCGAGTATGCAGATACGCAGTATTTT 797 CAWSVPPGTEYADTQYF TGTGCCAGCAGCCAAGAAGGGCAGAACTATGGCTACACCTTC 794 CASSQEGQNYGYTF TGCGCCAGCAGCTTTGCGGGAGGCGGCACAGATACGCAGTATTTT 793 CASSFAGGGTDTQYF TGTGCCAGCAGCTTAGAGACAGGGAAGCAGTACTTC 793 CASSLETGKQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCCCGGGCTCCTATAATTCACCCCTCCACTTT 4530 CASSPGSYNSPLHF TGTGCCAGCAGCCCCCGCTTTCGCTACACCTTC 4500 CASSPRFRYTF TGTGCCAGCAGCCCTGGTGGGACTTTATACGAGCAGTACTTC 3591 CASSPGGTLYEQYF TGCAGTGCTTCCGGGACTAGCGGGCGCAGCTACGAGCAGTACTTC 3217 CSASGTSGRSYEQYF TGTGCCAGCAGCTTAGAGCTGGACTATGGCTACACCTTC 3184 CASSLELDYGYTF TGTGCCAGCAGCCGGGGGCCTTATAATTCACCCCTCCACTTT 3082 CASSRGPYNSPLHF TGTGCCAGCAGCTTAGGGCAGGGCCATGGCTACACCTTC 3015 CASSLGQGHGYTF TGTGCCAGCAGCCGACAACAGCCTGGCACCGGGGAGCTGTTTTTT 2898 CASSRQQPGTGELFF TGTGCCCGGGGGGTCTACCCGAACACCGGGGAGCTGTTTTTT 2779 CARGVYPNTGELFF TGTGCCAGCAGCGATACCGGGACTAGCGGGATGCCCTACGAGCAGTACTTC 2674 CASSDTGTSGMPYEQYF TGTGCCAGCAGCCCCATGCGCACAGATACGCAGTATTTT 2601 CASSPMRTDTQYF TGTGCCAGCAGGACGGCCTCCCAAGAGACCCAGTACTTC 2576 CASRTASQETQYF TGTGCCAGCAGCTTAGTTGGCGGTTCTAGCACAGATACGCAGTATTTT 2343 CASSLVGGSSTDTQYF TGTGCCACCAGCAGAGGAATATACGACACTGAAGCTTTCTTT 2296 CATSRGIYDTEAFF TGTGCCAGCAGCTACAACATGAACACTGAAGCTTTCTTT 2280 CASSYNMNTEAFF TGTGCCAGCACTCGGGGGTTAAGGGCTGAAGCTTTCTTT 2143 CASTRGLRAEAFF TGTGCCTGTCAGGGACAGAGCTACAATGAGCAGTTCTTC 2071 CACQGQSYNEQFF TGCGCCAGCAGCTTTGCGGGAGCTGGGACAGATACGCAGTATTTT 1980 CASSFAGAGTDTQYF TGTGCCAGCAGCCCGACAGGCACTGGGGCCAACGTCCTGACTTTC 1797 CASSPTGTGANVLTF TGTGCCAGCAGCCCCCCCACTAGCGGGCCTCACAATGAGCAGTTCTTC 1761 CASSPPTSGPHNEQFF TGTGCCTGGTCGGAGAGGCCAACTGACAATGAGCAGTTCTTC 1733 CAWSERPTDNEQFF TGTGCCAGCAGCTCCGGGACAGGGGGAGGAGAAGCTTTCTTT 1711 CASSSGTGGGEAFF TGTGCCTGGGGAACGACAGGGTCCCATGGCTACACCTTC MANUSCRIPT1674 CAWGTTGSHGYTF TGCAGCGTTGAAGGGCTAGCGGGAGGGCCTCGAGAGACCCAGTACTTC 1659 CSVEGLAGGPRETQYF TGTGCCTGGTCTCCCTCCGGGTCCCCTTATGGCTACACCTTC 1650 CAWSPSGSPYGYTF TGTGCCTGGAGCGGAGCCGGGCTCAATCAGCCCCAGCATTTT 1604 CAWSGAGLNQPQHF TGTGCCAGCAGCCGGACGGTCGCGGGAGGACCCACAGATACGCAGTATTTT 1483 CASSRTVAGGPTDTQYF TGTGCCTGGAGCCCTACCCCCGGTGGCTACACCTTC 1453 CAWSPTPGGYTF TGTGCCTGGAGTACTTTTACAGGGGGAAGCACTGAAGCTTTCTTT 1300 CAWSTFTGGSTEAFF TGTGCCACCAGCAGAGATAGGAGACAGGGGGTAGAGCAGTTCTTC 1253 CATSRDRRQGVEQFF TGCGCCAGCAGCTTTGCGGGAGGCGGCACAGATACGCAGTATTTT 1166 CASSFAGGGTDTQYF TGTGCCACCAGCAGAAATGTGGCAGAGACCCAGTACTTC 1118 CATSRNVAETQYF TGTGCCAGCAGCCCCTACCAAGAGACCCAGTACTTC 1102 CASSPYQETQYF TGTGCCTGGAGCTCCTCTGCGAACACAGATACGCAGTATTTT 1089 CAWSSSANTDTQYF TGTGCCAGCAGTCCAACCCTTAAATCCTACGAGCAGTACTTC 1082 CASSPTLKSYEQYF TGTGCCAGCAGCCCGGGACTCAGCACCGGGGAGCTGTTTTTT 1005 CASSPGLSTGELFF TGTGCCAGCAGCTGGGGTTGGACAGGCAATGAGCAGTTCTTC 987 CASSWGWTGNEQFF TGTGCCAGCAGCAAATATGAGGTGCAGTACTTC 976 CASSKYEVQYF TGTGCCAGCAGCTTATTCGCTAGCGGGAGCACAGATACGCAGTATTTTACCEPTED 941 CASSLFASGSTDTQYF TGTGCCAGCAGCTCCCAAGGCGGGTCCAACACCGGGGAGCTGTTTTTT 941 CASSSQGGSNTGELFF TGCAGCGTGGGTGAGGCCGCTAATCAGCCCCAGCATTTT 940 CSVGEAANQPQHF TGTGCCAGCAGCTTATCGGCTAGCGGGCATACCGGGGAGCTGTTTTTT 917 CASSLSASGHTGELFF TGTGCCAGCAGCACCGGACAGTGGGGCTACACCTTC 913 CASSTGQWGYTF TGTGCCAGCAGCTTAGCAGGGCTGTACAATGAGCAGTTCTTC 875 CASSLAGLYNEQFF TGTGCCTGGAGTCGACAGGGAAACGAGCAGTACTTC 837 CAWSRQGNEQYF TGTGCCAGCAGCCAAGTTTGGGGACAGGGTCGGTTCTTC 835 CASSQVWGQGRFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCCGATCTTCGGCCCCAGGGGACAGCGAAAAACTGTTTTTT 33006 CASSRSSAPGDSEKLFF TGCAGCGTTGGCAGCCCCATTACCTACGAGCAGTACTTC 16214 CSVGSPITYEQYF TGTGCCAGCAGCTTCGACAGGGGTTCACCCCTCCACTTT 14121 CASSFDRGSPLHF TGTGCCTGGAGAGTTGGGACACCGAACACTGAAGCTTTCTTT 11011 CAWRVGTPNTEAFF TGCGCCAGCAGCTTGCAGGCCGGCTACACCGGGGAGCTGTTTTTT 9585 CASSLQAGYTGELFF TGCAGTGGGAAACGGGAGTCCGACAATGAGCAGTTCTTC 6010 CSGKRESDNEQFF TGTGCCAGCAGCGATGAGCCAGGGGTCTATGGCTACACCTTC 5129 CASSDEPGVYGYTF TGTGCCAGCAGCTCCCCTAGGGCTAACAGGAGGTCGGGCTACACCTTC 5061 CASSSPRANRRSGYTF TGTGCCAGCAGCTTGTGGGGACAGGGGAGTCAGCCCCAGCATTTT 4748 CASSLWGQGSQPQHF TGTGCCAGCCAGCCCGGCTATGGCTACACCTTC 4288 CASQPGYGYTF TGTGCCACCAGCAGAGAGGGACTAGCGGGAGGCACAGATACGCAGTATTTT 3584 CATSREGLAGGTDTQYF TGTGCCAGCAGCTTAAGAGCGGACTCCTACAATGAGCAGTTCTTC 3247 CASSLRADSYNEQFF TGTGCCAGCAGCCGAACACGTCAGCATTCACCCCTCCACTTT 2931 CASSRTRQHSPLHF TGTGCCAGCAGCTTAGGGCCCGCGGGAGCCAAAGAGACCCAGTACTTC 2865 CASSLGPAGAKETQYF TGTGCCAGCAGCTTAGAGATCTTACTAGGGGGTATCGAGACCCAGTACTTC 2806 CASSLEILLGGIETQYF TGTGCCAGCTGCCCCGGGACAGGGAGCTCACCCCTCCACTTT 2526 CASCPGTGSSPLHF TGCGCCAGCAGCCGTACTAGCGGGAGATCCGATACGCAGTATTTT 2379 CASSRTSGRSDTQYF TGTGCCAGCAGTTACTCCCCGCCAGCCAATGAGCAGTTCTTC 2235 CASSYSPPANEQFF TGTGCCAGCAGCTTCGGGGGCGGTGGCTACACCTTC 2020 CASSFGGGGYTF TGTGCCAGCAGCCCACGGACTAGCCTTGACATTCAGTACTTC 1864 CASSPRTSLDIQYF TGTGCCTGGAGGTCAGGGACACCCCCGGTAGAGCTGTTTTTT 1785 CAWRSGTPPVELFF TGTGCCTGGAGGGTAGGGACACCGAACACTGAAGCTTTCTTT 1684 CAWRVGTPNTEAFF TGTGCCAGCAGCCAAGGACAGCACCATGGCTACACCTTC MANUSCRIPT1602 CASSQGQHHGYTF TGTGCCAGCAGCTCCCTGACAGGTAACACTGAAGCTTTCTTT 1465 CASSSLTGNTEAFF TGTGCCACCAGCAGAGAGGGACTAGCGGGAGGGACAGATACGCAGTATTTT 1387 CATSREGLAGGTDTQYF TGCAGTGCTGGGCGGGAGCAGGATAATGAGCAGTTCTTC 1355 CSAGREQDNEQFF TGTGCCAGCACCCTCCTAGACCGGGGTGAGCAGTTCTTC 1303 CASTLLDRGEQFF TGTGCCAGCAGCTTCGGGTTCTTC 1298 CASSFGFF TGTGCCAGCAGCTTAAGTGACAGGGGACACGAGCAGTACTTC 1261 CASSLSDRGHEQYF TGTGCCAGCAGCCTTGGTGGAGGCACTGAAGCTTTCTTT 1193 CASSLGGGTEAFF TGTGCCAGCAGCTTAATTAGAGTCAGGGGATTGGAGACCCAGTACTTC 1054 CASSLIRVRGLETQYF TGTGCCAGCAGTTTATATCGGGGAGGTGAAGCTTTCTTT 1004 CASSLYRGGEAFF TGCGCCAGCAGATCGGGACTAGCGGGAGATGTGCATACGCAGTATTTT 984 CASRSGLAGDVHTQYF TGCAGTGCTAGCCTTAGCGGGAGAGATGCGGGTGAGCAGTTCTTC 943 CSASLSGRDAGEQFF TGTGCCAGCAGAACCTCCGCCCACGGGGACGAGCAGTACTTC 930 CASRTSAHGDEQYF TGCGCCAGCAGCCAAGCCAGGAACCCACGTCGGTTCTTC 904 CASSQARNPRRFF TGTGCCTATGACCGGGGTCCTAATCAGCCCCAGCATTTT 840 CAYDRGPNQPQHF TGTGCCAGCAGCTTTAGCAGGTTCGAGCAGTACTTCACCEPTED 801 CASSFSRFEQYF TGTGCCAGCACCTTGTTGAGGAGCACAGATACGCAGTATTTT 775 CASTLLRSTDTQYF TGCGCCAGCAGCTTGGCACCGAACCCTAACAATGAGCAGTTCTTC 744 CASSLAPNPNNEQFF TGTGCCAGTGTCCGGGACAGGGGACGTTCTGGGGCCAACGTCCTGACTTTC 726 CASVRDRGRSGANVLTF TGTGCCTGGAGGTTGGACAGGCGAAACTATGGCTACACCTTC 725 CAWRLDRRNYGYTF TGTGCCAGCAGCTTAACGTCGGCGGGAGAGCAGTTCTTC 722 CASSLTSAGEQFF TGCAGTGCTAGAAATATTCGGACAGGGGGCCTCCGAGCCTACGAGCAGTACTTC 704 CSARNIRTGGLRAYEQYF TGTGCCAGCAGCTCCGGGACAGCCGACTATTTTCATTTT 691 CASSSGTADYFHF TGTGCCAGCAGCTCCGGACTGAACACTGAAGCTTTCTTT 686 CASSSGLNTEAFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCCGTCAGTGGAACACTGAAGCTTTCTTT 28532 CASSRQWNTEAFF TGCAGTGCTAGAGATTGGAGCGGAAGAACAGATACGCAGTATTTT 18973 CSARDWSGRTDTQYF TGTGCCTGGAGTGTACTGGACACACATGGCTACACCTTC 9961 CAWSVLDTHGYTF TGTGCCTGGAACCGGGCCTTCGACGAGCAGTACTTC 9880 CAWNRAFDEQYF TGTGCCAGCAGCCAAGGACAGGGATACGGCTACACCTTC 7706 CASSQGQGYGYTF TGTGCCAGCAGCTTGGACTTATTAGCCAAAAACATTCAGTACTTC 7146 CASSLDLLAKNIQYF TGCGCCAGCAGCACTCTGACTAGCGGGAATCAGGAGCAGTTCTTC 5909 CASSTLTSGNQEQFF TGTGCCAGCAGTTACCGAGGCGGAGAAGCTTTCTTT 4891 CASSYRGGEAFF TGTGCCAGCAGCGGACTAGCACCTAGCTCCTACAATGAGCAGTTCTTC 4848 CASSGLAPSSYNEQFF TGTGCCAGTATCCAAGCCGGACTAGTTCAAGAGACCCAGTACTTC 3685 CASIQAGLVQETQYF TGCAGCGTTGGCAGCCCCATTACCTACGAGCAGTACTTC 2775 CSVGSPITYEQYF TGTGCCAGCGGACAGCGTGGGAATGAAAAACTGTTTTTT 2417 CASGQRGNEKLFF TGTGCCAGCAGCGCCGACGGCGGGCGCATGACCTCCTACAATGAGCAGTTCTTC 2183 CASSADGGRMTSYNEQFF TGCGCCAGCAGCTTGCGGACTAGTGGCAATACGCAGTATTTT 2061 CASSLRTSGNTQYF TGTGCCAGCAGCACTTGGACAGATCAGCCCCAGCATTTT 1756 CASSTWTDQPQHF TGTGCCTTGGGTTTTTC 1671 CAL_FF TGTGCCAGCAGCTTTTCTAGCACAGATACGCAGTATTTT 1670 CASSFSSTDTQYF TGTGCCAGCAGCCAGGGCAGTACTGCTGGCCAGCCCCAGCATTTT 1491 CASSQGSTAGQPQHF TGTGCCAGCACCCCGCGAAGGCGGAACACCGGGGAGCTGTTTTTT 1398 CASTPRRRNTGELFF TGTGCCTGGAGTTTGGGGCGGAATCAGCCCCAGCATTTT 1258 CAWSLGRNQPQHF TGTGCCAGCAGCGCCCGACAGGGGGAGAACACTGAAGCTTTCTTT 1200 CASSARQGENTEAFF TGTGCCAGCTGCCCCGGGACAGGGAGCTCACCCCTCCACTTT 1171 CASCPGTGSSPLHF TGTGCCAGCAGACTCACCAGACCTACGCCCTACGAGCAGTACTTC MANUSCRIPT1125 CASRLTRPTPYEQYF TGTGCCAGCAGTTCTGACAGGGTTGCACCCCTCCACTTT 1122 CASSSDRVAPLHF TGCAGTGCTAGAGGGACGCCGGCCTCCACAGATACGCAGTATTTT 1104 CSARGTPASTDTQYF TGTGCCTGGGGGGGACTAGCGGGAGAGGGGACCCAGTACTTC 1079 CAWGGLAGEGTQYF TGCGCCAGCAGCTTTATTGTAACGGGAGGAGGGAATGAGCAGTTCTTC 1061 CASSFIVTGGGNEQFF TGCAGCGTTGGTGAAGGGTGGGGTCGCTATGGCTACACCTTC 1003 CSVGEGWGRYGYTF TGTGCCAGCCGTGGGGGGACAGGGTCAAACACTGAAGCTTTCTTT 1003 CASRGGTGSNTEAFF TGTGCCAGCAGCTATGGAGCGCCAGTGTCCTACGAGCAGTACTTC 995 CASSYGAPVSYEQYF TGTGCCAGCAGCTTGACAGGGGGACTAACGCGCTCTGGGGCCAACGTCCTGACTTTC 940 CASSLTGGLTRSGANVLTF TGTGCCTGGAGTGCAAACGGGAAATACAATGAGCAGTTCTTC 925 CAWSANGKYNEQFF TGTGCCACCAGCAGCCGTCGGGGGGGCTGGGAATCGAACACTGAAGCTTTCTTT 913 CATSSRRGGWESNTEAFF TGTGCCACCTCCCTGGGACAGGGACGTTACACCTTC 912 CATSLGQGRYTF TGTGCCAGCAGCGACCAACCGAACACTGAAGCTTTCTTT 888 CASSDQPNTEAFF TGTGCCAGCAGCTCTCGCCTCGGCGAGCAGTACTTC 866 CASSSRLGEQYF TGTGCCTGGAGGCCTCGGGGGTTCGGGTACGAGCAGTACTTC 850 CAWRPRGFGYEQYF TGTGCCAGCAGCGTAGCCCAGACGGGTGAGCAGTTCTTCACCEPTED 823 CASSVAQTGEQFF TGTGCCAGCAGTTTACACGGGAATGAAAAACTGTTTTTT 809 CASSLHGNEKLFF TGTGCCAGCAGCTTAGATATCCGATCCTACGAGCAGTACTTC 805 CASSLDIRSYEQYF TGTGCCAGCATTCCTAGCGGGAGGTCCTACGAGCAGTACTTC 800 CASIPSGRSYEQYF TGTGCCAGCGGTTCCGCTGGCAATCAGCCCCAGCATTTT 792 CASGSAGNQPQHF TGCAGTGCTAGTGTCCTAGCGCACATATCTGTGCAGTTCTTC 785 CSASVLAHISVQFF TGTGCCAGCAGCTTCGCATATGGCTACACCTTC 772 CASSFAYGYTF TGTGCCAGCAGCTTATCCGGGGCGGGATCTTTCGACACGCAGTATTTT 740 CASSLSGAGSFDTQYF TGTGCCAGCAGCCCTCCGGGGGCGAGGCGAAACGAGCAGTACTTC 726 CASSPPGARRNEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGCGCCAGCAGCCGGGACAGAACTAATGGCTACACCTTC 12343 CASSRDRTNGYTF TGTGCCAGCAGCTTAGTAAGCGAGCAGTACTTC 11145 CASSLVSEQYF TGTGCCAGCAGCTTCTCCCGGACTAGCGGGAGTAAACGAGATACGCAGTATTTT 5901 CASSFSRTSGSKRDTQYF TGTGCCTGGAACCCGGACGGAGATGGCTACACCTTC 5516 CAWNPDGDGYTF TGTGCCTGGAGTGGTGGGATCTCTGGAAACACCATATATTTT 4140 CAWSGGISGNTIYF TGTGCCAGCAGGCAGACCGGGACAGGGACCGGGGAGCTGTTTTTT 4103 CASRQTGTGTGELFF TGTGCCAGCAGCCGGCCGGGACTGGCGCAGTATTTT 3850 CASSRPGLAQYF TGTGCCAGCAGCTTATTTCCGCGGGGGACCCAGTACTTC 3410 CASSLFPRGTQYF TGTGCCAGCAGGACGGGACAGGGGGGCACAGATACGCAGTATTTT 3123 CASRTGQGGTDTQYF TGTGCCAGCAGCTTTGGGGAGGGGTACAATGAGCAGTTCTTC 2965 CASSFGEGYNEQFF TGTGCCAGCAGCTTAGAGATCTTACTAGGGGGTATCGAGACCCAGTACTTC 2892 CASSLEILLGGIETQYF TGTGCCAGCAGCTTCAGCGGGAGCAGCCCAGATACGCAGTATTTT 2087 CASSFSGSSPDTQYF TGTGCCTGGAGTGAAGGTAGCGGGAGCGTTGATACGCAGTATTTT 2072 CAWSEGSGSVDTQYF TGTGCCAGCAGCTTAGCAAGGGGTCGGGGAGCTTTCTTT 2068 CASSLARGRGAFF TGTGCCAGCAGCTTAGCAACCTTCACCGGGGAGCTGTTTTTT 2013 CASSLATFTGELFF TGTGCCAGCTCGACTAGCGGGAGTTACGAGCAGTACTTC 1838 CASSTSGSYEQYF TGTGCCAGCAGCTTAGGTGTGGCTCGCAATCAGCCCCAGCATTTT 1814 CASSLGVARNQPQHF TGTGCCAGCAGCCAAGAGCTCAGCTTCGATACGCAGTATTTT 1749 CASSQELSFDTQYF TGTGCCAGCAGCTTAGGCCCCAATCAGCCCCAGCATTTT 1638 CASSLGPNQPQHF TGTGCCAGCAGCTTAGGCGGACAGGGATTTAAGCCCCAGCATTTT 1553 CASSLGGQGFKPQHF TGTGCCAGCAGCTTAGGAAGCACAGATACGCAGTATTTT 1495 CASSLGSTDTQYF TGTGCCAGCAGCCAAGACCGCCGAGCGGGGTTAGACGAGCAGTACTTC 1471 CASSQDRRAGLDEQYF TGCGCCAGCAGCCAAGGACAGGGGCCGGATTCACCCCTCCACTTTMANUSCRIPT1235 CASSQGQGPDSPLHF TGTGCCAGCACCTCCCCCCGGACAGGGGGTAACGAGCAGTACTTC 1210 CASTSPRTGGNEQYF TGTGCCAGCAGCTTGAACGGGGGAAAAGGGAATCAGCCCCAGCATTTT 1185 CASSLNGGKGNQPQHF TGTGCCAACCTTGACTAATGGTTTCCAGATACGCAGTATTTT 1161 CANLD*WFPDTQYF TGCAGTGCTAGTTTGGGGTTTTCCAGCTATGGCTACACCTTC 1068 CSASLGFSSYGYTF TGCAGTGCTAGGGCAGGGATCGGGAGCAATCAGCCCCAGCATTTT 1054 CSARAGIGSNQPQHF TGTGCCAGCAGCTTAACGGGACAGGGGCCCCAGTTCTTC 1052 CASSLTGQGPQFF TGCGCCAGCAGCCACCTACTAGCGGGCTACGAGCAGTACTTC 992 CASSHLLAGYEQYF TGTGCCAGCAGCGCCGGACAGGGCCCCTACGAGCAGTACTTC 958 CASSAGQGPYEQYF TGCAGCGTTGGTGGCAAGGAGGGACGGTGGACTGAAGCTTTCTTT 937 CSVGGKEGRWTEAFF TGTGCCAGCAGCGATCGAGGGGGCGGGTTCTTC 867 CASSDRGGGFF TGTGCCAGCAGCTCCTCTACCGGGGTACAGGTAGCTTTCTTT 845 CASSSSTGVQVAFF TGTGCCAGCAGCCTACGGACAGGGATCCTCCCGGAGACCCAGTACTTC 838 CASSLRTGILPETQYF TGCGCCAGCAGCCATAGTCCTAGCGAGAGGGAGACCCAGTACTTC 835 CASSHSPSERETQYF TGTGCCAGCAGTTACGGACAGGGAGGAACAGATACGCAGTATTTT 811 CASSYGQGGTDTQYF TGCAGTGCTTTGGACAGGGGGCTTCGAGAGACCCAGTACTTCACCEPTED 700 CSALDRGLRETQYF TGTGCCAGCAGTTACAGACAGGGAGACTACGAGCAGTACTTC 700 CASSYRQGDYEQYF TGTGCCAGAGGGACGGCTCCCACAGATACGCAGTATTTT 681 CARGTAPTDTQYF TGTGCCAGCAGTTCCCAGGACCGAGCGGGGGTTCGGGGTGAGCAGTTCTTC 653 CASSSQDRAGVRGEQFF TGTGCCAGCAGCTTAAATCCTAGCGGGACATACACAGATACGCAGTATTTT 624 CASSLNPSGTYTDTQYF TGCACCAGCAGCCGGGACAGAACTAATGGCTACACCTTC 604 CTSSRDRTNGYTF TGCAGTGCTAGAGGGACAGTGTTTAACACTGAAGCTTTCTTT 601 CSARGTVFNTEAFF TGTGCCTGGAGTGGTGAGAGGAGTGCTGAAGCTTTCTTT 571 CAWSGERSAEAFF TGTGCCAGCAGTCCAGTCTTGGGGTGGCCCAATGAGCAGTTCTTC 569 CASSPVLGWPNEQFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCTAGCAGCTTAGATGGGGGCCGGAACAATGAGCAGTTCTTC 61638 CASSLDGGRNNEQFF TGTGCCAGCAGCTCCCCCCCGACAGGGGTCTACGAGCAGTACTTC 25157 CASSSPPTGVYEQYF TGTGCCTGGAGGATCGGTTCTGGGGCCAACGTCCTGACTTTC 13979 CAWRIGSGANVLTF TGTGCCAGCAGCCAAGAGACTAGCGGGAGAAGAGATACGCAGTATTTT 12753 CASSQETSGRRDTQYF TGTGCCAGCAGCCTTCGACAGGGGCGGAATGAAAAACTGTTTTTT 9797 CASSLRQGRNEKLFF TGTGCCAGCAGCCCTAGCAACTCCTACGAGCAGTACTTC 8664 CASSPSNSYEQYF TGTGCCTGGAGTACAGGGAGTCTCAACGAGCAGTACTTC 5454 CAWSTGSLNEQYF TGCGCCAGCAGCTTGACGGAGGGGATGAACACTGAAGCTTTCTTT 4830 CASSLTEGMNTEAFF TGCAGTGCCCTAGGCAATGAGCAGTTCTTC 4359 CSALGNEQFF TGTGCCTGGAGTTTTTCGACGGCGGGAGGCTACACCTTC 3789 CAWSFSTAGGYTF TGTGCCTGGAGGTCGGGACGAATAATCACACCCCTCCACTTT 3344 CAWRSGRIITPLHF TGTGCCAGCAGCTTAGGGGGAAGATACGAGCAGTACTTC 3327 CASSLGGRYEQYF TGTGCCTGGAGTGAAGGGACCCACTACGAGCAGTACTTC 3103 CAWSEGTHYEQYF TGTGCCAGCAGCTTAGCCTACAGGATAAACTCTGGGGCCAACGTCCTGACTTTC 2728 CASSLAYRINSGANVLTF TGTGCCTGGAGTTCCCAGTGGACCGGGCAGTTCTTC 2710 CAWSSQWTGQFF TGTGCCAGCAGCCTTGACCGCTCGAGGGAGCAGTACTTC 2620 CASSLDRSREQYF TGTGCCTGGAGTTCCGAAACGACAGGGAGGGGTGAAAAACTGTTTTTT 2592 CAWSSETTGRGEKLFF TGTGCCAGCAGCACAACAGCACCCGGCGAGCAGTACTTC 2301 CASSTTAPGEQYF TGTGCCTGGAGGAGACTAGCCGCCACCGTTACGCAGTATTTT 2159 CAWRRLAATVTQYF TGTGCCAGCAGCTTGGGACAGGGAGGAGGCTACACCTTC 2097 CASSLGQGGGYTF TGCGCCAGCAGCCAAAGTTACTCCGAGAAAGCGTACTTC 1915 CASSQSYSEKAYF TGTGCCAGCAGTTTAGCGGGGGGCTATGGCTACACCTTC 1816 CASSLAGGYGYTF TGTGCCTGGAGTGTAGGGCTGATCTCCTACGAGCAGTACTTC MANUSCRIPT1414 CAWSVGLISYEQYF TGTGCCTGCCGACAGGGTTGGGCAGATACGCAGTATTTT 1383 CACRQGWADTQYF TGTGCCAGCAGCTTAGTGGCGTCTACCTACGAGCAGTACTTC 1321 CASSLVASTYEQYF TGTGCCTGGAGTGGCCCCACAGGGAGCGGTGGCTACACCTTC 1030 CAWSGPTGSGGYTF TGTGCCAGCAGCCTAGACTCAGGGAAGCAGTATTTT 956 CASSLDSGKQYF TGTGCCAGCAGTTCGGGGGGCTCCTACAATGAGCAGTTCTTC 855 CASSSGGSYNEQFF TGTGCCAGCAGCTTTTTGCCACAAGAGACCCAGTACTTC 841 CASSFLPQETQYF TGCGCCAGCAGCCAAGCCCCGGGGAATGAGCAGTTCTTC 830 CASSQAPGNEQFF TGTGCCAGCAGCTTAGGAGGGAGCTCAGATACGCAGTATTTT 763 CASSLGGSSDTQYF TGTGCCAGCAGTTATACAGGCTATAATTCACCCCTCCACTTT 762 CASSYTGYNSPLHF TGTGCCAGCAGTTACGGCGGTTCTAACTATGGCTACACCTTC 727 CASSYGGSNYGYTF TGTGCCTGCTCGACTTTTAACACCGGGGAGCTGTTTTTT 702 CACSTFNTGELFF TGTGCCAGCAGCTTATATGACAGGGGAGGCGAGCAGTTCTTC 676 CASSLYDRGGEQFF TGTGCCAGCAGAACCTCCGCCCACGGGGACGAGCAGTACTTC 622 CASRTSAHGDEQYF TGTGCCAGCAGCTTGCCCACGGATCCTGAAAAACTGTTTTTT 528 CASSLPTDPEKLFF TGTGCCAGCAGTGACCCCCAAGAGCCCCAGTTCTTCACCEPTED 511 CASSDPQEPQFF TGTGCCAGCAGTACTAGCGGCTTTTACGAGCAGTACTTC 506 CASSTSGFYEQYF TGTGCCTGGAGTCCTTTAGCGGCCAACGTCCTGACTTTC 455 CAWSPLAANVLTF TGTGCCAGCAGCCAAGATGAGGGCGGCGGGAGTAGACAAGATACGCAGTATTTT 449 CASSQDEGGGSRQDTQYF TGTGCCAGCGGCCGCGGGGGGAGCTCCTACAATGAGCAGTTCTTC 406 CASGRGGSSYNEQFF TGTGCCAGCAGCTTAGTCGGTGGCACCGGGGAGCTGTTTTTT 385 CASSLVGGTGELFF TGTGCCAGCAGCTCCCCTCGACGTTACAATGAGCAGTTCTTC 368 CASSSPRRYNEQFF TGCGCCAGCAGCTTGCGGACTAGTGGCAATACGCAGTATTTT 365 CASSLRTSGNTQYF TGTGCCAGCAGCTTAACGGTTACCTACGAGCAGTACTTC 348 CASSLTVTYEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAGAAGGCTCAGGGGGAACACTGAAGCTTTCTTT 2797 CAWRRLRGNTEAFF TGTGCCTGGAGCGTCCGGCGAGCGGCGGATGAGTTCTTC 2135 CAWSVRRAADEFF TGTGCCTGGAGCGCCGGGCCCGCTGGAAAACTGTTTTTT 1934 CAWSAGPAGKLFF TGTGCCTGGAGTGTACTGGCGGGGGGTCAGCCCCAGCATTTT 1839 CAWSVLAGGQPQHF TGTGCCAGCAGCTTAGCATCGGGACAGATGAACACTGAAGCTTTCTTT 1800 CASSLASGQMNTEAFF TGTGCCTGGAGTGTTGGGTTAGGCCCCTATAGCAATCAGCCCCAGCATTTT 1623 CAWSVGLGPYSNQPQHF TGTGCCCTTATAAGGGAGGGCCGTGAGACCCAGTACTTC 1526 CALIREGRETQYF TGTGCCTGGAGTGGCCGGACAGTCTATGGCTACACCTTC 1503 CAWSGRTVYGYTF TGTGCCTGGCAAACTGGGGGCAGGTTTCAGCCCCAGCATTTT 1257 CAWQTGGRFQPQHF TGTGCCTGGACGAGCGGCAACTCTGGAAACACCATATATTTT 1008 CAWTSGNSGNTIYF TGTGCCTGGAGAAACGCCGGGACTAGCGGGGACAATGAGCAGTTCTTC 992 CAWRNAGTSGDNEQFF TGTGCCTGGAGTGTAAGAAGCACAGATACGCAGTATTTT 988 CAWSVRSTDTQYF TGTGCCTTACGTCGGGGAGGCGGCGGCTACACCTTC 975 CALRRGGGGYTF TGTGCCTGGAGTCCGCGGAGGGCCGATACGCAGTATTTT 940 CAWSPRRADTQYF TGTGCCTGGAGTTACGGTTACGAGCAGTACTTC 933 CAWSYGYEQYF TGTGCCTGGAGTAAAGATCGCTCCTACGAGCAGTACTTC 906 CAWSKDRSYEQYF TGTGCCTGGAGTGAGGGGCTTACCTACGAGCAGTACTTC 877 CAWSEGLTYEQYF TGTGCCTGGAGTGCGGGGTATGGCTACACCTTC 862 CAWSAGYGYTF TGTGCCAGCAGCCCTAAGGGGCGGGAGGGCCAGCGCCTACAATGAGCAGTTCTTC 855 CASSPKGRE_ASAYNEQFF TGTGCCTGGAGTGTTCGGACAGGGACAGGTCAGCCCCAGCATTTT 855 CAWSVRTGTGQPQHF TGTGCCTGGAGTCGGAACAGGGGGACCTCAGGCTACACCTTC 832 CAWSRNRGTSGYTF TGTGCCTGGAGGGGGGACAGTGGGGCCTACGAGCAGTACTTC 824 CAWRGDSGAYEQYF TGTGCCAGCAGCCCCCGGAAATGGTGACGTTCAAGGTGGCCACCCCTCCACTTTMANUSCRIPT823 CASSPRKW*RSRWPPLHF TGTGCCAGCAGCCCCTCCTGGACCCCAGAAAAAACACTGAAGCTTTCTTT 794 CASSPSWT_RKNTEAFF TGTGCCTGGACCGGGACAGGGGTAGGCTACACCTTC 776 CAWTGTGVGYTF TGTGCCTGGAGTTGGCAGCTGGGCGGCTACACCTTC 765 CAWSWQLGGYTF TGTGCCTGCACACGAGCGGAATCCTATAATTCACCCCTCCACTTT 751 CACTRAESYNSPLHF TGTGCCTGGAGTGAAGCGATTAACTATGGCTACACCTTC 740 CAWSEAINYGYTF TGTGCCTGGAGTTCGGCTATGGATACGCAGTATTTT 735 CAWSSAMDTQYF TGTGCCTGGAGGGAACTAGCGGGAAGAAGCTATAATGAGCAGTTCTTC 723 CAWRELAGRSYNEQFF TGTGCCGGCGGAGGGGATTGCACTGAAGCTTTCTTT 717 CAGGGDCTEAFF TGTGCCTGGAGCAGGGGGACAGCGAAGAATTCACCCCTCCACTTT 705 CAWSRGTAKNSPLHF TGTGCCTGGAATCCCGACTACCGCTATGGCTACACCTTC 677 CAWNPDYRYGYTF TGCGCCAGCAGCCAACTCGCCAGTGAGACCCAGTACTTC 661 CASSQLASETQYF TGTGCCTGGAGTGATGGGCGCCCCAATCAGCCCCAGCATTTT 643 CAWSDGRPNQPQHF TGTGCCAGCCGGGACAGGGTATATGGCTACACCTTC 642 CASRDRVYGYTF TGTGCCTGGAGTTTGGGAGGGACCTATAATTCACCCCTCCACTTT 638 CAWSLGGTYNSPLHF TGTGCCTGGAGTAGGTCAGATTCACTGTTGACTGAAGCTTTCTTTACCEPTED 627 CAWSRSDSLLTEAFF TGTGCCTGGAGTACCGACTGGGTGCAGTACTTC 622 CAWSTDWVQYF TGTGCCTACGTGCGGACAGGGGTCTACGAGCAGTACTTC 621 CAYVRTGVYEQYF TGTGCCTGGGAACTACAGAGGAACACTGAAGCTTTCTTT 620 CAWELQRNTEAFF TGTGCCCGGAAACCAGACAGGGCAGGGTGCAATCAGCCCCAGCATTTT 607 CARKPDRAGCNQPQHF TGTGCCTGGAGTGTCCCAGGGATTGGGAACTATGGCTACACCTTC 600 CAWSVPGIGNYGYTF TGTGCCGGAGGGGGCAGACTCCAAGAGACCCAGTACTTC 592 CAGGGRLQETQYF TGTGCCTGGTCGGGTGAGGCCAACGTCCTGACTTTC 580 CAWSGEANVLTF TGTGCCAGAAGGCCTCCAGCCGCAGGGGAAGGTCAGCCCCAGCATTTT 570 CARRPPAAGEGQPQHF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGTTATTATCAGGGGGCGACTGAAGCTTTCTTT 7459 CASYYQGATEAFF TGTGCCAGCAGTTTGAGCGGGGGAGCCGGGGAGCTGTTTTTT 1003 CASSLSGGAGELFF TGTGCCTTAATTCCGGGACAGGGGGATGAAAAACTGTTTTTT 797 CALIPGQGDEKLFF TGTGCCTGGAGTGTGTATGCGGCAGAGACCCAGTACTTC 656 CAWSVYAAETQYF TGTGCCAGCAGCAACGGCTGGGAGCAGTTCTTC 635 CASSNGWEQFF TGTGCCAGCAGCCCAAGAAACAATGAGCAGTTCTTC 531 CASSPRNNEQFF TGTGCCAGCAGCAAACTAGCGGGAGGGGTAGGCAATGAGCAGTTCTTC 508 CASSKLAGGVGNEQFF TGTGCCAGCAGCTTAGGACAGGGCTCTGGGGCCAACGTCCTGACTTTC 495 CASSLGQGSGANVLTF TGTGCCTGGAGTGTACGGCTCAGGAATGAGCAGTTCTTC 456 CAWSVRLRNEQFF TGTGCCAGCAGCTTCGGACAGTCAGGTGGCTACACCTTC 424 CASSFGQSGGYTF TGTGCCTGGATCGGACAGAACTATGGCTACACCTTC 405 CAWIGQNYGYTF TGTGCCACCAGCAGCCGGGACACGGAACGGGGCTACACCTTC 401 CATSSRDTERGYTF TGTGCCAGCCACTTCAGGAACACTGAAGCTTTCTTT 400 CASHFRNTEAFF TGTGCCAGCAGCTTAGGGCTGAGCAATCAGCCCCAGCATTTT 386 CASSLGLSNQPQHF TGTGCCTGGAGTTGGGGTAGTAAGGATCAGCCCCAGCATTTT 385 CAWSWGSKDQPQHF TGTGCCAGCAGCTTACCCTCGGTGGGTAACTATGGCTACACCTTC 382 CASSLPSVGNYGYTF TGTGCCAGCAGCGACGGGGGCCACAGGGGGCGTCAGCCCCAGCATTTT 379 CASSDGGHRGRQPQHF TGTGCCAGCAGCCAAGATCCCAATCAGCCCCAGCATTTT 376 CASSQDPNQPQHF TGTGCCAGCAGCCCGCGGACAGGGAATCAGCCCCAGCATTTT 372 CASSPRTGNQPQHF TGTGCCAGCAGCTCGACCGGGACTAGCGGGAGGAGGGACCAGTACTTC 363 CASSSTGTSGRRDQYF TGTGCCAGCAGCTTAAAAAAGACCCAGTACTTC 356 CASSLKKTQYF TGTGCCAGCAGCTCTCAGCAGGGGTACAATGAGCAGTTCTTC 355 CASSSQQGYNEQFF TGCGCCAGCAGCCAAGATCGGTCTAGCGGGAGGGTCGATGAGCAGTTCTTC 352 CASSQDRSSGRVDEQFF TGTGCCAGCGCCTCGGATAGGGTAGGCACTGAAGCTTTCTTT MANUSCRIPT351 CASASDRVGTEAFF TGTGCCTGGAGTTACGCGCGGGGGATCATGAACACTGAAGCTTTCTTT 346 CAWSYARGIMNTEAFF TGTGCCAGCAGCTTAGGGCTGGTGGCTGAAGCTTTCTTT 329 CASSLGLVAEAFF TGTGCCTGGAGACTACAGGGACCCGGGGAGCTGTTTTTT 326 CAWRLQGPGELFF TGTGCCAGCAGCTGGCGGGAGGACGCATCCAATCAGCCCCAGCATTTT 321 CASSWREDASNQPQHF TGTGCCAGCAGCTTGGGACAGGGGTCGACCGGGGAGCTGTTTTTT 309 CASSLGQGSTGELFF TGTGCCTGGACACAAGGCTCCTACTATGGCTACACCTTC 299 CAWTQGSYYGYTF TGTGCCAGCAGCGCAGACAGTGCCGTTAGCTATGGCTACACCTTC 297 CASSADSAVSYGYTF TGTGCCAGCAGGCTACAGGGGGCCACTGAAGCTTTCTTT 293 CASRLQGATEAFF TGTGCCAGCAGCTTGGGGACAGGGTATTCACCCCTCCACTTT 290 CASSLGTGYSPLHF TGTGCCAGCAGCTCTTGGAGCTCGAGGGGCAGGGACGAGCAGTACTTC 289 CASSSWSSRGRDEQYF TGTGCTGGGGGAGTAGCGACGGGTGAGCAGTTCTTC 289 CAGGVATGEQFF TGTGCCTGGACCGACGGAGGGACTGAAGCTTTCTTT 284 CAWTDGGTEAFF TGTGCCAGCAGCTTAGGTCTCAACACTGAAGCTTTCTTT 283 CASSLGLNTEAFF TGTGCCTGGAGTGTCCGGGAAAGAGATACGCAGTATTTT 278 CAWSVRERDTQYF TGTGCTAGCAGTCTAACAACTAGCGGGCCCAACACCGGGGAGCTGTTTTTTACCEPTED 277 CASSLTTSGPNTGELFF TGTGCCAGCAGCTTAGACGGCTCTGGGGCCAACGTCCTGACTTTC 275 CASSLDGSGANVLTF TGTGCCAGCAGCCAATCTTCCCCCTACGAGCAGTACTTC 272 CASSQSSPYEQYF TGTGCCAGCAGCTTAGGGGCAGGGAGGCATTCACCCCTCCACTTT 266 CASSLGAGRHSPLHF TGTGCCAGCAGCTTGACAGGGGGACTAACGCGCTCTGGGGCCAACGTCCTGACTTTC 264 CASSLTGGLTRSGANVLTF TGTGCCACCAGCAGAGATTACGGTTCGGACCAGGTGGGCGAGCAGTACTTC 262 CATSRDYGSDQVGEQYF TGTGCTAGCAGCTTTCAGGGGCAAGAGACCCAGTACTTC 257 CASSFQGQETQYF TGTGCCAGCAGCTTAACGGCCGAGGGGGAAGCTTTCTTT 257 CASSLTAEGEAFF TGTGCCAGCAGCTTAGGGGGGGCAGGAGAAGGCTACACCTTC 256 CASSLGGAGEGYTF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCCCCCTAGCGGGAGTAGGTTACAATGAGCAGTTCTTC 23724 CASSPLAGVGYNEQFF TGTGCCAGCAGCTTGCGGCGGCAGCGAGGAGATACGCAGTATTTT 19583 CASSLRRQRGDTQYF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 12924 CSVLRDPNSYEQYF TGTGCCTGGAGCGCCGGGCCCTACGAGCAGTACTTC 10982 CAWSAGPYEQYF TGTGCCAGCAGCACGTTTGGGGGAGGCTACACCTTC 7151 CASSTFGGGYTF TGTGCCAGCAGGCGAAGCGGGCGGGGATACACCGGGGAGCTGTTTTTT 5211 CASRRSGRGYTGELFF TGTGCCAGCAGCTTAGGCAGATTCAATCAGCCCCAGCATTTT 4553 CASSLGRFNQPQHF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 3477 CSVHRDPNSHEQFF TGTGCCAGCAGCCAGGGTGGGGGAGTGCTTGATACGCAGTATTTT 3476 CASSQGGGVLDTQYF TGTGCCAGCAGCTTAACGGGGGAGCCCGCCTACGAGCAGTACTTC 3181 CASSLTGEPAYEQYF TGTGCCAGCAGCTTAAGGGACGCCGGGTATGGCTACACCTTC 3074 CASSLRDAGYGYTF TGCGCCAGCAGCCTAATTAGCGGGAGGGCCGGAAGAACCGGGGAGCTGTTTTTT 2845 CASSLISGRAGRTGELFF TGTGCCAGCAGCTTACAAAGCCCCTCAATGAACACTGAAGCTTTCTTT 2719 CASSLQSPSMNTEAFF TGTGCCAGCAGTTACTCGCGACAGGGGGCTTTTGAAGCTTTCTTT 2662 CASSYSRQGAFEAFF TGTGCCTGGAGTCTCGGAGACTATCAGCCCCAGCATTTT 2474 CAWSLGDYQPQHF TGTGCCAGCAGCCTTAGGGGGGGACGGCAGAACACTGAAGCTTTCTTT 2228 CASSLRGGRQNTEAFF TGTGCCAGCAGCCTTCTATTGGCCCAGTACTTC 1895 CASSLLLAQYF TGTGCCAGCAGCCGACCCCGAGGTGACTCCTCCTACGAGCAGTACTTC 1605 CASSRPRGDSSYEQYF TGTGCCCTGGAGGGGGGGGCAGTAGGGACCCAGTACTTC 1604 CALEGGAVGTQYF TGCGCCAGCAGGAGGACAGGGGAAGGCAATCAGCCCCAGCATTTT 1587 CASRRTGEGNQPQHF TGCGCCAGCAGCCTGACGTCCGGGACCTCCTACGAGCAGTACTTC 1412 CASSLTSGTSYEQYF TGTGCCCGCCAGGGGGAAGGCCCAGATACGCAGTATTTT 1307 CARQGEGPDTQYF TGCGCCAGCAGCCAAGATTGGGCAGGGAGAGGTCATGGCTACACCTTCMANUSCRIPT1305 CASSQDWAGRGHGYTF TGCGCCAGCAGCTCGACAGGGACGAACACTGAAGCTTTCTTT 1269 CASSSTGTNTEAFF TGTGCCAGCAGCTTAGCACAGGGTCCCCACGAGCAGTACTTC 1260 CASSLAQGPHEQYF TGCGCCAGCAGCTTGACAGGCACTGGAGGGGCCAACGTCCTGACTTTC 1257 CASSLTGTGGANVLTF TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1178 CASSFTGAMNTEAFF TGTGCCAGCAGCTTAGAGGCGGGGGGTCCCCAGTACTTC 1163 CASSLEAGGPQYF TGTGCCAGCAGCTTAATCGGGTACGAGCAGTACTTC 1127 CASSLIGYEQYF TGCAGTGCCGCCCCCGGGTCGGGAGGAGGGGAGACCCAGTACTTC 1074 CSAAPGSGGGETQYF TGTGCCAGCAGCACCCACCTGTGGGAGACCCAGTACTTC 1026 CASSTHLWETQYF TGTGCCAGCAGCTTAGCTGTCGGACGACAGGGGAATGAGCAGTTCTTC 1026 CASSLAVGRQGNEQFF TGTGCCACCACACCGGGACAGGGGGCGCGCGGCTACACCTTC 1007 CATTPGQGARGYTF TGTGCCAGCAGCTTAGGGCTGAGGAGCGGGAGGGACAATGAGCAGTTCTTC 1004 CASSLGLRSGRDNEQFF TGTGCCAGCAGCCAACGTCCTCAGGGAGCTAATGAAAAACTGTTTTTT 977 CASSQRPQGANEKLFF TGTGCCAGCAGCCAAAGATCAGCGGATTACACCGGGGAGCTGTTTTTT 932 CASSQRSADYTGELFF TGTGCCAGCAGCTTAGTTGCGGGAAAAGAGACCCAGTACTTC 915 CASSLVAGKETQYF TGTGCCAGCAGCCTAGGGGGGAACACTGAAGCTTTCTTTACCEPTED 883 CASSLGGNTEAFF TGTGCCTGGAGTCCCGGGTGGAACATTCAGTACTTC 873 CAWSPGWNIQYF TGCAGTGCTACACCCACCGGGGTCGCTGGTAAACTGTTTTTT 870 CSATPTGVAGKLFF TGCAGCGTTTCGACAGGGGGCCAGCCCCAGCATTTT 806 CSVSTGGQPQHF TGCAGTGCTAGAGAAGGGTATAATGAAAAACTGTTTTTT 741 CSAREGYNEKLFF TGCGCCAGCAGCCAAGATAGAGTGGGCTCCTACAATGAGCAGTTCTTC 735 CASSQDRVGSYNEQFF TGTGCCAGCAGCCAAACATCTAGCGGTTCTACAGATACGCAGTATTTT 712 CASSQTSSGSTDTQYF TGTGCCACCAGCAGTGAGCCGTTGGGCGAGCAGTACTTC 697 CATSSEPLGEQYF TGTGCCAGCACCTTGACCGGGACGGCGAACACTGAAGCTTTCTTT 656 CASTLTGTANTEAFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGTTACTCGCGACAGGGGGCTTTTGAAGCTTTCTTT 33215 CASSYSRQGAFEAFF TGTGCCAGCAGCTTAAAGGGGCGACTAGCGGGAGGGCGGGAGCAGTTCTTC 9603 CASSLKGRLAGGREQFF TGCGCCAGCAGGGCCCAGGGGCAGGGGAATGAGCAGTTCTTC 7217 CASRAQGQGNEQFF TGTGCCAGCAGCCTGGGGACAGGCCAAGAGACCCAGTACTTC 4168 CASSLGTGQETQYF TGTGCCTCCGAGAGGGGTCAGGGAAACATTCAGTACTTC 4025 CASERGQGNIQYF TGTGCCAGCAGCTTAGCTTACAATCAGCCCCAGCATTTT 3651 CASSLAYNQPQHF TGTGCCAGCAGCGTGGGACAGGGGGAAGGCTACACCTTC 3272 CASSVGQGEGYTF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 3244 CSVLRDPNSYEQYF TGTGCCTCTGCGACTAGCGGGTTCCCAGATACGCAGTATTTT 2871 CASATSGFPDTQYF TGTGCCCGCCAGGGGGAAGGCCCAGATACGCAGTATTTT 2801 CARQGEGPDTQYF TGCGCCAGCAGCCAAGGTACCGCGAGCTCCTACAATGAGCAGTTCTTC 2693 CASSQGTASSYNEQFF TGTGCCAGCAGCTTAGTCGGAGTTCAGTTTAATGAAAAACTGTTTTTT 2506 CASSLVGVQFNEKLFF TGTGCCAGCAGCTTCCGAATAACCGGGGAGCTGTTTTTT 2426 CASSFRITGELFF TGTGCCTGGAGTGTACTCGACGGGGGGAGAAATGGCTACACCTTC 2302 CAWSVLDGGRNGYTF TGTGCCAGCAGCTTATTTGGGACAGGGGACAATCAGCCCCAGCATTTT 2011 CASSLFGTGDNQPQHF TGTGCCTGGAGTGTACGCCAGGGGGACGAGCAGTACTTC 2004 CAWSVRQGDEQYF TGTGCCAGCAGCTTTTCGGGACAGGGACCCTATGGCTACACCTTC 1815 CASSFSGQGPYGYTF TGTGCCTGGAGTGTACGCGTTAGAGAGACCCAGTACTTC 1772 CAWSVRVRETQYF TGTGCCTGGAGTCGGCAGGGAGATGGCTACACCTTC 1723 CAWSRQGDGYTF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 1637 CSVHRDPNSHEQFF TGTGCCAGCAGCTTAGTGGGGGACAGGGCTTACGAGCAGTACTTC 1600 CASSLVGDRAYEQYF TGTGCCAGCAGCTTCACCGCCACGGGGTCCCAGTACTTC 1490 CASSFTATGSQYF TGTGCCAGCAGCTACCCGGGACTTTACACCGGGGAGCTGTTTTTT 1462 CASSYPGLYTGELFF TGTGCCTGGAGCGCCGGGCCCTACGAGCAGTACTTC MANUSCRIPT1453 CAWSAGPYEQYF TGTGCCAGCAGCCTGTGGTCCCACCAAGAGACCCAGTACTTC 1442 CASSLWSHQETQYF TGTGCCAGCAGCTTAACTGGACAGGGACATAGTGGCTACACCTTC 1346 CASSLTGQGHSGYTF TGTGCCAGCTCACCGGGACTAGCGGGAGGTGGAGAGACCCAGTACTTC 1344 CASSPGLAGGGETQYF TGTGCCGTCGGCGGTTTAGCCTCCTACAATGAGCAGTTCTTC 1326 CAVGGLASYNEQFF TGTGCCAGCAGCTTAGAGGCGGGACGCACAGATACGCAGTATTTT 1247 CASSLEAGRTDTQYF TGCGCCAGCAGCCGGGACCGACAGGGAGAGACGGATGAGCAGTTCTTC 1240 CASSRDRQGETDEQFF TGTGCCAGCAGCTTAGTTGCGGGAAAAGAGACCCAGTACTTC 1192 CASSLVAGKETQYF TGTGCCTGGAGTGCCCAAATTTCGGGTCAGCCCCAGCATTTT 1171 CAWSAQISGQPQHF TGTGCCAGCAGCCCCCAGACAGGGACGAGCAATCAGCCCCAGCATTTT 1059 CASSPQTGTSNQPQHF TGTGCCACCAGCAGAGCCCCGGACGGACCCTACGAGCAGTACTTC 1037 CATSRAPDGPYEQYF TGCAGCGTAGCCGGGACAGGGGCCGAGACCCAGTACTTC 990 CSVAGTGAETQYF TGCGCCAGCAGCCAAGGGGGGTCCACTGCTTTCTTT 979 CASSQGGSTAFF TGTGCCAGCAGCCCATACAGGATTCGGACGAACACCGGGGAGCTGTTTTTT 890 CASSPYRIRTNTGELFF TGTGCCTGGAGTGTAGCGGGAGAGACCCAGTACTTC 853 CAWSVAGETQYF TGTGCCAGCAGCCGCCAGACAGGGCGGAACACTGAAGCTTTCTTT 799 CASSRQTGRNTEAFF TGTGCCAGCAGCTTAGTCCGATCTGGAAACACCATATATTTTACCEPTED 766 CASSLVRSGNTIYF TGCAGCTCAGCTCGGGACACAGATACGCAGTATTTT 759 CSSARDTDTQYF TGCAGCCCCCTGGCTAAAGAGACCCAGTACTTC 755 CSPLAKETQYF TGTGCCAGCAGCTTAACCCCGTTAGGGGGAGGCACAGATACGCAGTATTTT 730 CASSLTPLGGGTDTQYF TGTGCCAGCAGCCGACCCCGAGGTGACTCCTCCTACGAGCAGTACTTC 713 CASSRPRGDSSYEQYF TGTGCCAGCAGCCCCGGGGGAGCGGGATTTCGGGAGACCCAGTACTTC 707 CASSPGGAGFRETQYF TGCAGTGCTAGGGACAACATGAACACTGAAGCTTTCTTT 707 CSARDNMNTEAFF TGTGCCAGCAGCTTAGTACCTAGCGGGAACTCCTACGAGCAGTACTTC 705 CASSLVPSGNSYEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGGGGGGACTAGCGGGAGAGGGGACCCAGTACTTC 22581 CAWGGLAGEGTQYF TGCAGCGCTAGCGGGAGGCGCTACAATGAGCAGTTCTTC 18305 CSASGRRYNEQFF TGTGCCAGCAGCGCCGGACAGGGCCCCTACGAGCAGTACTTC 14208 CASSAGQGPYEQYF TGTGCCAGCAGCTCTCGCCTCGGCGAGCAGTACTTC 12246 CASSSRLGEQYF TGCGCCAGCACCTTAACGGCCGCGGGAGGAGGCACAGATACGCAGTATTTT 9508 CASTLTAAGGGTDTQYF TGTGCCAGCAGCCCCTTGACAGGGGGGATGACTGAAGCTTTCTTT 6217 CASSPLTGGMTEAFF TGCAGTGCCCCGACTAGCAGCACAGATACGCAGTATTTT 5813 CSAPTSSTDTQYF TGCGCCAGCAGCCAAGATCTCAACCCAACTAATGAAAAACTGTTTTTT 5129 CASSQDLNPTNEKLFF TGCAGTGCTAGTTTGGATAATCTAAAATATGGCTACACCTTC 4446 CSASLDNLKYGYTF TGTGCCAGCAGACCTTCGTCCGCAGGGGGATCTGGAAACACCATATATTTT 4055 CASRPSSAGGSGNTIYF TGTGCCTGGAACGCAGGGGGCATAAACACTGAAGCTTTCTTT 3904 CAWNAGGINTEAFF TGCGCCAGCAGCTCTCCGTCTAGCGGGATAACGGATACGCAGTATTTT 3171 CASSSPSSGITDTQYF TGTGCCAGCAGCTCCTATAGCGGGAAGAACACCGGGGAGCTGTTTTTT 2912 CASSSYSGKNTGELFF TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 2741 CASSLDPGTGGYGYTF TGTGCCAGCACTTGGGGAGGAAACACAGATACGCAGTATTTT 2710 CASTWGGNTDTQYF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 2503 CSVHRDPNSHEQFF TGTGCCAGCAGTGGCGGACAGGTATCGCGCCACGAGCAGTACTTC 2341 CASSGGQVSRHEQYF TGCAGTGCTAGAGTTCCCCCGAACACTGAAGCTTTCTTT 2006 CSARVPPNTEAFF CGTGCCAGCAGCTTAGGACTAGCGGACAATGAGCAGTTCTTC 1791 RASSLGLADNEQFF TGTGCCAGCAGCCTTGGGACAGGGGCGTTCTATGGCTACACCTTC 1658 CASSLGTGAFYGYTF TGCAGTGCTACCACCCACAGTAGACAGGGTTACACTGAAGCTTTCTTT 1554 CSATTHSRQGYTEAFF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 1512 CSVLRDPNSYEQYF TGTGCCAGCAGCTTATCGGGGGAGACCCAGTACTTC MANUSCRIPT1430 CASSLSGETQYF TGTGCCAGCAGCTCCCGGACAGGGCTTTTACAGACCCAGTACTTC 1258 CASSSRTGLLQTQYF TGTGCCTGGAGTGTAACGTCGTCCCCTCCTTTTAACTATGGCTACACCTTC 1247 CAWSVTSSPPFNYGYTF TGCGCCAGCAGCCAAGATCGGGGAGGCTCCTACAATGAGCAGTTCTTC 1206 CASSQDRGGSYNEQFF TGTGCCAGCAGCGGGGGGACTAGCGGGAGTTTGGGTGAGCAGTTCTTC 1168 CASSGGTSGSLGEQFF TGTGCCAGCAGCTCATGGGGGACCTCTGGAAACACCATATATTTT 1092 CASSSWGTSGNTIYF TGTGCCGGCAGGCCAGGGGGTTACTTGAGCACTGAAGCTTTCTTT 846 CAGRPGGYLSTEAFF TGTGCCAGCAGCCACCGGAGCGGGACCGGTTACGAGCAGTACTTC 759 CASSHRSGTGYEQYF TGCAGTGCTAGGGGGGGGTCCACTGAAGCTTTCTTT 725 CSARGGSTEAFF TGTGCCAGCAGGGGGGCCTACGAGCAGTACTTC 619 CASRGAYEQYF TGTGCCAGCAGCTTAACACAGGGGTCACAGCAGTACTTC 611 CASSLTQGSQQYF TGTGCCAGCAGCTTAGTGGGGGCACTGAACACTGAAGCTTTCTTT 578 CASSLVGALNTEAFF TGTGCCAGCAGCTTGACAGTTAACTATGGCTACACCTTC 555 CASSLTVNYGYTF TGTGCCAGCAGCCCTTTACGGGAGAAAGGCGGGGAGCTGTTTTTT 511 CASSPLREKGGELFF TGTGCCAGCAGCCCATCGGGACAGGGGTCCTACGAGCAGTACTTC 438 CASSPSGQGSYEQYF TGTGCCAGCAGTTGGAAGAAAGAGTCAATTGAAGAGACCCAGTACTTCACCEPTED 432 CASSWKKESIEETQYF TGTGCCAGCAGCGACAGGGAAAGGGCGCCCCAGCATTTT 397 CASSDRERAPQHF TGTGCCAGCAGCTCGGGACAGGGGAGGCCCCAGCATTTT 394 CASSSGQGRPQHF TGTGCCAGCAGCTTAGGAGTGGGATTCACAAATGAGCAGTTCTTC 389 CASSLGVGFTNEQFF TGTGCCAGCAGTCCAGACAGGGAATGGGAGCAGTACTTC 338 CASSPDREWEQYF TGTGCCTGCCCCTCCGGGTCGCAAGGCTATGGCTACACCTTC 333 CACPSGSQGYGYTF TGTGCCAGCAGCCCCCGCCTGGGCACCTACGAGCAGTACTTC 327 CASSPRLGTYEQYF TGCAGCGTTGGAGGTCCTGAACAGCCCCAGCATTTT 309 CSVGGPEQPQHF TGTGCCTGGAGTGTAGGGACTGCCCTTGGAGAGCAGTACTTC 292 CAWSVGTALGEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAATCCTGGTGTAGTTACAGATACGCAGTATTTT 28173 CAWNPGVVTDTQYF TGCAGTGCTAGAAGGGGCGACACTGAAGCTTTCTTT 27297 CSARRGDTEAFF TGTGCCAGCAGCTTAGTGGGGGCACTGAACACTGAAGCTTTCTTT 17916 CASSLVGALNTEAFF TGTGCCAGCACTTTATACGGGACAGGGGGCTATGGCTACACCTTC 9190 CASTLYGTGGYGYTF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 8937 CSVHRDPNSHEQFF TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 7799 CASSFTGAMNTEAFF TGTGCCAGCACCAGGACTAGCGATACGCAGTATTTT 7085 CASTRTSDTQYF TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 6352 CASSLDPGTGGYGYTF TGTGCCAGCAGTTACAGGGGGCCGGTTAGCGAGACCCAGTACTTC 4778 CASSYRGPVSETQYF TGTGCCAGCAGCCCTAGTGGAGGCACTGAAGCTTTCTTT 4232 CASSPSGGTEAFF TGTGCCAGCAGTTCAGGACAGGGGGTTACCGGGGAGCTGTTTTTT 3976 CASSSGQGVTGELFF TGCAGTGCTAGAGCCCCGTGGGGGGGAGATACGCAGTATTTT 3044 CSARAPWGGDTQYF TGCGCCAGCACCTTAACGGCCGCGGGAGGAGGCACAGATACGCAGTATTTT 2092 CASTLTAAGGGTDTQYF TGTGCCTGGAGTGTACGATACGAGCAGTACTTC 1540 CAWSVRYEQYF TGTGCCAGCAGCTCCCGGGTGGGACAGGGCAGTCAGCCCCAGCATTTT 1278 CASSSRVGQGSQPQHF TGCAGTGCTACGACAGGGGGCTCGCAGGTTAATGGCTACACCTTC 1264 CSATTGGSQVNGYTF TGTGCCAGCAGCGCTAGCGTAGCGGGGGTATACGAGCAGTACTTC 1106 CASSASVAGVYEQYF TGTGCCAGCACCGCTCGAGGGAACTATGGCTACACCTTC 897 CASTARGNYGYTF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 736 CSVLRDPNSYEQYF TGTGCCAGCAGCTTAGGAGGGCAGAGACCCAGTACTTC 718 CASSLG_AETQYF TGCAGCGTTTTAAAGGACCCAAACTACAATGAGCAGTTCTTC 614 CSVLKDPNYNEQFF TGTGCCTGGAGTGGATCCAATCAGCCCCAGCATTTT 531 CAWSGSNQPQHF TGTGCCAGCAGTCGGGACAGGGGGGATCAGCCCCAGCATTTT MANUSCRIPT443 CASSRDRGDQPQHF TGTGCCAGCAGTGACTGGTCCTACACCTTC 261 CASSDWSYTF TGTGCCTGCCAAGTCGCGGGAGGTTTAGGTCATGATGAGCAGTTCTTC 204 CACQVAGGLGHDEQFF TGTGCCAGCAGCAATCTCGGGGGCCCCAGAAGCGAGCAGTTCTTC 201 CASSNLGGPRSEQFF TGTGCCAGCAGCCTATACAGGAACACCGGGGAGCTGTTTTTT 167 CASSLYRNTGELFF TGTGCCAGCGGGAGCAGGGGAGTTGCAACTAATGAAAAACTGTTTTTT 144 CASGSRGVATNEKLFF TGCAGTGCTAGAGATGGGGGGACAGGGTTTTCACCCCTCCACTTT 144 CSARDGGTGFSPLHF TGCAGTGCTAGAGTGGGCGGAGCTTGGGGTGGCTACACCTTC 132 CSARVGGAWGGYTF TGTGCCAGCAGCCCCAGAACCGGGACTAGCGGGAGAAGAATGGAAGAGACCCAGTACTTC 112 CASSPRTGTSGRRMEETQYF TGTGCCGCGAGGGGGAGTGATACGCAGTATTTT 111 CAARGSDTQYF TGTGCCAGCAGTTTAACGGCCCATGCTAGCAATCAGCCCCAGCATTTT 104 CASSLTAHASNQPQHF TGCAGCGTTGGAAGGGACCCGAACACCGGGGAGCTGTTTTTT 93 CSVGRDPNTGELFF TGCGCCAGCAGCCAGCCTTGCCAGTGCAGTACTTC 88 CASSQP_PVQYF TGTGCCAGCAGCGCGTTAGGTGCGGGAGCTTCAGATACGCAGTATTTT 84 CASSALGAGASDTQYF TGTGCCAGCAGATGGGCTAGCACAGATACGCAGTATTTT 50 CASRWASTDTQYF TGTGCCAGCAGCCAAGATGGTCGGCAGGGGGGCACTGAAGCTTTCTTT 42 CASSQDGRQGGTEAFF TGTGCCAGCAGTTCCCCTAGCGGGAGTGGATACAATGAGCAGTTCTTCACCEPTED 41 CASSSPSGSGYNEQFF TGCAGTGCTAGTTCATACCGCGAGCAGTACTTC 35 CSASSYREQYF TGCAGTGCTCCGGGACAGGGAAATCAGCCCCAGCATTTT 30 CSAPGQGNQPQHF TGTGCCAGCAGCCTAGGACTAGCGGGAGATTACGAGCAGTACTTC 30 CASSLGLAGDYEQYF TGTGCCAGCAGCTTAGCTCGCACCGGACTAGGAAACAATACAATGAGCAGTTCTTC 30 CASSLARTG_RKQYNEQFF TGTGCCAGCAGCTTAGTTCTCCAAGAGGCCCCTTATTTT 29 CASSLVLQEAPYF TGCAGTGCTAGAGAGAGCTTATCCTACGAGCAGTACTTC 28 CSARESLSYEQYF TGTGCCAGCATTGCCCACCAAGGCGGAGGGCAGTACTTC 28 CASIAHQGGGQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGCAGTGCTAGAGGCACCGGTGGCGCCAGGGATTACACTGAAGCTTTCTTT 16754 CSARGTGGARDYTEAFF TGTGCCAGCAGCCCCCAGGGGGCTATCGAGCAGTACTTC 13946 CASSPQGAIEQYF TGTGCCAGCAGCCAAAGGCGGGAGGGGATCTACGAGCAGTACTTC 12405 CASSQRREGIYEQYF TGTGCCTGGAGTGTACAGGGGAACTATGGCTACACCTTC 11075 CAWSVQGNYGYTF TGTGCCTGGAGGGACCGGACAGGGGGCCGTAGCAATCAGCCCCAGCATTTT 9891 CAWRDRTGGRSNQPQHF TGTGCCAGCAGCTCCTATAGCGGGAAGAACACCGGGGAGCTGTTTTTT 9331 CASSSYSGKNTGELFF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 6818 CSVHRDPNSHEQFF TGTGCCAGCAGCCCAAAAGGGGGTTCCGGGGCCAACGTCCTGACTTTC 5603 CASSPKGGSGANVLTF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 5304 CSVLRDPNSYEQYF TGTGCCAGCAGCGGGCCCGGGACCGGGGAGACCCAGTACTTC 4166 CASSGPGTGETQYF TGTGCCTGGAGTGGGGGGTGGTACGAGCAGTACTTC 3082 CAWSGGWYEQYF TGCAGCGGGGGACAGGGGAACTATGGCTACACCTTC 2902 CSGGQGNYGYTF TGTGCCAGCAAATCACAGGGAGGGGAGACCCAGTACTTC 2838 CASKSQGGETQYF TGTGCCAGCAGCTCATCGCCCAACGAGGGGTATGGCTACACCTTC 2275 CASSSSPNEGYGYTF TGCGCCAGCAGCCAAGACTCCAGGGAAAAACTGTTTTTT 2197 CASSQDSREKLFF TGCAGCGTTGGACGGGACCCGAACACCGGGGAGCTGTTTTTT 1948 CSVGRDPNTGELFF TGCGCCAGCAGCCAAGAACGGGGGCTGCCCTACGAGCAGTACTTC 1892 CASSQERGLPYEQYF TGTGCCAGCAGCTTAGCGCTTAGCGGGAAGGGTCAAGAGACCCAGTACTTC 1832 CASSLALSGKGQETQYF TGTGCCAGCAGCTTAAGGGACGCCGGGTATGGCTACACCTTC 1822 CASSLRDAGYGYTF TGTGCCAGCAGATTCTCTGGGGCCAACGTCCTGACTTTC 1739 CASRFSGANVLTF TGTGCCAGCACCCGAGGCCCTGAAGCTTTCTTT 1649 CASTRGPEAFF TGTGCCAGCCAAAATCAGGCGGCGAAGGTTGAGACCCAGTACTTC 1539 CASQNQAAKVETQYF TGCAGTGCTGGGGGGGACAGGGGGTTTAGAGGTGGCACTGAAGCTTTCTTTMANUSCRIPT1425 CSAGGDRGFRGGTEAFF TGTGCCAGCAGCCTAGACTCAAACACCGGGGAGCTGTTTTTT 1312 CASSLDSNTGELFF TGTGCCTGGGCGGCGGGGACAGCTTTGTACGAGCAGTACTTC 1248 CAWAAGTALYEQYF TGCGCCAGCAGCCAAGATCTTCCCGGGACAGGGTATTACGCTGAGCAGTTCTTC 1238 CASSQDLPGTGYYAEQFF TGTGCCAGCAGTCGGACTAGCGGGAACGGCGAGCAGTACTTC 1156 CASSRTSGNGEQYF TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1095 CASSFTGAMNTEAFF TGTGCCAGCAGGGGAGGTGGGGCCAACGTCCTGACTTTC 1019 CASRGGGANVLTF TGTGCCAGCAGCTTAGGACAGGGAGGTCAGCCCCAGCATTTT 995 CASSLGQGGQPQHF TGTGCCAGCAGCCCAAGTAGCGGGAGAGTGAACACCGGGGAGCTGTTTTTT 959 CASSPSSGRVNTGELFF TGCGCCAGCAGTTCGGACCCTTCCTCCTCAGATACGCAGTATTTT 835 CASSSDPSSSDTQYF TGTGCCAGCAGCCACACGGGTCCGGGGGAGCAGTACTTC 769 CASSHTGPGEQYF TGTGCCAGCAGCTTCCTAGCGGGTACCGTTAGGGGCGAGCAGTACTTC 749 CASSFLAGTVRGEQYF TGTGCCAGCAGCTTAGGACAGGGACTAGCCAAAAACATTCAGTACTTC 730 CASSLGQGLAKNIQYF TGTGCCAGCAGCCTCTCGAGGCTAGCGGGAGGGAGTAACACCGGGGAGCTGTTTTTT 576 CASSLSRLAGGSNTGELFF TGTGCCAGCAGCTTAGACCCCGGGACAGGAGGCTATGGCTACACCTTC 570 CASSLDPGTGGYGYTF TGTGCCTGGAGTCGGCAGGGGGCGAGTAAGAAACTGTTTTTTACCEPTED 552 CAWSRQGASKKLFF TGTGCCAGCAGGGATGCGGGGGGTGGGGCTTACAATGAGCAGTTCTTC 547 CASRDAGGGAYNEQFF TGTGCCAGCACCCGGGACAGGGGGCGGTGGGAGCAGTACTTC 531 CASTRDRGRWEQYF TGCGCCAGCAGCCAAGGGAGCGGTGGATTTTATGGCTACACCTTC 529 CASSQGSGGFYGYTF TGTGCCAGCAGCTACTCCAATACAGGGAGCTACGAGCAGTACTTC 487 CASSYSNTGSYEQYF TGTGCCAGCAGCTTAGCAGGTGAAGCTTTCTTT 441 CASSLAGEAFF TGTGCCAGCAGCTTATTCACAGATACGCAGTATTTT 402 CASSLFTDTQYF TGTGCCAGCAGCTTATTTCAGGACACCGGGGAGCTGTTTTTT 392 CASSLFQDTGELFF TGTGCCAGCAGCGTAGGTGTCAGGGGGGGCGAGCAGTACTTC 359 CASSVGVRGGEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCTTGATGTGGGAGCAGTACTTC 27446 CASSLMWEQYF TGTGCCTGGAGTGGAACAGGGAGGAGTGAGCAGTTCTTC 22505 CAWSGTGRSEQFF TGTGCCAGCAGCTTGACTAGCGGGGGCTCCTACGAGCAGTACTTC 9382 CASSLTSGGSYEQYF TGTGCCAGCAGTTGGACAGGGGGCGTTTACGAGCAGTACTTC 8999 CASSWTGGVYEQYF TGTGCCAGCAGCTGGGGACAGGGGCCCTATGGCTACACCTTC 8588 CASSWGQGPYGYTF TGTGCCAGCCTAAACAGGGGGCGCGGCACTGAAGCTTTCTTT 8536 CASLNRGRGTEAFF TGCAGCGTACTCCGGGACCCTAATTCCTACGAGCAGTACTTC 8032 CSVLRDPNSYEQYF TGCGCCAGCAGCCAAGGCAGGAACACCGGGGAGCTGTTTTTT 7069 CASSQGRNTGELFF TGTGCCAGCAGCTTTTCATCATCATACTCTGGAAACACCATATATTTT 6826 CASSFSSSYSGNTIYF TGCGCCAGCAGCCAAGAGGGCGGCGAGCGCTTCAATGAGCAGTTCTTC 4006 CASSQEGGERFNEQFF TGCGCCAGCAGCTTGGCCGGGACAGGGAACACTGAAGCTTTCTTT 3059 CASSLAGTGNTEAFF TGTGCCTGGAAACTAGCGGGAGTCGAGCAGTACTTC 3050 CAWKLAGVEQYF TGCAGCGTACACCGGGACCCGAACTCCCATGAGCAGTTCTTC 2305 CSVHRDPNSHEQFF TGTGCCAGCAGCTTAGGACCTTACACCGGGGAGCTGTTTTTT 2144 CASSLGPYTGELFF TGTGCCTGGAGTTCAGGGACAGGGCTTCGCGGCTACACCTTC 1942 CAWSSGTGLRGYTF TGTGCCAGCAGCTTTTACACAGATACGCAGTATTTT 1884 CASSFYTDTQYF TGTGCCTGGAACCTCAGGTTGGCAGAGACCCAGTACTTC 1830 CAWNLRLAETQYF TGTGCCAGCAGCTTTACAGGAGCAATGAACACTGAAGCTTTCTTT 1758 CASSFTGAMNTEAFF TGTGCCAGCAGCTTAGAGGGGTCTAGCGGGAGGCAGACCCAGTACTTC 1656 CASSLEGSSGRQTQYF TGCAGTGCTAGAAGGGGCGACACTGAAGCTTTCTTT 1606 CSARRGDTEAFF TGTGCCAGCAGCCGGGCATATCGGGGGTATGGCTACACCTTC 1432 CASSRAYRGYGYTF TGTGCCAGCAGCGAGAGACAGGGTCTCTTCAGTGGCTACACCTTC 1257 CASSERQGLFSGYTF TGTGCCAGCCAAAATCAGGCGGCGAAGGTTGAGACCCAGTACTTC 1121 CASQNQAAKVETQYF TGTGCCTGGAGCCCAGGGGGGGCTATCAGTCAGCCCCAGCATTTT 1120 CAWSPGGAISQPQHF TGCGCCAGCAGCCAAGAACGTGGGACTAGTGGCTCTTGGCAGTTCTTC MANUSCRIPT1091 CASSQERGTSGSWQFF TGTGCCAGCACCCCCCAAGGGACGGGAGAAAAAGCTTTCTTT 985 CASTPQGTGEKAFF TGTGCCAGCAGCTTGGACTACCAAGAGACCCAGTACTTC 952 CASSLDYQETQYF TGTGCCTGGAGCCCTCCAGGGCCAGATACGCAGTATTTT 940 CAWSPPGPDTQYF TGTGCCAGCAGCAGACAGGGGGCCGTTCACGAGCAGTACTTC 940 CASSRQGAVHEQYF TGTGCCTGGAGTGTTCGGCGCACAGATACGCAGTATTTT 925 CAWSVRRTDTQYF TGTGCCAGCAGCTCACAGGGGGCGATGGAGACCCAGTACTTC 883 CASSSQGAMETQYF TGCGCCAGCAGCTTGGAGACAGGGCAATGGGGTTCACCCCTCCACTTT 838 CASSLETGQWGSPLHF TGTGCCAGCAGCTCCTCTGGAAACACCATATATTTT 764 CASSSSGNTIYF TGCGCCAGCCGAAGACAGAATATGAACACTGAAGCTTTCTTT 737 CASRRQNMNTEAFF TGCAGTGCTAGTGGACAGGCCGGCGAGCAGTACTTC 712 CSASGQAGEQYF TGTGCCAGCAGCTTGAATTCGCGGGAGAGGGAAGAGACCCAGTACTTC 710 CASSLNSREREETQYF TGTGCCAGCAGCGATACAGGGGCCTATAATTCACCCCTCCACTTT 668 CASSDTGAYNSPLHF TGTGCCAGCAGCCTGACTAGCGGGTCCTACAATGAGCAGTTCTTC 639 CASSLTSGSYNEQFF TGCAGTCTAGCGGGAGAGGTGTGGGAGACCCAGTACTTC 606 CSLAGEVWETQYF TGTGCCACCAGCAGAGAAGGACAGGTGTATGGCTACACCTTC 587 CATSREGQVYGYTF TGCAGTGCTAGAAAGGAGGCGTTTAGCTCCTACAATGAGCAGTTCTTC 572 CSARKEAFSSYNEQFF TGTGCCAGCAGCAATGGACTAGCCAGCACGAATGAGCAGTTCTTCACCEPTED 559 CASSNGLASTNEQFF TGTGCCAGCGCTCCCCCCCGAGCTTCGGCTAATCAGCCCCAGCATTTT 526 CASAPPRASANQPQHF TGCAGCGTTGCTGGACAGGGGGTGGGCTACACCTTC 505 CSVAGQGVGYTF TGCGCCAGCAGACCAGGGACTATGAACACTGAAGCTTTCTTT 473 CASRPGTMNTEAFF TGCGCCAGCAGCCAAGCCCTAACCGGTGAGCAGTTCTTC 425 CASSQALTGEQFF TGCGCCAGCAGCCGCTTCCCGGGACTAGCGGGAGAGCCTAAGTCTAGCACAGATACGCAGTATTTT 424 CASSRFPGLAGEPKSSTDTQYF TGTGCCAGCAGGCGACAGGACAATCAGCCCCAGCATTTT 421 CASRRQDNQPQHF TGTGCCAGCAGCTTAAAGGGGCGACTAGCGGGAGGGCGGGAGCAGTTCTTC 350 CASSLKGRLAGGREQFF TGCAGCGTTTTAAAGGACCCAAACTACAATGAGCAGTTCTTC 348 CSVLKDPNYNEQFF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCTGGAGTGTTGGGTCAAACAGCAATCAGCCCCAGCATTTT 1690 CAWSVGSNSNQPQHF TGTGCCTGGAGTGTACGGGAGGCGTCCCAACCCAGTTTT 1396 CAWSVREASQPSF TGTGCCTGGAGTCTGCCGGACACTGAAGCTTTCTTT 1157 CAWSLPDTEAFF TGTGCCTGGAGTGTCAGAAGCCAAGATACGCAGTATTTT 896 CAWSVRSQDTQYF TGTGCCTGGAGTGTGAGGGGCAGGGGATGGACTGAAGCTTTCTTT 867 CAWSVRGRGWTEAFF TGTGCCAGCAGCCATGACACGAAGCTTTCTTT 820 CASSH_HEAFF TGTGCCTGGAGCAAAACAGGGGGCAGTGAGCAGTTCTTC 806 CAWSKTGGSEQFF TGTGCCTGGACCCAGGCTAGGAGGCAGCGGGAGACCCAGTACTTC 728 CAWTQARRQRETQYF TGTGCCAGCAGCTTAGCGAAGAGCGGGAGGGTTAACGAGCAGTACTTC 707 CASSLAKSGRVNEQYF TGTGCCTCCTCCCACGGCGGAGATCAGCCCCAGCATTTT 659 CASSHGGDQPQHF TGTGCCTGGAGTGTACAACAGGGGATGGAGTACTTC 650 CAWSVQQGMEYF TGTGCCAGCAGTTTCAAGGGGTTTTTT 618 CASSFKGFF TGTGCCTCGACTTCCGGGACAGGGATTGGAAACACCGGGGAGCTGTTTTTT 614 CASTSGTGIGNTGELFF TGTGCCTGGAGCGGGGGCAGCCTCGGAGGGAATCAGCCCCAGCATTTT 608 CAWSGGSLGGNQPQHF TGTGCCTGGGGCAGGGGCAGCTATGGCTACACCTTC 600 CAWGRGSYGYTF TGTGCCTGGAGTACTAGCGGGCACGCGAACACCGGGGAGCTGTTTTTT 597 CAWSTSGHANTGELFF CGTGCCAGCAGCCCCCGCCGGGCTAGCGGGGCCAGCTCCTACAATGAGCAGTTCTTC 592 RASSPRRASGASSYNEQFF TGTGCCTGGAGTGTGGGAGGCTACACCTCTGGAAACACCATATATTTT 590 CAWSVGGYTSGNTIYF TGTGCCTGGAGTGTATCGGGTTTGGGGTCTACGGAGACCCAGTACTTC 578 CAWSVSGLGSTETQYF TGTGCCTGGAGTGTCCGGGAGGCAGATACGCAGTATTTT 576 CAWSVREADTQYF TGTGCCTGGAGGGGGGCGGTACAAGAGACCCAGTACTTC 559 CAWRGAVQETQYF TGTGCCTGGAGTGTGGGGAGGGCGTGGGAGACCCAGTACTTC 551 CAWSVGRAWETQYF TGTGCCAGCAGCTTAGGGGTCGGGACAGGGTTCAGTCAGCCCCAGCATTTT 548 CASSLGVGTGFSQPQHF TGTGCCAGCAGCTTAGGCGGCTTCCGGGATCACTGAAGCTTTCTTT 543 CASSLGGF_GITEAFF TGTGCCAGCACCCCGGGACTAGCGGGAGGAACCGGGGAGCTGTTTTTT 528 CASTPGLAGGTGELFF TGTGCCTGGTCAACGGGAGCGGTCGAGCAGTACTTC MANUSCRIPT510 CAWSTGAVEQYF TGTGCCTGGAAACCACCGCCCCAGCGGTACTACGAGCAGTACTTC 507 CAWKPPPQRYYEQYF TGTGCCTGGAGTGTACGGGACAGGGTAGGCTATGGCTACACCTTC 506 CAWSVRDRVGYGYTF TGTGCCTGGAGTGTACGTCAGCGGGGACTCCCCTACGAGCAGTACTTC 500 CAWSVRQRGLPYEQYF TGTGCCTGGAGTGTACGGGCAACAGGGAGGGCCTATGGCTACACCTTC 500 CAWSVRATGRAYGYTF TGTGCCAGCGCGCCCCCCTCGTCAGCCCCAGCATTTT 496 CASAPP_RQPQHF TGTGCCTGGAGAGCGGCAGGGGCTCACTACGAGCAGTACTTC 491 CAWRAAGAHYEQYF CGTGCCAGCAGCTTAACCTGGGCGAGCACAGATACGCAGTATTTT 489 RASSLTWASTDTQYF TGTGCCTGGCGCAGGGAGGCTGAAGCTTTCTTT 485 CAWRREAEAFF TGTGCCTGCCCGGTGGGGGACCTAGCTGGGGTGAACACTGAAGCTTTCTTT 476 CACPVGDLAGVNTEAFF TGTGCCTGGAGTGATCCCCTAGCGGGAGGCCACGAGTACTTC 466 CAWSDPLAGGHEYF TGTGCCACCAGCAGATTAGGGACGGCAGTATATGGCTACACCTTC 465 CATSRLGTAVYGYTF TGTGCCTGGTGGCAGGGTGAATCCTACGAGCAGTACTTC 451 CAWWQGESYEQYF TGTGCCTGGAGTGGTCCAGGGACCAACGTCCTGACTTTC 443 CAWSGPGTNVLTF TGTGCCTGGAGCCCCGGGACCTCACCCCTCCACTTT 441 CAWSPGTSPLHF TGTGCCTGCTCAAGTCGCTACGAGCAGTACTTC 435 CACSSRYEQYF TGTGCCAGCAGCTACTCCAATACAGGGAGCTACGAGCAGTACTTCACCEPTED 434 CASSYSNTGSYEQYF TGTGCCTGGACCACGGGACAGGCCTCCTACGAGCAGTACTTC 431 CAWTTGQASYEQYF TGTGCCTGGAGTGTCTTGGGGACCCAGTACTTC 427 CAWSVLGTQYF TGTGCCTGGAGTGTTTTACAGGGATTCGCGAACACCGGGGAGCTGTTTTTT 409 CAWSVLQGFANTGELFF TGTGCCTGGAGTCAGAGAAGGGCCACTGAAGCTTTCTTT 400 CAWSQRRATEAFF TGTGCCTGGGGCCTCGGGGACAGACCCTCCTACGAGCAGTACTTC 384 CAWGLGDRPSYEQYF TGTGCCTGGACACCAGGAGCGCTTATTGAAGCTTTCTTT 380 CAWTPGALIEAFF TGTGCCTGTCTTCGGGACAGGGCACCCAACTATGGCTACACCTTC 378 CACLRDRAPNYGYTF TGTGCCTGGAGGCCCGGACAGGACCCTACCTACGAGCAGTACTTC 365 CAWRPGQDPTYEQYF ACCEPTED MANUSCRIPT
Clone count CDR3 AA Seq CDR3 DNA Seq TGTGCCAGCAGCTTTCGGACTAGCGGCGAGCAGTACTTC 2677 CASSFRTSGEQYF TGTGCCAGCAGCCCCCCCCGCGGGGTCCGGGAGCTGTTTTTT 1392 CASSPPRGVRELFF TGTGCCTGGAGTGTTTGGGGGACCCGGAACACTGAAGCTTTCTTT 979 CAWSVWGTRNTEAFF TGTGCCTGGCAGAAACAGGACAACTATGGCTACACCTTC 885 CAWQKQDNYGYTF TGTGCCAGCAGCCAAGAGCCAGGGCACAATCAGCCCCAGCATTTT 840 CASSQEPGHNQPQHF TGTGCCTGGAGTGTGGACAGTGGCAATCAGCCCCAGCATTTT 769 CAWSVDSGNQPQHF TGTGCCTGGAGTGTACTGGCTGCAGGCCAAGAGACCCAGTACTTC 706 CAWSVLAAGQETQYF TGTGCCAGCAACAAAGCGGCAAACCAAGAGACCCAGTACTTC 694 CASNKAANQETQYF TGTGCCTGGAGTGTACGTGACAGGGAGGTCATTGAAGCTTTCTTT 647 CAWSVRDREVIEAFF TGTGCCTGGAGCAAAACAGGGGGCAGTGAGCAGTTCTTC 635 CAWSKTGGSEQFF TGCGCCAGCAGCCAGTTAGGGCAGTACACCGGGGAGCTGTTTTTT 558 CASSQLGQYTGELFF TGTGCCAGCAGCTTAGGAACAGAACCATCCTACGAGCAGTACTTC 478 CASSLGTEPSYEQYF TGTGCCTGGAGTACCCGGACCTACCAGGGAGAATTC 442 CAWSTRTYQGEF TGTGCCAGCAGCTTAGCGAGCCTAGCGGGAGAGCAGTACTTC 440 CASSLASLAGEQYF TGTGCCAGCAGCCCCGGGACAGTCACAGATACGCAGTATTTT 412 CASSPGTVTDTQYF TGTGCCTGGAGCGTCGGGCCCGGCCTGAACACTGAAGCTTTCTTT 408 CAWSVGPGLNTEAFF TGTGCCTGGACAAGACTCGGAAACACCATATATTTT 406 CAWTRLGNTIYF TGTGCCTGGAGACCTAGGACCAGCGGGGAGCTGTTTTTT 404 CAWRPRTSGELFF TGTGCCTGGGCGACAGGGGAAAATCAGCCCCAGCATTTT 403 CAWATGENQPQHF TGTGCCTGGAGTGTCCGCGGAGAGACCCAGTACTTC 391 CAWSVRGETQYF TGTGCCAGCAGCTCAGGGGCTAGCCCTCAAGAGACCCAGTACTTC 372 CASSSGASPQETQYF TGTGCCTGGAGTGTCGTTCTAGCGGGATTAGAGACCCAGTACTTC 355 CAWSVVLAGLETQYF TGTGCCAGCAGTCCCTTACTGAACACTGAAGCTTTCTTT 343 CASSPLLNTEAFF TGTGCCAGCAGCTCCTCGAATGAAAAACTGTTTTTT 331 CASSSSNEKLFF TGCGCCAGCAGCCAAATTGGGGAGACCCAGTACTTC MANUSCRIPT325 CASSQIGETQYF TGTGCCAGCAGCCGGCCAGCAACGAACACTGAAGCTTTCTTT 311 CASSRPATNTEAFF TGTGCCTGCCGTGGCGGACAGGGGAACTATGGCTACACCTTC 307 CACRGGQGNYGYTF TGTGCCTGGATAGCGGCGGGGAGAGAGACCCAGTACTTC 306 CAWIAAGRETQYF TGTGCCTGGAGTGGGGTTTCCAACACTGAAGCTTTCTTT 300 CAWSGVSNTEAFF TGTGCCAGCAGCCCTCGTCCCCGTGAGCAGTTCTTC 299 CASSPRPREQFF TGTGCCAGCAGCGAGGGGACAGGGAACTATGGCTACACCTTC 299 CASSEGTGNYGYTF TGTGCCTGGAGGGTAGCGGGAGGCCTTCTAGAGCAGTACTTC 295 CAWRVAGGLLEQYF TGTGCCAGCAGCCCCCCGGACAGCGGCTTACCCAATCAGCCCCAGCATTTT 288 CASSPPDSGLPNQPQHF TGTGCCTGGAGTGTCCTACAAGAGACCCAGTACTTC 288 CAWSVLQETQYF TGTGCCAGCAGCTTAAACAGGGGCTCCTACGAGCAGTACTTC 282 CASSLNRGSYEQYF TGTGCCTGGAGTAGCGGGCTAGCGGGCGGGGAGCTGTTTTTT 275 CAWSSGLAGGELFF TGCAGCGTTGAAAGGGGGTCCAATGAGCAGTTCTTC 267 CSVERGSNEQFF TGTGCCTGGCGTGGGGACAGATACTACGAGCAGTACTTC 267 CAWRGDRYYEQYF TGTGCCTGGAGTGTAGGCGGGGGAGGAGATACGCAGTATTTT 262 CAWSVGGGGDTQYF TGTGCCAGCAGCTCCCGACTAGCCTCCGAGCAGTACTTC 257 CASSSRLASEQYF TGTGCCAGCGGGACTAGCGGAGGCACAGATACGCAGTATTTTACCEPTED 255 CASGTSGGTDTQYF TGTGCCTGGAGACCTACCGGTGCGCGAGATACGCAGTATTTT 254 CAWRPTGARDTQYF TGTGCCTGGATGGTGTCCTATGGCTACACCTTC 254 CAWMVSYGYTF TGTGCCAGCAGCCAAGCGGAAAGCAATCAGCCCCAGCATTTT 254 CASSQAESNQPQHF TGTGCCAGCAGCGCCCGACTAGCGGAAGAGACCCAGTACTTC 248 CASSARLAEETQYF TGTGCCTGGAGAGACTACCGGGACAGGGGGAACACCTTC 247 CAWRDYRDRGNTF TGTGCCAGCAGCTTCGGGGTGTGGAATTCACCCCTCCACTTT 242 CASSFGVWNSPLHF TGTGCCAGCAGCTTGGGGCTAGCGGAAGATACGCAGTATTTT 241 CASSLGLAEDTQYF TGTGCCAGCAGCTTAGCGGGGGGACTAACCTACGAGCAGTACTTC 240 CASSLAGGLTYEQYF ACCEPTED MANUSCRIPT Supplementary Table 5. Clinical characteristics of Oxford cohort patients assessed in this study for gene expression analysis.
Characteristic Control UC CD (n=13) (n=31) (n=27) Male/female 3/9 13/17 11/16 Median (IQR) age at sampling (years) 58 48 37 (41–65) (35–62) (24–61) Median (IQR) age at diagnosis (years) n/a 31 25 (24–39) (18–46) Median (IQR) disease duration (years) n/a 9 9 (3–23) (6–13) Median (IQR) C-reactive protein (mg/l) n/a 6.0 11.1 (1.1–14.9) (3.8–46.3) Median (IQR) peripheral blood leukocytes 8.8 8.3 8.0 (10^9/l) (8.3–9.0) (7.0–9.7) (6.5–10.7) Current medication at sampling 5-Aminosalicylates 21 1 Corticosteroids 8 1 Azathioprine/6-mercaptopurine 7 10 Infliximab/adalimumab 1 9 Unknown 4 7 Demographic and clinical characteristics of IBD patients analysed in Figure 5E and 7D
MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT Supplementary Table 6. Clinical characteristics of Oxford cohort patients assessed in this study for the analysis of microbiota-specific CD4 T cells.
Characteristic All patients UC CD (n=44) (n=20) (n=24) Male/female 24/20 10/10 14/10 Median (IQR) age at sampling (years) 41 42 ( 38.5 (31.5-54.75) 34-55.75) (31-52) Median (IQR) age at diagnosis (years) 31 39 26.5 (21-47) (25-55) (19.5-37.75) Median (IQR) disease duration (years) 7 4 12 (2-15) (1-12) (6-15.75) Median (IQR) C-reactive protein (mg/l) 2.1 1.3 2.6 (0.9-4.425) (0.85-3.56) (0.92-4.65) Median (IQR) peripheral blood 7.065 7.1 6.9 leukocytes (10^9/l) (5.4-8.11) (5.35-9.11) (5.33-8) Current medication at sampling 5-Aminosalicylates 10 2 Corticosteroids 4 2 Azathioprine/6-mercaptopurine 3 12 Infliximab/adalimumab 2 10 Unknown 3 1 Demographic and clinical characteristics of IBD patients analysed in Figure 7A, B and Supplementary 6A-H MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT Supplementary Table 7. Clinical characteristics of Oxford cohort patients assessed in this study for cell accumulation in the mucosa.
Characteristic Control UC CD (n=13) (n=8) (n=9) Male/female 6/7 6/2 4/5 Median (IQR) age at sampling (years) 32.5 29.5 24 (25–45.5) (25.75–39.5) (18.5–29.5) Median (IQR) age at diagnosis (years) 25 25 23.5 (22-46) (17.5–28.5) (19.5–33.75) Median (IQR) disease duration (years) n/a 4 1 (1–10.5) (0–6) Median (IQR) C-reactive protein (mg/l) 1.75 0.9 20.7 (0.15-8.3) (0.4–6.3) (6.3–43.2) Median (IQR) peripheral blood 7.02 6.73 6.12 leukocytes (10^9/l) (5.8–8.3) (6.018–8.54) (5.35–11.68) Current medication at sampling 5-Aminosalicylates 4 2 Corticosteroids 1 0 Azathioprine/6-mercaptopurine 1 4 Infliximab/adalimumab 0 1 Unknown or no treatment 2 4 Demographic and clinical characteristics of IBD patients analysed in Figure 3B, C and Figure 7C MANUSCRIPT
ACCEPTED ACCEPTED MANUSCRIPT
A Firmicutes Actinobacteria B **** * Clostridiaceae Bifidobacteriaceae 10 Clostridium difficile Bifidobacterium animalis Lactobacillaceae Lactobacillus acidophilus Mycobacteriaceae 1 Mycobacterium tuberculosis Lachnospiraceae
cells (%) 0.1 Roseburia intestinalis Bacteroidetes + ns Ruminococcus obeum Bacteroidaceae 0.01 Ruminococcaceae Bacteroides vulgatus
Faecalibacterium prausnitzii of CD4 + 0.001 Staphylococcaceae Fungi Staphylococcus aureus Saccharomycetaceae Candida albicans 0.0001 CD154 Proteobacteria 0.00001 Enterobacteriaceae Obligate aerobe Escherichia coli Facultative anaerobe LPS Salmonella typhimurium Obligate anaerobe C. difficile no microbe S. typhimurium C gated on live, CD3+ E. coli L. acidophilus B. vulgatus S. aureus no microbe 5 10 0.123 105 0.0146 105 2.63e-3 105 0.118 105 0 + D CD154 CD4+ T cells 4 - 10 104 104 104 104 CD154 100 3 10 103 103 103 103
2 80 10 102 102 102 102
0 0 0 0 0 60
3 4 5 0 10 10 10 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 S. typhimurium F. prausnitzii R. intestinalis C. albicans LPS 40 5 10 0.1 105 4.08e-3 105 4.17e-3 105 0.0752 105 4.84e-4 20
4 10 104 104 104 104 0 2 3 4 5 3 0 10 10 10 10 10 103 103 103 103 CD69 2 10 102 102 102 102 CD154 100 0 0 0 0 0
3 4 5 % of maximum 80 0 10 10 10 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
B. animalis C. difficile R. obeum M. tuberculosis SEB 60 105 0.0241 105 0.024 105 3.92e-3 105 0.0113 105 4.78
40 104 104 104 104 104
103 103 103 103 103 20
102 102 102 102 MANUSCRIPT102 0 2 3 4 5 0 0 0 0 0 0 10 10 10 10 ICOS 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CD4
S. typhimurium B. animalis L. acidophilus S. typhimurium C. difficile E G 0.08 0.03 100 ** 100 *** 100 ** *** ** 80 80 80 F 5 0.06 10 0.02 60 60 60 **** * 0.04 4 40 40 40 10 0.01 T cells (%)
+ microbe 0.02 20 20 20 Adult blood
microbe 3 cells (%) cells 10 +
+ Cord blood CD4 0 0 0 + α-MHCII 0 0.00 + R. obeum C. difficile F. prausnitzii S. aureus C. albicans 2 B. vulgatus R. intestinalis CD4 10 0.015 0.008 0.008 *** ** **** 6 ** 100 ** 100 100 100 * * of CD4 + /10 80 80 80 80 1 0.006 0.006 10 0.010
60 60 60 60 Microbiota-reactive cells
CD154 0.004 0.004 40 40 40 0 40 10 0.005 0.002 0.002 20 20 20 20 Reduction in CD154
0 0 ACCEPTED0 0 0.000 0.000 0.000 C. difficile
S. typhimurium
Supplementary Figure S1 E. coli S. typhimurium B. animalis L. acidophilus (3.84x) (6.6x) (2.5x)ACCEPTED(3.7x) MANUSCRIPT A 1 **** 1 **** 1 **** 1 **** B **** **** 0.1 0.1 0.1 0.1 100 90 0.01 0.01 0.01 0.01 80
0.001 0.001 0.001 0.001 70 60 0.0001 0.0001 0.0001 0.0001 50 F. prausnitzii C. difficile B. vulgatus R. intestinalis 40
(3.5x) (6.2x) (3.2x) (5.8x) Memory (%) 1 1 1 1 30 **** **** **** **** 3.5x 20 0.1 0.1 0.1 0.1 (%) 10 +
0.01 0.01 0.01 0.01 0
CD154 0.001 0.001 0.001 0.001 C. difficile
0.0001 0.0001 0.0001 0.0001 S. typhimuriumTotal CD4 T cells R. obeum S. aureus C. albicans M. tuberculosis SEB (4.3x) (10x) (6.2x) (32.5x) (0.6x) 1 1 1 1 100 **** **** **** **** **** 10 0.1 0.1 0.1 0.1 **** 1 0.01 0.01 0.01 0.01 0.1 0.001 0.001 0.001 0.001 0.01
0.0001 0.0001 0.0001 0.0001 0.001 CD154+ of CD154+ of naive T cells (CD45RA+) memory T cells (CD45RA-)
Memory CD4 T cells + + + - C (live CD3 CD4 CD154 CD45RA ) D CD4+ CD154+ CD45RA- T cells 100 CD4+ CD154- CD45RA- T cells B. animalis stimulation 90 105 93.6 2.6 95.1 1.3 51.8 44.7 62 2.7 55 1.1 33.4 31.6 80 104
70 103 60 0 50 MANUSCRIPT34.7 0.5 43.2 0.6 32 3.0 3.7 0.1 3.5 0.1 3.1 0.28 40 0 102 103 104 105 0 102 103 104 105 0 103 104 105 TNF- α (%) 30 Integrin β7 CCR9 CCR2
105 20 76.6 6.0 90.3 96.1 0.4 20 20.8 44.4 4.0 60.8 54.5 2.6 10 104
0 103 CCR6 80 0 9.6 25.1 2.1 33.1 39.6 3.2 70 1.6 1.6 0.07 3.6 3.4 0.06
2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 60 CCR4 CCR7 CCR10
50 105 87.6 9.1 83.7 10 17.4 79.3 55.6 9.0 56.6 7.6 16.5 48.7 40 104
3 IL-2 (%) 30 10 20 0 28.2 6.1 31.6 4.1 28.4 6.3 2.25 0.9 3.5 2.8 1.6 1.5
10 0 103 104 105 0 103 104 105 0 102 103 104 105 0 CXCR3 CXCR5 CD161 s
E. coli C. difficile R. obeumS. aureus B. animalis B. vulgatus C. albican F. prausnitzii R. intestinalis S. typhimurium L. acidophilus ACCEPTEDM. tuberculosis
Supplementary Figure S2 ACCEPTED MANUSCRIPT 105 37.4 50.6 105 82.3 1.13 105 55.6 3.11 105 87.1 3.09 105 12.1 0.16 A 4 4 4 4 4 10 10 10 10 10
3 3 3 3 10 3 10 10 10 10 LPMCs
2 102 10 102 + 0 0 0 CD4 T cells 0 0 2.89 9.12 14.5 2.05 41.2 0.06 9.62 0.16 84.7 3.02 0 102 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
105 105 105 105 105 CD69 CD69 0.1 0.9 0.26 0.42 CCR7 32.2 61.6 32.6 60.7 3.5 24.4 CD127 4 4 104 104 10 10 104 Integrin β 7
3 3 3 3 10 10 3 PBMCs 10 10 10 + 2 102 CD4 T cells 10 102 0 0 0 0 0 11.6 87.3 37.7 61.6 5.79 0.39 5.09 1.61 34.5 37.6 0 102 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 CCR7 CD45RA B D LPMCs S. typhimurium C. difficile
250K 5 250K 6000 5 1600 10 105 10 80.3 **** 200K 200K 4 1400 10 104 79.2 104 56.5 96.9 5000
150K 150K cells 3 1200 10 3 + 10 103 100K 85.4 4000 100K CD3 CD4 1000 2 SSC-A 10 FSC-H 102 0 0 50K 50K 0 CD4 3000 800
6 0 0
Live dead (EF780) 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 600 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 2000 FSC-A FSC-A FSC-A FSC-A FSC-A 400 1000 200 cells/10 Microbiota-reactive 105 8.44 4.38 105 40.3 9.06 0 0
4 Control mucosa 10 104 + E. coli LPS 3 5 5 Peripheral blood 10 10 10 103 CD154 0.36 0.007 IL-22 IFN- γ + 104 104 0 TNF-α 0
103 103 70.9 16.2 39.1 11.6 3 4 5 3 4 5
0 10 10 10 0 10 10 10 TNF- α TNF- α 102 102 0 0 IL-17A 2.24 0.731 Control mucosa 5 5 10 0 0 10 0 0 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 E Peripheral blood 4 10 104 CD154 CD154 + IL-17A 3 γ 3 IFN- 10 CD154 10 IL-22 + 60 100 15 - IFN- γ IL-22 TNF-α 0 0 90 * ns **** *** 50 100 0 100 0 80 0 103 104 105 0 103 104 105 CD154 IL-17A + 40 70 10 ns ns 60 gated on Live, 30 50 cells (%) PBMCs lymphocyte, singlets, + 40 + + C of TNF- α 20 5 5 5 10 10 MANUSCRIPTCD3 , CD4 + 1.57 0 48.8 7.09 30 CD4 4 10 104 E. coli LPS 20 + 105 105 10 103 3 CD154 10 4 4 10 + 10 10 IL-22 IFN- γ 102 TNF-α 0 3 0.028 3 0.0008 0 10 10 Cytokine 0 0 0
81.1 17.3 33.9 10.2 2 2 TNF- α 10 TNF- α 10 0 103 104 105 0 103 104 105 0 0 IL-17A 0.10 0.02 0 102 103 104 105 0 102 103 104 105 105 0 0 105 0.204 0 C. difficile C. difficile C. difficile CD154 CD154 4 10 104 + S. typhimurium S. typhimurium S. typhimurium 3 CD154 10 103 - IL-22 2 IFN- γ TNF-α 10 0 0 99.8 0.204 99.6 0.204 0 103 104 105 0 103 104 105 IL-17A
F IL-2 GMCSF IL-4
+ 100 60 20 Control mucosa * * ** ns ns ns ns ns ns ns ns ns ns **** ns ns ns ns ns ns 90 Peripheral blood 80 50
CD154 15 + 70 40 60 50 30 10 * * ns * ns ns ns ns * ns
cells (%) ACCEPTED + 40 of TNF- α + 30 20 CD4 5 20 10 10
Cytokine 0 0 0 s s s SEB SEB SEB E. coli E. coli E. coli C. difficileS. aureus C. difficileS. aureus C. difficileS. aureus B. animalis C. albican B. animalis C. albican B. animalis C. albican F. prausnitzii L. acidophilusF. prausnitzii F. prausnitzii L. acidophilus S. typhimurium M. tuberculosis S. typhimurium L. acidophilus M. tuberculosis S. typhimurium M. tuberculosis
Supplementary Figure S3 ACCEPTED MANUSCRIPT B E. coli 5 5 5 A ** ns * ** ** ** ** 105 17.7 1.1 10 30.2 7.7 10 5.86 0.9 10 0.28 0.4 50 4 4 4 104 10 10 10
3 3 10 103 10 ** *** ns ns ns ns ns 103 40 0 102 102 0 0 0 E. coli Nissle 24 57.2 11.5 50.6 35.9 57.4 41.4 57.9 IL-4 IL-17A IFN- γ IL-22 2 3 4 5 2 3 4 5 2 3 4 5 30 0 102 103 104 105 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CFSE 20 B. animalis
105 3.78 2.4 105 4.16 14.5 105 1.23 2.9 105 0.9 0.8
4 4 4 10 104 10 10 10
3 3 3 3 10 10 10 10
cells (%) 102 102 102
+ 0 0 0 0 0 8.2 85.7 6.3 75 10.2 85.7 11.1 87.2 IL-17A IFN- γ IL-22 IL-4 ** ** ** ** * ** * 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 0 102 103 104 105 80 CFSE of CD4
low ns ns ns ** ns ns ns F. prausnitzii 60 105 1.6 2.5 105 3.6 40.1 105 0.5 2.4 105 0.4 1.4
B. animalis 4 4 4 4 10 10 10 10 CFSE
3 3 3 3 10 10 10 10
40 2 10 2 2 0 10 10 0 0 0 IL-4 4.2 91.7 2.2 54.1 5.3 91.7 2.2 96 IL-17A IFN- γ IL-22 2 3 4 5 2 3 4 5 2 3 4 5 2 3 4 5 20 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 CFSE
0 D no microbe S. aureus Flu FMO control 5 5 5 10 0.07 2.95 10 48.2 0.14 10 0.25 0.6 105 0 0.02
4 4 4 10 10 10 104 E. coli C. difficile 3 3 3 no microbe B. animalis 10 10 10 103 E. coli Nissle L. acidophilusF. prausnitzii 2 2 2 S. typhimurium 10 10 10 102 FMO PE adherent invasive ROR γ t 0 0 0 0
0.09 96.9 40.2 11.4 28.1 71.1 86.7 13.3 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105
5 5 5 IL-17A IFN-γ 10 0.07 1.32 10 32.5 0.19 10 18.3 2 105 0.02 0 4 4 4 10 10 10 104
3 3 3 ns ns ns ns 10 10 10 103 ** *** 2 2 2 2 **** **** **** 10 10 10 **** C T-bet 10
100 100 FMO PB 0 0 0 0 90 90 0.08 98.5 55.9 11.4 9.91 69.7 86.5 13.5 2 3 4 5 2 3 4 5 2 3 4 5 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 80 80 5 5 5 10 0 0.3 10 0.1 0.03 10 0 0.4 105 0 0
70 70 4 4 4 10 MANUSCRIPT10 10 104 3 3 3 60 60 10 10 10 103
50 50 0 0 0 0 GATA-3 FMO APC FMO
40 40 0.2 99.4 88.4 11.5 28.6 71 86.9 13.1 2 3 4 5 2 3 4 5 2 3 4 5 30 30 0 10 10 10 10 0 10 10 10 10 0 10 10 10 10 0 102 103 104 105 CFSE 20 20 cells (%) + 10 10 E S. typhimurium L. acidophilus F. prausnitzii C. difficile 0 0 CD4
low IL-22 IL-4
ns ns ns * ns ns ns ns **** 50 **** 25 of CFSE + Flu S. aureus 40 20 RORγt + + + + - - - - Cytokine 30 15 T-bet + + - - + + - - GATA-3 + - + - + - + - 20 10 10 ns ns ns ** ** *** 10 5 105 30.1 7.34
104 8 ACCEPTED + F. prausnitzii 3 IL-10 0 0 10 F 5 10 high high 6 0 104 (%) 56.7 5.87 + 0 CFSE S. aureus CFSE S. aureus 103 C. albicans C. albicans 105 1 21.7 6.99 - IL-10 4 2 10 L
Tetanus toxoid Tetanus toxoid I M. tuberculosis M. tuberculosis 104 Influenza vaccine Influenza vaccine 0
3 - 0 102 103 104 105 10 IL-10 2 CD3 0 65.1 6.2 IFN- γ 0 103 104 105 0 IL-17A SEB E. coli B. animalis C. difficile F. prausnitzii S. typhimuriumL. acidophilus
Supplementary Figure S5 ACCEPTED MANUSCRIPT
S. typhimurium A S. typhimurium C. difficile B S. typhimurium C. difficile E 0.08 0.03 0.6 0.08 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2 ns ns * **** of ns **** 90 *** 90
**** + 60 80 80 0.06 0.06 30 cells (%) 0.02 0.4 50 70 70 + 60 60 0.04 40
0.04 TNF- α
cells (%) 50 50 + 20 + 0.01 0.2 30 40 40
of CD4 0.02 0.02 20 30 30 + CD4 10 20 20
CD154 10 0.00 0.00 0.0 0.00 10 10 Control (n=30) Control mucosa (n=17-20) 0 0 0 0 CD154 IBD (n=38) Inflamed mucosa (n=10-15) B. animalis 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2 90 ns 90 60 *** ** *** 80 80 30 C 50 70 70 15 40 60 60 14 *** E. coli SEB 50 20 50 D 13 + 100 100 30 40 40 *** * 12 90 90 20 30 30 10 11 80 80 20 20 10 10 70 70 10 10 CD154 + 9 60 60 0 0 0 0 8 50 50 cells (%)
+ L. acidophilus 7 40 40 cells (%)
+ 70 IL-17A 70 IFN-γ 40 IL-22 100 IL-2 6 30 30 CD4 5 of TNF- α ns ns 90
+ 20 20 60 **** 60 ****
(norm. to controls) 80 4 10 10 CD4
+ 30 50 50 70 3 IL-2 0 0 2 40 40 60
Memory CD4 T cell abundance T Memory CD4 0 Control (n=23) 20 50 30 30 IBD (n=33) CD154 40 Control Inflamed + 20 20 30 10 mucosa mucosa 20 10 10 10 0 0 0 0 of TNF- α S. aureus + F. prausnitzii F 100 IL-17A 100 IFN-γ 70 IL-22 100 IL-2 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2 ns 90 90 ** *** 90 * 90 ns ns 90 60 60 * ** 80 80 80 Cytokine 80 80 30 70 70 50 70 50 70 70 60 60 60 40 MANUSCRIPT40 60 60 50 50 50 50 20 50
cells (%) 30 30 + 40 40 40 40 40 30 30 20 30 20 30 30 10 20 20 20
CD4 20 20
+ 10 10 10 10 10 10 10 0 0 0 0 0 0 0 0 M. tuberculosis
CD154 C. difficile
+ IL-17A IFN-γ IL-22 IL-2 70 100 40 100 70 IL-17A 100 IFN-γ 40 IL-22 100 IL-2 ns ns ns *** 90 90 90 ns ns 90 60 60 ** **** 80 80 80 80 30 30 50 70 70 50 70 70 of TNF- α
+ 60 60 40 40 60 60 50 20 50 50 20 50 30 40 40 30 40 40 20 30 30 20 30 30 10 10 Cytokine 20 20 20 20 10 10 10 10 10 10 0 0 0 0 0 0 0 0 Control (n=23) Control (n=9-17) IBD (n=33) IBD (n=15-22)
G+ 50 50 50 *** **** ** ns ns *** E. coli B. animalis ns H 4 I Control (n=4) CD4 *** S. typhimurium L. acidophilus + **** ACCEPTED*** 40 40 40 ns IBD (n=6) 2.5 2.5 3 IL-17A ns IFN-γ *
CD154 30 30 30 ns 2.0 * 2.0 + * 2 1.5 1.5
cells (%) 20 20 20
IL-10 (%) 1.0 1.0 of TNF- α + 10 10 10 1 0.5 0.5
IL-17A 0 0 0 0 0.0 0.0 Control Control Cytokine (% of control) Control Control (n=9) − + + − + + Ulcerative colitis Active IBD Aminosalicylates IL-1β Crohn’s disease IBD remission Purine synthesis IBD (n=11) IL-6 − + + − + + inhibitors IL-23 − + + − + + Biologics
Supplementary Figure S6 ACCEPTED MANUSCRIPT Tc Tc Tc B cells Dendritic cells Epithelial cells Stromal cells Microbiota Circulation Bc Dc Tc Tc 1 T cells T T cells T T cells T + + + T cells T 4 3 Recruitment of Activated CD4 Activated CD4 (in inflammation) Naive CD4 Neutrophils Macrophages microbiota-specific 2 Activation of Tc Tc Tc T cells in tissue T Accumulation of tissue-resident cells Tc phenotype 5 Tc Tc Tc Tc Change in T cell T Change in Tc CCL2/ CCL7 Tc CXCL2/5/8/9/10/11 Tc Tc Dc Tc Recruitment of myeloid cells IL-6 Tc IL-1 β IL-23 Tc Tc Tc IFN- γ TNF- α Dc Epithelial leakage IL-17A Inflammation Dc layer Inner mucus Dysbiosis MANUSCRIPT layer Outer mucus Symbiosis layer Inner mucus Dc Tc Tc Tc IL-10 IFN- γ Tc IL-17A TNF- α Tc Tc Tc Dc Bc Tc Steady state Bc Bc Tc Dc Tc
ACCEPTEDTc Tc mLNs Tc Tc Tc Tc Tc Tc CCR6 CCR4 CCR7 CCR9 Tc Tc Tc Circulation Integrin α 4 β 7
Supplementary Figure S7