Costa-Bento DF

Gallimore A

Godkin A

Keita ÅV

Scurr MJ

Smart K

Somerville MS

Thomson A

English

Newcastle University E-Prints

ARTICLEMolecular DiagnosticsPrognostic significance of interleukin-17A-producingcolorectal tumour antigen-specific T cellsAmanda Thomson1, Diana F. Costa Bento1, Martin J. Scurr 1, Kathryn Smart1, Michelle S. Somerville1, Åsa V. Keita2,Awen Gallimore1 and Andrew Godkin1,3BACKGROUND: The T cell cytokine profile is a key prognostic indicator of post-surgical outcome for colorectal cancer (CRC). WhilstTH1 (IFN-γ+) cell-mediated responses generated in CRC are well documented and are associated with improved survival, antigen-specific TH17 (IL-17A+) responses have not been similarly measured.METHODS: We sought to determine the cytokine profile of circulating tumour antigen-(5T4/CEA) specific T cells of 34 CRC patientsto address whether antigen-specific IL-17A responses were detectable and whether these were distinct to IFN-γ responses.RESULTS: As with IFN-γ-producing T cells, anti-5T4/CEA TH17 responses were detectable predominantly in early stage (TNM I/II) CRCpatients. Moreover, whilst IL-17A was always produced in association with IFN-γ, this release was mainly from two distinct T cellpopulations rather than by ‘dual producing’ T cells. Patients mounting both tumour-specific TH1+/TH17+ responses exhibitedprolonged relapse-free survival.CONCLUSIONS: Tumour antigen-specific TH17 responses play a beneficial role in preventing post-operative colorectal tumourrecurrence.British Journal of Cancer (2021) 124:1552–1555; https://doi.org/10.1038/s41416-021-01283-3BACKGROUNDThe adaptive immune system plays a critical role in the control ofcolorectal cancer (CRC). Our previous work identified that thepresence of pre-operative circulating TH1 responses specific for5T4, an oncofoetal antigen not expressed in healthy adult tissue,has a positive association with post-operative relapse-free survival,1in keeping with studies demonstrating that tumour infiltrationof TH1-type (IFN-γ-producing) T cells associate with favourableoutcome.2 Unexpectedly, the presence of TH1 responses directedtowards carcinoembryonic antigen (CEA), an auto-antigenexpressed at low levels in normal intestinal epithelium, associatedwith poor relapse-free survival in CRC patients.1 Whilst both CEAand 5T4 are upregulated in the majority of colorectal tumours,there is a potential that tumour-selective T cell responses impactclinical outcome differently to auto-antigen T cell responses.3Since TH1 responses are usually associated with control ofcancer progression, we postulated that CEA-specific IFN-γ-producing T cells were not the cause of the observed associationwith poor survival, but a surrogate marker for skewed tumour-specific T cell functionality. In particular, tumour-infiltrating TH17cells have previously been associated with CRC progression.2 IL-17A itself has been implicated in tumour progression throughSTAT3 activation, promoting colonic epithelial cell transformation,tumour cell proliferation and invasion.4 Contrastingly, thepresence of colorectal tumour-infiltrating TH17 cells was recentlyreported to correlate with improved survival,5 implying that IL-17Ahas important context and tissue-dependent roles in enactingpro- or anti-tumour immune mechanisms.6This current study was designed to identify the presence ofCEA-specific and 5T4-specific IL-17A- and IFN-γ-producing T cellswithin the blood of CRC patients and healthy controls andinvestigate their role on post-operative outcome and colonicepithelial barrier function.METHODSParticipantsBlood and colon samples were obtained from 34 patients under-going colorectal cancer resection or investigative colonoscopyprocedures at the University Hospital of Wales, Cardiff. Patientcharacteristics are summarised (Supplementary Table 1). Additionalblood samples were obtained from age-matched healthy donors(age range 48–62; 5 male, 4 female). All participants gave written,informed consent personally prior to inclusion. The Wales ResearchEthics Committee granted ethical approval for this study.AntigensThe CEA protein was split into seventy 20mer peptides and5T4 split into forty-one 20mers, each overlapping by 10 aminoacids and synthesised to >90% purity (GLBiochem, Shanghai,www.nature.com/bjcReceived: 4 December 2020 Revised: 8 January 2021 Accepted: 13 January 2021Published online: 5 March 20211Division of Infection & Immunity, Henry Wellcome Building, Cardiff University, Cardiff, UK; 2Division of Surgery, Orthopedics & Oncology, Department of Clinical andExperimental Medicine, Linköping University, Linköping, Sweden and 3Department of Gastroenterology & Hepatology, University Hospital of Wales, Heath Park, Cardiff, UKCorrespondence: Andrew Godkin (godkinaj@cardiff.ac.uk)These authors contributed equally: Amanda Thomson, Diana F. Costa Bento, Martin J. ScurrThese authors contributed equally: Awen Gallimore, Andrew Godkin© Crown 2021 Published by Springer Nature on behalf of Cancer Research UKChina). Peptides were dissolved in minimal DMSO and incorpo-rated into peptide pools, used at a final concentration of1.45 μg/ml/peptide. Phytohaemagglutinin (PHA; Sigma Aldrich)was used as a positive control.Peripheral blood mononuclear cell (PBMC) extraction and culturePBMCs were extracted from whole blood as previously described.15 × 105 PBMCs were seeded per well in a 96-well plate in 100 μl R+media supplemented with 5% human AB serum (Welsh BloodService, Pontyclun, UK), 2 mM L-glutamine, 1 mM sodium pyruvateand 50 μg/ml penicillin/streptomycin (Gibco, Paisley, UK). PBMClines were cultured for 14 days in the presence or absence ofpeptide pool, supplemented with 10 μl CellKine (purified T cellgrowth factors derived from pooled PHA-stimulated T cells;Helvetica Healthcare, Geneva, Switzerland), on day 3 and freshmedia containing 40 IU/ml IL-2 on days 6 and 9.FluoroSpot assaysThe human IFN-γ/IL-17A FluoroSpot kit (MabTech, Sweden) wasused and performed according to the manufacturer’s instruc-tions. Duplicate lines of cultured PBMCs were pooled together,washed and plated at 5 × 105 cells/well with or without thecorresponding peptide pool for direct comparison. FluoroSpotplates were incubated at 37 °C, 5% CO2 for 48 h. In someinstances, blocking antibodies to MHC class-I (anti-HLA-A/B/B(W6/32); BioLegend, UK) and MHC class-II (anti-HLA-DR (L243);anti-HLA-DQ (1A3); BioLegend) were added at a final concentra-tion of 10 μg/ml, incubated for 30 min before addition ofantigen. Cytokine-producing PBMCs were enumerated usingan automated FluoroSpot reader (CTL, Germany). Dual cytokine-secreting cells were visualised and enumerated by a compu-terised overlay. Positive IFN-γ and IL-17A responses wereidentified by a minimum of 10 and 5 spot-forming cells (SFC)90300100No 5T4 responseNo CEA responseCEA+ IFN-γ responseCEA+ IFN-γ & IL-17A response5T4+ IFN-γ & IL-17A response5T4+ IFN-γ response80604020020010005T4 IL-17A SFC/105 cultured PBMC5T4 IFN-γ SFC/105 cultured PBMC300 504030201002001000CEA IFN-γ SFC/105 cultured PBMCCEA IL-17A SFC/105 cultured PBMCPercentage of donors100806040No response (n=18)IFN-γ+ only (n=10)IFN-γ+ IL-17A+ (n=6)No response (n=14)IFN-γ+ only (n=11)IFN-γ+ IL-17A+ (n=9)2000 1 2 3Time, Years4 50 1 2 3Time, Years4 5Relapse-free survival, %100806040200Relapse-free survival, %100a b cd eg hf806040200Percentage of donors803020100HD l/ll lllTNM StageHD l/ll lllTNM StageHD l/ll lllTNM Stage5T4CEAHD l/ll lllTNM StageHD CRCTNM l/llCRCTNM llln=15n=19n=9HD CRCTNM l/llCRCTNM llln=15n=19n=9Fig. 1 CEA- and 5T4-specific TH1/TH17 responses prevent colorectal tumour recurrence. a The percentage of donors mounting a CEA-specific IFN-γ response (dark grey), a dual CEA-specific IFN-γ and IL-17A response (light grey), or no positive CEA-specific response (black), asindicated by results from the IFN-γ/IL-17A FluoroSpot assay. The magnitude of CEA-specific T cell response, normalised to spot-forming cells(SFC)/105 cultured PBMC, and associated with healthy donor (HD) or early-stage (TNM I/II) or late-stage (TNM III) CRC is shown for IFN-γ (b) andIL-17A (c). Error bars indicate median with interquartile range. d The percentage of donors mounting a 5T4-specific IFN-γ response (dark grey),a dual 5T4-specific IFN-γ and IL-17A response (light grey), or no positive 5T4-specific response (black), as indicated by results from the IFN-γ/IL-17A FluoroSpot assay. The magnitude of 5T4-specific T cell response, normalised to spot-forming cells (SFC)/105 cultured PBMC, andassociated with healthy donor (HD) or early-stage (TNM I/II) or late-stage (TNM III) CRC is shown for IFN-γ (e) and IL-17A (f). Error bars indicatemedian with interquartile range. The CEA-specific (g) or 5T4-specific (h) TH1/TH17 response measured in all CRC patients (n= 34) wasassociated with relapse-free survival.Prognostic significance of interleukin-17A-producing colorectal tumour. . .A Thomson et al.15531234567890();,:per 1 × 105 cultured PBMC, respectively, and a minimum 2-foldincrease above background.Intestinal permeability measurementsElectrophysiological resistance and paracellular flux in humancolonic biopsies was measured using an Ussing chamber, aspreviously described.7 Readouts of intestinal permeability wereassociated with the presence of IFN-γ and IL-17A CEA- and 5T4-specific T cell responses, with patients differentiated based on themedian magnitude of response generated to each antigen.Statistical analysisGraphPad Prism (v.8) was used for all statistical analyses. Datasetnormality was tested using the Shapiro-Wilk test. Significance wasdetermined using one-way ANOVA with corrections for multiplecomparisons made using the Tukey test; a P-value < 0.05 wasconsidered significant. For survival analyses using Kaplan–Meiercurves, relapse-free survival was calculated from the date ofsurgery to the date of relapse, as determined by follow-up CT scanand/or endoscopy. Non-relapsing patients were censored at thetime of last follow-up assessment (September 2019). Survivalcurves were compared using the log-rank test.RESULTSCEA-specific and 5T4-specific IFN-γ, IL-17A and IFN-γ/IL-17A dual-secreting T cell responses from pre-operative CRC patient andage-matched healthy donor PBMC were investigated. CulturingPBMC for 14 days in the presence of a particular cytokine cocktailwas necessary to expand low frequency antigen-specific TH17responses to detectable levels (Supplementary Fig. 1A). WhilstTH17 responses were identified, IFN-γ/IL-17A dual secretion fromthe same cell was rarely detected; example FluoroSpot wellimages and results are shown for CEA and 5T4 (SupplementaryFig. 1B). CEA- and 5T4-specific TH1 and TH17 cell responses werereadily identifiable amongst healthy donors and cancer patients(Supplementary Fig. 1C); these responses were predominantlyMHC class II-restricted (Supplementary Fig. 1D). The proportion ofcancer patients mounting detectable CEA-specific IFN-γ T cellresponses diminished in comparison to healthy age-matchedcontrols (Fig. 1a) and further declined as the tumour became moreadvanced (Fig. 1b). No overall change in CEA-specific IL-17Aproduction was noted between healthy donors and CRC patients(Fig. 1c).Similarly, 5T4-specific TH1 responses significantly declined asthe colorectal tumour advanced (HD v III, P= 0.033; I/II v III, P=0.031; Fig. 1d, e), validating previous findings with 5T41,8 and otherwidely-expressed upregulated tumour antigens.9 5T4-specificTH17 responses were rarely detectable in healthy donors, andonly significantly increased in earlier stage CRC patients (P= 0.036,Fig. 1f).The prognostic impact of pre-operative CEA and 5T4-specificTH1 and TH17 responses was determined by associating responseswith 5-year relapse-free survival. Patients were separated based onwhether they mounted both an IFN-γ and IL-17A response or notto CEA (Fig. 1g) and 5T4 (Fig. 1h); one patient that mounted ananti-5T4 IL-17A response only was included as a non-responder.Although the size of the groups did not allow statisticalsignificance to be reached, it is clear that in both cases the mostfavourable immune profile pre-operatively for prolonged disease-free survival is the presence of both TH1 and TH17 responses(Fig. 1g, h).From this data we conclude that release of IL-17A by CEA-specific T cells is not responsible for the poorer survival of patientswith IFN-γ-producing CEA (but not 5T4)-specific T cells. Unlike 5T4,CEA is expressed at low levels in normal epithelium. A highermagnitude of IFN-γ+ CEA-specific T cell response, i.e. above themedian of all anti-CEA responses tested in patients undergoingendoscopy (Supplementary Fig. 2A, B), was associated withdecreased electrical resistance in ascending colonic epithelium(P= 0.037; Supplementary Fig. 2C) and increased paracellularpassage (i.e. leak) of Lucifer yellow probe (P= 0.11; SupplementaryFig. 2D), implying that the integrity of the epithelial barrier issubtly compromised in patients with relatively high CEA TH1response, a finding not replicated when separating responsesbased on IL-17A+ CEA responders, nor 5T4 responders from non-responders. This observation, specific to the right-sided ascendingcolonic epithelium, is worthy of further investigation.DISCUSSIONMounting pre-operative tumour antigen-specific TH17 and TH1combined responses appears to be associated with less post-operative CRC relapse. Despite the relatively small sample size, thereis a clear indication that IL-17A production by tumour antigen-specific T cells has a positive influence on anti-tumour immunity,warranting further investigation. In particular, the emergence of 5T4-specific TH17 response is indicative of early stage CRC and mayprovide diagnostic/prognostic information. This study also highlightsthe possibility for detrimental clinical outcomes when using IL-17/TH17-targeted treatments; indeed it has been suggested that clinicalresponse to anti-PD-1 may be reliant on TH17 responses andtreatment success is compromised by IL-17A blockade.6,10,11We have previously shown an adverse effects of ex vivomeasured anti-CEA TH1 responses on post-operative CRC patientoutcome.1 In this previous study, we did not measure IL-17Aresponses, which require a period of in vitro culture andexpansion. This study, albeit on smaller patient numbers, stronglysuggests that TH17 responses are beneficial when combined withTH1 responses. It is possible that an IFN-γ-driven chronic, sub-clinical enteropathy which facilitates tumorigenesis is reversed oralleviated by IL-17A production.In conclusion, tumour antigen-specific TH17 responses play abeneficial role in preventing colorectal tumour recurrence aftersurgical resection.ACKNOWLEDGEMENTSWe thank Mr. Simon Phillips, Mr. Michael Davies, Ms. Rachel Hargest, Mr. Leigh Daviesand Mr. James Horwood for provision of CRC patient bloods. We thank Dr. TomPembroke, Dr. Gautham Appanna, Dr. Lawrence Sunder Raj, Dr. Brijesh Srivastava andDr. Dharmaraj Durai for provision of patient blood samples and colonic epithelialbiopsies from endoscopy patients.AUTHOR CONTRIBUTIONSAn.G. and Aw.G. conceived, designed and were the principal investigators of thisstudy; A.T. and D.F.C.B. recruited the cohorts. A.T., D.F.C.B. and K.S. performed theimmunological assays; A.T., K.S. and Mi.S. performed the Ussing chamberexperiments; A.T. and D.F.C.B. performed data management, A.T., D.F.C.B. and M.J.S.interpreted the data and performed statistical analyses. A.T., M.J.S. and Aw.G. wrotethe original draft of the paper. A.T., D.F.C.B., M.J.S., K.S., Mi.S., Aw.G. and An.G.reviewed and/or edited the paper.ADDITIONAL INFORMATIONEthics approval and consent to participate All patients and healthy donorsgave their signed informed consent, and the protocol was approved by the WalesResearch Ethics Committee (reference:15/WA/0291). The study was conducted inaccordance with the Declaration of Helsinki.Consent to publish Not applicable.Data availability The datasets generated during this study are available from thecorresponding author on reasonable request.Competing interests The authors declare no competing interests.Prognostic significance of interleukin-17A-producing colorectal tumour. . .A Thomson et al.1554Funding information This work was supported by Cancer Research Wales and aCancer Research UK programme grant (C16731/A21200).Supplementary information The online version contains supplementary materialavailable at https://doi.org/10.1038/s41416-021-01283-3.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claimsin published maps and institutional affiliations.REFERENCES1. Scurr, M. J., Brown, C. M., Costa Bento, D. F., Betts, G. J., Rees, B. I., Hills, R. K. et al.Assessing the prognostic value of preoperative carcinoembryonic antigen-specificT-cell responses in colorectal cancer. J. Natl. Cancer Inst. 107, djv001 (2015).2. Tosolini, M., Kirilovsky, A., Mlecnik, B., Fredriksen, T., Mauger, S., Bindea, G. et al.Clinical impact of different classes of infiltrating T cytotoxic and helper cells(Th1, Th2, Treg, Th17) in patients with colorectal cancer. Cancer Res. 71, 1263–1271(2011).3. Parkhurst, M. R., Yang, J. C., Langan, R. C., Dudley, M. E., Nathan, D. A., Feldman, S.A. et al. T cells targeting carcinoembryonic antigen can mediate regression ofmetastatic colorectal cancer but induce severe transient colitis. Mol. Ther. 19,620–626 (2011).4. Hurtado, C. G., Wan, F., Housseau, F. & Sears, C. L. Roles for Interleukin 17 andAdaptive Immunity in Pathogenesis of Colorectal Cancer. Gastroenterology 155,1706–1715 (2018).5. Amicarella, F., Muraro, M. G., Hirt, C., Cremonesi, E., Padovan, E., Mele, V. et al. Dualrole of tumour-infiltrating T helper 17 cells in human colorectal cancer. Gut 66,692–704 (2017).6. McGeachy, M. J., Cua, D. J. & Gaffen, S. L. The IL-17 Family of Cytokines in Healthand Disease. Immunity 50, 892–906 (2019).7. Thomson, A., Smart, K., Somerville, M. S., Lauder, S. N., Appanna, G., Horwood, J.et al. The Ussing chamber system for measuring intestinal permeability in healthand disease. BMC Gastroenterol. 19, 98 (2019).8. Scurr, M., Bloom, A., Pembroke, T., Srinivasan, R., Brown, C., Smart, K. et al. Escalatingregulation of 5T4-specific IFN-γ(+) CD4(+) T cells distinguishes colorectal cancerpatients from healthy controls and provides a target for in vivo therapy. CancerImmunol Res. https://doi.org/10.1158/2326-6066.cir-13-0035 (2013).9. Laheurte, C., Dosset, M., Vernerey, D., Boullerot, L., Gaugler, B., Gravelin, E. et al.Distinct prognostic value of circulating anti-telomerase CD4+ Th1 immunityand exhausted PD-1+/TIM-3+ T cells in lung cancer. Br. J. Cancer 121, 405–416(2019).10. Dulos, J., Carven, G. J., van Boxtel, S. J., Evers, S., Driessen-Engels, L. J., Hobo, W.et al. PD-1 blockade augments Th1 and Th17 and suppresses Th2 responses inperipheral blood from patients with prostate and advanced melanoma cancer.J. Immunother. 35, 169–178 (2012).11. Esfahani, K. & Miller, W. H. Reversal of Autoimmune Toxicity and Loss of TumorResponse by Interleukin-17 Blockade. N. Engl. J. Med 376, 1989–1991 (2017).Open Access This article is licensed under a Creative CommonsAttribution 4.0 International License, which permits use, sharing,adaptation, distribution and reproduction in anymedium or format, as long as you giveappropriate credit to the original author(s) and the source, provide a link to the CreativeCommons license, and indicate if changes were made. The images or other third partymaterial in this article are included in the article’s Creative Commons license, unlessindicated otherwise in a credit line to the material. If material is not included in thearticle’s Creative Commons license and your intended use is not permitted by statutoryregulation or exceeds the permitted use, you will need to obtain permission directlyfrom the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.© Crown 2021Prognostic significance of interleukin-17A-producing colorectal tumour. . .A Thomson et al.1555

Prognostic significance of interleukin-17A-producing colorectal tumour antigen-specific T cells

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Prognostic significance of interleukin-17A-producing colorectal tumour antigen-specific T cells

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