Soluble forms of the coinhibitory receptors programmed death 1 (PD-1) and Tim-3 exist, but their relationship with T-cell surface expression remains unclear. When measured by an enzyme-linked immunosorbent assay in plasma, soluble PD-1, and soluble Tim-3 were elevated during primary HIV infection, decreased on antiretroviral therapy to levels found in controls, and correlated with cell surface expression. We conclude that soluble PD-1 and soluble Tim-3 are easy to measure biomarkers of immune exhaustion which potentially eliminate the need for flow cytometry

CHERUB Investigators

Fidler, S

Fox, J

Frater, AJ

Martin, GE

Nwokolo, N

Willberg, CB

Zilber, E

Frater, John

Martin, Genevieve E

Willberg, Christian B

Zilber, Eva

Fox, Julie

Nwokolo, Nneka

Fidler, Sarah

Investigators, CHERUB

Oxford University Research Archive

1Research LetterSoluble Plasma PD-1 and Tim-3 in Primary HIV InfectionEva ZILBER1*, Genevieve E MARTIN1*, Christian B WILLBERG1,2, Julie FOX3, Nneka NWOKOLO4, Sarah FIDLER5,6, John FRATER1,2,7, on behalf of the CHERUB Investigators 1University of Oxford, Nuffield Department of Medicine, Oxford, United Kingdom 2National Institute of Health Research Biomedical Research Centre, Oxford, United Kingdom 3Guys and St Thomas’ NHS Trust, Department of Genitourinary Medicine and Infectious Disease, London, United Kingdom 4Chelsea and Westminster Hospital, London, United Kingdom 5Imperial College, Division of Medicine, Wright Fleming Institute, London, United Kingdom 6Imperial College National Institute of Health Research Biomedical Research Centre, London, United Kingdom7Oxford Martin School, Oxford, United Kingdom * these authors contributed equallyCorresponding authors:John Frater and Genevieve Martin, Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, John Radcliffe Hospital, Oxford, UK. Email: john.frater@ndm.ox.ac.uk; genevieve.martin@ndm.ox.ac.ukShort title: Soluble PD-1 and Tim-3 in PHIWord Count: Introduction 69/75; Text 971/1000123456789101112131415161718192021222324252SummarySoluble forms of the co-inhibitory receptors PD-1 and Tim-3 exist, but their relationshipwith T cell surface expression remains unclear. When measured by ELISA in plasma,sPD-1 and sTim-3 were elevated  during PHI,  decreased on ART to levels  found incontrols, and correlated with cell surface expression. We conclude that sPD-1 and sTim-3 are easy to measure biomarkers of immune exhaustion which potentially eliminate theneed for flow cytometry. 26272829303132333Main textAntiretroviral therapy (ART) dramatically improves the life expectancy of people livingwith  HIV  [1,  2],  and prevents  onward viral  transmission  [3,  4].  However,  in  sometreated  individuals  the  co-inhibitory  receptors  PD-1  and  Tim-3  [5] are  persistentlyelevated  in  primary  and  chronic  HIV  infection,  and  are  associated  with  T  cellexhaustion  [6-9]. This T cell dysfunction is increasingly recognised as a limitation tocurative  strategies  such  as  “kick  and  kill”  which  will  rely  on  an  effector  immuneresponse. Furthermore, PD-1 expression during primary HIV infection (PHI) correlateswith viral reservoir size and predicts time to viral rebound after treatment interruption[10]. Importantly, the success of cancer immunotherapy to target T cell exhaustion [11]has prompted investigation into whether these pathways can be targeted as part of anHIV cure strategy [12].Both PD-1 and Tim-3 also have soluble forms which can interact with these signallingpathways.  Soluble  PD-1  (sPD-1)  is  an  alternative  splice  form of  PD-1  [13],  whilesoluble Tim-3 (sTim-3) is produced when Tim-3 is cleaved from the cell surface by thesheddase ADAM10 [14]. sTim-3 is increased during acute, early, and chronic untreatedHIV infection, and correlates with plasma viral load (pVL) [14]. Recently, sPD-1 wasshown to be increased in viraemic patients when compared to both healthy controls andpatients on long-term ART  [15]. The relationship between plasma levels and T cellexpression of these markers is unknown. Here, we measured plasma sPD-1 and sTim-3pre-treatment and one year after ART initiation in PHI. We investigated the relationshipof soluble co-inhibitory receptor plasma concentration with key clinical parameters andwith the respective receptor surface expression on CD4+ and CD8+ T cells. 343536373839404142434445464748495051525354555657448 participants  from the HEATHER cohort were selected based on sample and dataavailability.  All  were male,  with  the median  time  from estimated  seroconversion toART initiation of 46 [IQR 28–70] days. The median baseline pVL was 5.50 [IQR 4.49–6.55]  log10 HIV  RNA  copies/ml,  median  CD4  cell  count  526  [IQR  405.2–667.8]cells/mm3. 10 healthy controls (HCs) (REC 16/YH/0247) were all male, with medianage  34.5  [IQR 30.5-42.5]  years.  sPD-1  and  sTim-3  in  plasma  was  measured  usingHuman PD-1 (PDCD1) ELISA kit (Thermo Fisher Scientific, Waltham, MA USA) andQuantikine ELISA Human TIM-3 Immunoassay kit (R&D Systems, Minneapolis, MNUSA)  in  accordance  with  manufacturers’  instructions.  T  cell  PD-1  and  Tim-3expression  was  measured  using  flow  cytometry.  Cell  surface  staining  was  withLiveDead Near IR, Tim-3 PE, PD-1 PE-eFluor610, CD3 BV570, CD4 BV605, CD8BV650 as detailed elsewhere [16]. Data were acquired on an LSR II (BD) and analysedusing FlowJo Version 10.0.8r1 (Treestar). HIV DNA was quantified following a protocol described in detail elsewhere [17]. Allsamples  were  run  in  triplicate  at  two  dilutions,  and  assay  standards  were  run  induplicate. Groups  were  compared  using  Kruskal-Wallis  tests  with  pairwise  comparison  usingDunn’s  test.  Linear  mixed-effects  models  were  fitted  with  data  transformations  asappropriate.  Analysis  was  performed  with  GraphPad  Prism  (version  7.0b)  and  R(version  3.4.0)  with  MASS  (version  7.3-47),  nlme  (version  3.1-131),  and  corrplot(version 0.84) packages. 5859606162636465666768697071727374757677787980815We initially aimed to determine if soluble PD-1 and Tim-3 expression was perturbed inPHI and was associated with their respective surface co-inhibitory receptor expression.PD-1 and Tim-3 expression was quantified in samples from the 48 HIV+ participantsand 10 HCs. sPD-1 and sTim-3 were significantly elevated in PHI when compared toHCs. Following 1 year of ART, both decreased to levels not different from those foundin HCs (Figure 1A,B). This demonstrates elevation of sPD-1 during PHI for the firsttime, and confirms previous findings for sTim-3 [14]. Soluble forms of the co-inhibitoryreceptors behaved similarly to surface forms: PD-1 and Tim-3 expression was increasedon both CD4+ and CD8+ T cells in PHI compared to HCs, and decreased significantlywith  ART  (Figure  1C-F).  Furthermore,  soluble  co-inhibitory  receptor  expressioncorrelated with respective cell surface expression (Figure 1G,H). Considering the known relationship between T cell exhaustion, HIV clinical parametersand reservoir size, we investigated the relationship of soluble co-inhibitory receptorswith pVL, CD4 T cell count, CD4/CD8 ratio (all at baseline), and reservoir size afterone year on ART. sPD-1 correlated significantly with all four, and pVL and CD4 countwere more strongly correlated with this soluble form than surface PD-1 expression onCD4 or CD8 T cells (Figure 1G). To investigate the nature of these relationships further, we used linear mixed effects(LME)  modelling  to  determine  which  variables  were  independently  associated  withsPD-1  levels.  The  use  of  LME  models  allowed  for  consideration  of  longitudinalsampling of HIV-infected participants. The optimal model included the time-point, CD4and  CD8  PD-1%,  baseline  pVL  and  CD4/CD8  ratio  (Supplementary  Table  1).8283848586878889909192939495969798991001011021031041056Importantly,  there were significant  relationships  between sPD-1 levels  and pVL andCD4/CD8 ratio, and their interaction (Supplementary Table 1). As expected, there was arelationship between CD4+ PD-1% expression and sPD-1 concentration, although thiswas not statistically significant (p=0.059) when these measures of clinical progressionwere also considered. Overall, sPD-1 expression is linked to CD4 T cell expression ofthis marker, and measures of disease progression during PHI. Further investigation ofsPD-1 as an easily obtainable and clinically relevant measure of T cell  functionalityduring PHI is warranted. Similarly to Tim-3 expression on CD8 T cells, sTim-3 correlated with pVL, CD4/CD8ratio, and reservoir size (Figure 1H). In the optimal LME model, sTim-3 was associatedwith  the  time-point  and  CD8  Tim-3%  only.  Therefore,  sTim-3  might  be  a  goodsurrogate for surface Tim-3 expression. In conclusion, sPD-1 and sTim-3 are associated with their respective surface receptorexpression and with key clinical parameters. Importantly, they are cheap and easy tomeasure and do not require viable fresh or cryopreserved cells, or the access to flowcytometry  facilities.  These  soluble  markers  could  therefore  be  useful  adjuncts  forstudying immune exhaustion and potential immunotherapy development, and may havevalue for screening or stratification within clinical trials.1061071081091101111121131141151161171181191201211221231241251267ACKNOWLEDGMENTSWe thank the participants of HEATHER. The HEATHER study is conducted as part of the CHERUB (Collaborative HIV Eradication of Reservoirs: UK BRC) collaboration. CHERUB Steering Committee: Andrew Lever (University of Cambridge), Mark Wills (University of Cambridge), Jonathan Weber (Imperial College, London), Sarah Fidler (Imperial College, London), John Frater (University of Oxford), Lucy Dorrell (University of Oxford), Mike Malim (King’s College, London), Julie Fox (King’s College London), Ravi Gupta (University College London), Clare Jolly (University College London). We thank Dr Daniel Lunn for statistical advice. AUTHORSHIP CONTRIBUTIONSThe experiments were conceived and designed by GEM, EZ, CBW, and JFr. Experiments were performed by GEM and EZ. Data were analyzed by GEM and EZ. Design and recruitment of the trials was performed by JFr, NN, JF and SF. The paper was written by GEM, EZ, JFr and CBW with input from all authors.DISCLOSURE OF CONFLICTS OF INTERESTThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.1271281291301311321331341351361371381391401411421431441451468REFERENCES1. Wada N, Jacobson LP, Cohen M, French A, Phair J, Munoz A. Cause-specific mortality among HIV-infected individuals, by CD4(+) cell count at HAART initiation, compared with HIV-uninfected individuals. AIDS 2014; 28(2):257-265.2. Trickey A, May MT, Vehreschild J-J, Obel N, Gill MJ, Crane HM, et al. Survival of HIV-positive patients starting antiretroviral therapy between 1996 and 2013: a collaborative analysis of cohort studies. The Lancet HIV 2017; 4(8):e349-e356.3. Rodger AJ, Cambiano V, Bruun T, Vernazza P, Collins S, van Lunzen J, et al. 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Jones M, Williams J, Gartner K, Phillips R, Hurst J, Frater J. Low copy target detection by Droplet Digital PCR through application of a novel open access bioinformatic pipeline, 'definetherain'. J Virol Methods 2014; 202:46-53.19219319419519619719819920020120211Figure LegendFigure  1. Soluble  PD-1  and  Tim-3  expression  are  perturbed  in  PHI  and  areassociated with respective surface co-inhibitory receptor expression and clinicalparameters. (A-B),  soluble  PD-1  and  Tim-3  concentration  in  plasma.  (B-F)  Co-inhibitory  receptor  expression  on  CD4+ and  CD8+  T  cells.  All  three  groups  werecompared using Kruskall-Wallis tests (overall p<0.001 for all tests), with subsequentpairwise  comparisons  performed  using  Dunn’s  test.  *:  p<0.05,  **:  p<0.01,  ***:p<0.001, ****: p<0.0001. (G-H) Corrplots showing Spearman’s correlations betweenpairs of variables  at  baseline where the colour and size of circle  corresponds to thecorrelation  coefficient  between  these  two  variables;  only  significant  (p<0.05)correlations shown.203204205206207208209210211212213

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Soluble plasma programmed death 1 (PD-1) and Tim-3 in primary HIV infection

Soluble plasma programmed death 1 (PD-1) and Tim-3 in primary HIV infection

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