RSV-specific airway resident memory CD8 þ T cells and differential disease severity after experimental human infection

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1 Received Oct 15 Accepted 1 Nov 15 Pulished 1 Dec 15 DOI: 1.138/ncomms1 OPEN RSV-specific irwy resident memory CD8 þ T cells nd differentil disese severity fter experimentl humn infection Agnieszk Jozwik 1, Mximillin S. Hii 1, Alln Prs 1, Jie Zhu 1, Aleks Guvenel 1, Jideep Dhriwl 1, Mrk Almond 1, Ernie H.C. Wong 1, Annemrie Sykes 1, Mtthew Myeno, Jerico Del Rosrio 1, Mri-Belen Trujillo-Torrlo 1, Ptrick Mlli 1, John Sidney, Bjoern Peters, Onn Min Kon 1, Alessndro Sette, Sestin L. Johnston 1, Peter J. Openshw 1 & Christopher Chiu 1 In niml models, resident memory CD8 þ T (Trm) cells ssist in respirtory virus elimintion ut their importnce in mn hs not een determined. Here, using experimentl humn respirtory syncytil virus (RSV) infection, we investigte systemic nd locl virus-specific CD8 þ T-cell responses in dult volunteers. Hving defined the immunodominnce hierrchy, we nlyse phenotype nd function longitudinlly in lood nd y seril ronchoscopy. Despite rpid clinicl recovery, we note surprisingly extensive lower irwy inflmmtion with persistent virl ntigen nd cellulr infiltrtes. Pulmonry virus-specific CD8 þ T cells disply CD9 þ CD13 þ Trm phenotype nd ccumulte to strikingly high frequencies into convlescence without continued prolifertion. While these hve more highly differentited phenotype, they express fewer cytotoxicity mrkers thn in lood. Nevertheless, their undnce efore infection correltes with reduced symptoms nd virl lod, implying tht CD8 þ Trm cells in the humn lung cn confer protection ginst severe respirtory virl disese when humorl immunity is overcome. 1 Ntionl Hert nd Lung Institute, Imperil College London, London W 1PG, UK. Centre for Infectious Disese, Division of Vccine Discovery, L Joll Institute of Allergy nd Immunology, 9 Athen Circle, L Joll, Cliforni 93, USA. Correspondence nd requests for mterils should e ddressed to C.C. (emil: c.chiu@imperil.c.uk). NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 1

2 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 CD8 þ T cells re essentil effectors tht eliminte intrcellulr pthogens nd confer protection ginst symptomtic reinfection vi immune memory 1. In niml models of respirtory virus infections such s respirtory syncytil virus (RSV) nd influenz, memory CD8 þ T cells reduce virl repliction, prevent infection or decrese disese severity, nd confer cross-protection ginst ntigeniclly distinct strins. However, while some vccine cndidtes ginst RSV nd influenz my hve the cpcity to induce CD8 þ T cells, they hve not yet een shown cliniclly to improve protection 3 5. RSV is glolly the commonest cuse of lower respirtory trct infection in children, leding to n estimted 3. million hospitliztions ech yer. It is lso mjor contriutor to mortlity in older nd immunosuppressed dults. Recurrent symptomtic RSV infection occurs throughout life even with helthy immune system nd limited virl ntigenic vrition 8. Therefore, chrcterizing immune responses required for roust protection hs een prolemtic nd effective vccines remin mjor clinicl need. We recently showed tht nti-rsv IgA in the nsl mucos correlted strongly with protection from infection, ut tht the high levels required for immunity re poorly mintined, llowing recurrent infection 9. Despite this, most older children nd young dults suffer only minor symptoms, implying tht when ntiodies fil to prevent infection, cell-medited immunity reduces disese severity. In mice, depletion of RSV-specific CD8 þ T cells leds to prolonged virl repliction, while doptive trnsfer of virus-specific memory cells enhnces virus clernce 1,11. However, the sence of T cells lso leds to reduced symptom severity nd trnsfer of RSV-specific memory T cells worsens disese, indicting tht hrmful immunopthology cn outweigh the enefits of cell-medited virl clernce under certin circumstnces 1,13. In humns, the role of CD8 þ T cells remins less cler with evidence of their protective role minly limited to oservtions of children with T-cell defects (who suffer more severe disese with prolonged virl shedding) 1. In influenz, correltions etween memory T cells in the lood nd reduced severity of disese on susequent infection hve een shown 15,1, ut no such evidence exists in RSV nd the extent to which T cells contriute to protection or pthology in this nd other respirtory virl infections remins unknown. Respirtory viruses re usully confined to the lung with systemic spred only in the worst cses 1. Virus-specific CD8 þ T cells in peripherl lood re therefore unlikely in most situtions to e directly relevnt to protection. Insted, studies of rnge of tissues hve recently defined suset of non-circulting memory T cells specilized to protect sites of pthogen entry 18. These resident memory T (Trm) cells re not only poised for rpid killing on virus re-encounter ut my lso exhiit innte-like sensing functions 19. In mouse models of influenz, CD þ nd CD8 þ Trm cells in the lung confer greter protection thn spleen-derived cells,1. However, restrictions on smpling of humn lungs men tht little is known out these Trm cells except tht they re undnt in non-inflmed lung from tumour excisions or donted orgn tissue,3. We investigted the CD8 þ T-cell response to experimentl RSV infection in 9 helthy dult volunteers, round hlf of who lso underwent seril ronchoscopy. While controlled for vritions in virl inoculum nd co-moridities, this cohort nevertheless represented genotypiclly diverse ntigenexperienced popultion tht llowed chrcteriztion of the redth of virus-specific CD8 þ T-cell responses nd identifiction of novel immunodominnt nd sudominnt epitopes. Anlysis using mjor histocomptiility complex (MHC)-peptide tetrmers reveled highly contrsted kinetics, phenotypes nd functionlity of RSV-specific CD8 þ T cells in the lower respirtory trct compred with lood, the diversity of which llowed us to infer specilised role in protection ginst RSV disese. Results Experimentl RSV infection cuses upper trct disese. We enrolled 9 helthy dults ged 18 5 yers (medin.5 yers; Supplementry Tle 1). Two weeks nd immeditely efore inocultion, they underwent lood nd nsl smpling (Fig. 1). All individuls were then inoculted with 1 plque-forming units of RSV A Memphis 3 (RSV M3) y intrnsl drops s previously descried 9, followed y sequentil lood nd nsl smples up to months lter. Twenty-six sujects (53%) developed PCR þ infection (Fig. 1). Of these, 1 (5%) suffered common cold ccording to stndrdized criteri (see Methods), while 9 (35%) reported ± ronchoscopy Totl symptom score Qurntine nd dily nsl smpling Dy 1 1 RSV M3 inocultion ± ronchoscopy Infected Uninfected Dy post inocultion Symptom score ± ronchoscopy RSV M3 inocultion n=9 c d e Upper trct Lower trct Systemic Dy post inocultion Nsl virl lod (log1 copies/ml) % 53% PCR -ve n=3 PCR ve n= 5% 35% Cold n=1 No cold n= Dy post inocultion Figure 1 Inocultion of helthy dults with RSV cuses upper respirtory trct infection. Forty-nine helthy dult volunteers were inoculted with RSV Memphis 3. () The study design is shown. () Clinicl outcomes re shown. (c) Self-reported symptoms scores of 9 sujects were recorded dily. Cumultive dt re shown s men±s.e.m. Infected individuls were defined y hving hd RSV detected from nsl lvge on t lest consecutive dys y qpcr of nsl lvge. (d) Symptoms from 9 sujects were ctegorized s originting from the upper respirtory trct (lue), lower respirtory trct (red) or s systemic (green) ccording to previously descried criteri (see Methods). Dt re shown s men± s.e.m. (e) Virl lod of 9 individuls ws determined y N gene qpcr from nsl lvge. Individul dt points nd mens re shown. NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

3 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE miniml or no symptoms. Symptoms in infected individuls peked round dy post inocultion (Fig. 1c). While lmost ll symptomtic individuls complined of upper respirtory symptoms, lower respirtory trct nd systemic symptoms were unusul (Fig. 1d). In infected individuls, virl lod peked t dys 8 post infection with men of. log 1 copies per ml (±s.e.m..3; Fig. 1e), correlting with self-reported symptoms (Supplementry Fig. 1). RSV cuses inflmmtion in the lower respirtory trct. Twenty-four volunteers dditionlly underwent seril ronchoscopy (Supplementry Tle ). Thus, endoronchil iopsies, ronchil rushings nd roncholveolr lvge () were otined 1 dys efore inocultion nd or 1 nd dys fter. Of these, 5% developed RSV infection. In view of the predominntly upper respirtory disese, we were surprised to find strikingly extensive mcroscopic inflmmtion involving the lower respirtory trct of infected individuls (Fig. ; Supplementry Tle 3). Lower irwys involvement ws supported y quntittive PCR (qpcr) of ronchil rushings, which detected RSV in ll dy smples nd /5 infected sujects t dy 1, gin peking round dys (men. log 1 ml 1 ±s.e.m..1; Fig. ). RSV could lso e mesured in in 5/ individuls t dy t lower copy numers (pek 1.8 log 1 ml 1 ±s.e.m..1). Interestingly, virus ws detected in ronchil rushings of n dditionl four sujects in whom no virus ws found in nsl lvge. These were found t low-to-moderte copy numers (.31 log 1 ml 1 t dy, nd.9,.1 nd 1. log 1 ml 1 t dy 1) nd were reproducile on repet ssy. To confirm the unexpected presence of RSV in the lower irwy, endoronchil iopsies were nlysed y immunohistochemistry (Fig. c). While RSV ntigen ws not found pre-inocultion, ll individuls with PCR-detectle virus in the nose hd t lest moderte ntigen stining t oth cute nd convlescent time points. Of these, 3/11 hd extensive RSV ntigen in ronchil iopsies t dy 1 nd 3/1 t dy (Supplementry Tle ). In contrst, PCR sujects hd, t most, stining of isolted epithelil cells, suggesting the presence of RSV ntigen ut no onwrd spred. RSV ntigen ws ssocited with infiltrtion of CD8 þ T cells (Fig. d). In those with PCR þ ronchil rushings, CD8 þ T cells incresed significntly in oth the epithelil nd suepithelil lyers t cute nd convlescent time points (Fig. e). In contrst, while CD8 þ T cells did increse trnsiently in the iopsies of PCR individuls, these were less consistently seen nd did not remin significntly elevted t dy. Thus, experimentl infection of dults with RSV cn led to virus repliction in the lower irwy, which cuses mrked inflmmtion ssocited with infiltrtion of CD8 þ T cells. RSV virl lod drives CD8 þ T-cell prolifertion. To further investigte the kinetics of the response, CD8 þ T cells induced y RSV were trcked y flow cytometry using mrkers of prolifertion (Ki-) nd ctivtion (CD38; Fig. 3). From dy post infection, ctivted CD8 þ T cells in lood expnded peking round dy 1 (men.1%±s.e.m..9 of CD8 þ lymphocytes), fter which Ki- þ CD38 þ CD8 þ T cells rpidly returned to seline frequencies (Fig. 3). No significnt prolifertive response ws seen in PCR individuls, indicting tht ntigen chllenge without virl repliction ws not sufficient to induce CD8 þ T-cell ctivtion (Fig. 3c). Similrly, frequencies of ctivted CD8 þ T cells incresed in (Fig. 3d). However, the verge frequency ws significntly higher thn in lood (P ¼.98 Wilcoxon mtched pirs test; Fig. 3e). The ppernce of ctivted CD8 þ T cells coincided with the fll in virl lod (Fig. 1e). Antigen vilility nd inflmmtion oth contriute to T-cell ctivtion, nd indeed, cumultive virl lod nd symptoms (y trpezoidl re under the curve) oth correlted with the pek frequency of ctivted CD8 þ T cells (Fig. 3f,g), suggesting tht incresed virus urden nd/or inflmmtory chnges in the irwy drive T-cell ctivtion, which in turn leds to virl clernce. HLA-ssocited immunodominnce hierrchies ginst RSV. Antigenic trgets of RSV-specific CD8 þ T cells hve not een systemticlly investigted nd only few isolted epitopes hve een identified,5. To define the redth of these responses, we generted peptide lirry from RSV M3 predicted to ind the most common humn leukocyte ntigen (HLA)-A nd HLA-B llelesusingtheimmuneepitopedtseconsensusprediction tools. The top 1% of predicted MHC-inding peptides for ech RSV protein ws pooled nd peptide pools used to screen peripherl lood mononucler cells (PBMCs) from sujects t dy 1 post infection y interferon-g (IFN-g) enzyme-linked immunospot (ELISpot). Pools contining peptides from M, N, P, NS1 nd NS (ll internl proteins) induced IFN-g production (Fig. ). Pools tht stimulted responses were susequently deconvoluted to individul epitopes (Fig. ; Supplementry Tle 5). In the context of A1:1, two epitopes (previously descried YLEKESIYY [YLE] 5 nd the novel VTDNKGAFKY [VTD]) tht elicited dominnt responses were identified in the M protein nd one sudominnt epitope (LSDSTMTNY [LSD]) in the NS1 protein (Supplementry Tle 5). A 1-mer differing from LSD in one dditionl mino cid lso induced responses ut hd 1- fold lower MHC-inding ffinity. YLE nd VTD were conserved with the prototypic RSV A strin. Only single A:1-restricted epitope (FLVNYEMKL [FLV]) ws identified, derived from the NS nd conserved with RSV A. Finlly, with B:, four epitopes were defined: NPKASLLSL (NPK, descried previously 5 ) nd QVMLRWGVL (QVM) from N protein (previously shown s prt of longer polypeptide to induce IFN-g in some individuls ); IPAYRTTNY (IPA) from L; nd KPNIRTTLL (KPN) from G (not conserved with RSV A). VTD, LSD nd FLV hve previously een predicted ut not confirmed s epitopes on the sis of HLAinding ffinity lone. Indeed, ll ut three epitopes exhiited moderte to good mesured MHC peptide-inding ffinity (o5 nm). Importntly, ech HLA llele presented epitopes from different RSV proteins with no overlp in the specificities of CD8 þ T cells trgeted ginst them. In sujects with sufficient PBMCs, RSV-specific CD8 þ T-cell responses were nlysed efore, during nd fter infection using IFN-g ELISpot. Before infection, epitope-specific cells were rre, with medin (interqurtile rnge (IQR)) of 5 ( 1) spot-forming units (SFU) per million PBMCs (A1:1); (13 5) SFU per million PBMCs (B:); nd o5 (o5 11) SFU per million PBMCs (A:1; Fig. c,d; Supplementry Tle ). At the pek of the CD8 þ T-cell response, these hd incresed y B1-fold to 19 (11 5) SFU per million PBMCs for A1:1; 31 (138 5) SFU per million PBMCs for B:; nd (18 5) SFU per million PBMCs for A:1. The extent of expnsion etween the three HLA-restricted responses ws similr (Fig. d; Supplementry Fig. ). By dy, these popultions hd contrcted ut remined enlrged compred with seline in most individuls. Throughout the course of infection, the immunodominnce hierrchy ws mintined oth etween individuls nd within sujects who expressed two or three of these HLA lleles. Thus, A1:1-restricted responses were consistently greter thn those ginst B:-restricted epitopes nd those ginst the NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 3

4 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 c Bseline Dy Dy Bronchil virl lod (log1 copies per ml) Bseline Dy Dy 5 Bronchil rush Dy post inocultion d e CD8 cells per.1 mm CD8 cells per mm Epithelil PCR Epithelil PCR 15 Bseline Dy 1 Dy Bseline Dy 1 Dy Suepithelil PCR CD8 cells per mm CD8 cells per.1 mm 1 5 Suepithelil PCR Bseline Dy 1 Dy Bseline Dy 1 Dy Figure RSV infection of helthy dults cuses widespred lower respirtory trct inflmmtion. Twenty-four volunteers underwent seril ronchoscopic procedures 1 dys efore inocultion with RSV M3, or 1 dys nd dys post inocultion. () The mcroscopic ppernce of the irwys ws noted y ronchoscopists linded to the infection sttus of the suject. One representtive suject is shown. () Virl lod ws determined from ronchil rushings (solid line) nd roncholveolr lvge (dshed line) y qpcr. Cumultive dt re shown s men±s.e.m. (c) RSV ntigen (rown) ws detected in ronchil iopsies y immunohistochemistry. One representtive donor is shown. (d) CD8 þ cells (rown) were identified in ronchil iopsies y immunohistochemistry. Scle r, mm. (e) CD8 þ cells were enumerted in ronchil iopsies of infected individuls (with RSV detected y qpcr in ronchil rushings) nd PCR individuls. Dt re presented s numer of positive cells per squre millimetre of suepithelium or per.1 mm of epithelium. Significnt P vlues for two-tiled Wilcoxon mtched pirs tests re shown (Po.5, Po.1, Po.1) compring (with seline) epithelil PCR þ smples t dy 1 (P ¼.1) nd dy (P ¼.), epithelil PCR smples t dy 1 (P ¼.9), suepithelil PCR þ smples t dy 1 (P ¼.) nd dy (P ¼.19), nd suepithelil PCR smples t dy 1 (P ¼.15). NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

5 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE 1 5 Dy Dy 1 Dy CD Men Ki-CD38 cells (% of CD8 lymphocytes) Infected Unifected 1 3 Dy post inocultion Ki- c Pek Ki-CD38 cells (% of CD8 lymphocytes) 1 8 Infected (PCR) Uninfected (PCR ) d Men Ki-CD38 cells (% of CD8 lymphocytes) f Pek Ki-CD38 (% CD8 lymphocytes) Infected Unifected 1 3 Dy post inocultion r s =.9 P= Cumultive virl lod (AUC) e Pek Ki-CD38 cells (% of CD8 lymphocytes) g Pek Ki-CD38 (% CD8 lymphocytes) r s =. P= Cumultive symptoms (AUC) Figure 3 RSV infection induces short-lived ctivtion nd prolifertion of CD8 þ T cells in peripherl lood. Forty-nine dult volunteers were inoculted with RSV Memphis 3, seril lood (n ¼ 9) nd (n ¼ ) smples were stined with nti-cd3, CD8, Ki- nd CD38, nd nlysed y flow cytometry. () Plots re gted on CD3 þ CD8 þ lymphocytes. Numers represent percentge of CD8 þ T cells. One representtive suject is shown. () Men nd s.e.m. of Ki- þ CD38 þ CD8 þ T-cell frequencies in the lood of infected (PCR þ ) nd chllenged ut uninfected (PCR ) re shown. The P vlue for two-tiled Wilcoxon mtched pirs test is shown (Po.1). (c) Frequencies of Ki- þ CD38 þ cells s proportion of CD8 þ T lymphocytes were determined t dy 1 post inocultion in infected nd uninfected individuls. Men±s.e.m. re shown with the P -vlue for two-tiled Mnn Whitney test (P ¼.1). (d) Men nd s.e.m. of Ki- þ CD38 þ CD8 þ T-cell frequencies in the of infected (PCR þ ) nd chllenged ut uninfected (PCR ) re shown. The significnt P vlue for two-tiled Wilcoxon mtched pirs test is shown (P ¼.31). (e) Frequencies of Ki- þ CD38 þ cells, s proportion of CD8 þ T lymphocytes were determined t dy 1 post inocultion in mtched lood nd smples. The significnt P vlue for the Wilcoxon mtched pirs tests is shown (P ¼.59). Correltions etween pek frequencies of Ki- þ CD38 þ CD8 þ Tcells 1 dys post infection in lood nd (f) cumultive virl lod nd (g) symptoms (trpezoidl re under the curve (AUC)) re shown using non-liner regression nd Spermn s rnk correltion. NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 5

6 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 1, NS1nd N NS nd P M SH nd G A1:1 A:1 B: NS 1, F M L_1 L_ L_3 L_ NS1_1 NS1_ NS1_3 NS1_ NS_1 NS_ NS_3 NS_ N_1 N_ N_3 N_ P_1 P_ M_1 M_ M_3 M_ M_5 M_ NS1 nd N NS1 nd P M SH nd G F M L_1 Peptide pools L_ L_3 L_ NS1_1 NS1_ NS1_3 NS_1 NS_ NS_3 NS_ N_1 N_3 N_ N_5 N_ P_1 P_ M_1 M_ M_3 M_ Peptides NS1 nd N NS1 nd P M SH nd G F M L_1 A1:1 A:1 B: L_ L_3 L_ 3 1 NS1_1 NS1_ N_ N_3 N_ N_5 N_ N_ N_8 N_9 N_1 N_11 N_1 M_1 M_ M_3 M_ M_5 M_ M_ M_8 M_9 M_1 SH_1 SH_ G_1 G_ G_3 G_ G_5 G_ G_ G_8 G_9 L_1 L_ L_3 L_ L_5 L_ L_8 L_9 c 3 1 1, Dy NS1_ NS1_ M_ M_ NS_3 N_ N_8 G_ L_3 A1:1 A:1 B: Dy Dy Suject d A1:1 Dy Dy 1 D A:1 Dy Dy 1 D NS B: Dy Dy 1 D Figure Immunodominnce hierrchies in MHC clss-i-restricted RSV epitope-specific CD8 þ T-cell responses. Epitopes were predicted in silico for inding nd used to stimulte dy 1 PBMCs from HLA-mtched infected volunteers with sufficient cells (HLA A1:1, n ¼ ; A:1, n ¼ 5; nd B:, n ¼ ) in interferon-g (IFN-g) ELISpot ssys. () Peptides were pooled ccording to their originting RSV protein. Two-tiled Mnn Whitney tests were crried out etween peptide pools nd negtive control wells (A1:1_NS1/N, P ¼.; A:1_NS1/N, P ¼.9; A:1_NS/P, P ¼.119; nd B:_NS1/N, P ¼.). () Individul peptides from positive pools were susequently tested seprtely. Dotted lines represent cutoff of 5 spots per 1 PBMCs. Mens±s.e.m. nd P vlues for unpired Mnn Whitney tests re shown (A1:1_NS1_, P ¼.; A1:1_NS1_, P ¼.; A1:1_M_, P ¼.9; A1:1_M_, P ¼.9; A:_NS_3, P ¼.; B:_N_, P ¼.9; B:_N_8, P ¼.9; B:_G_, P ¼.8; nd B:_L_3, P ¼.9). (c) The medin frequencies of IFN-g þ cells in response to individul RSV epitopes presented y HLA-A1:1 (lues), A:1 (reds) nd B: (greens) re shown in 1 HLA-mtched RSV-infected individuls t dy, 1 nd post infection. (d) The frequencies of medin totl IFN-g-producing cells t seline, dy 1 nd dy post infection in response to HLA-A1:1 (lue)-, A:1 (red)- nd B: (green)-restricted epitopes in 1 HLA-mtched infected individuls re shown. P vlues for Wilcoxon mtched pirs tests re shown (A1:1, P ¼.11; A:1, P ¼.39; nd B:, P ¼.15; NS (not significnt) ¼ P.5, Po.5, Po.1, Po.1). NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

7 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE A:1-restricted FLV epitope were B1-fold lower. However, the FLV response ws more prominent in individuls without the A1:1 or B: lleles, suggesting the effect of immunodomintion. Thus, we identified severl new RSV epitopes (oth immunodominnt nd sudominnt) for three common HLA lleles with potentil s vccine trgets. The immunodominnce hierrchies indicted mrked quntittive differences in responses determined y HLA type nd the influence of HLA lleles on ech other when co-expressed. RSV-specific CD8 þ T cells ccumulte in convlescent irwys. MHC peptide tetrmers were constructed using three immunodominnt epitopes (A1-M-YLE, A-NS-FLV nd B-N-NPK) to investigte RSV-specific CD8 þ T cells in further detil y flow cytometry in lood nd (Fig. 5). In lood t seline, tetrmer þ cells were found t low frequencies rnging from undetectle to.3% of CD8 þ lymphocytes (Fig. 5,d). After dy post infection, these expnded coincident with the fll in virl lod (Fig. 1e). They peked t dy 1 post infection with up to men 18-fold increse, fter which epitope-specific popultions contrcted rpidly. While tetrmer þ cells still remined elevted in most individuls dys post infection, their numers were not mintined long term, so tht y months post infection there ws no sttisticl difference compred with their preinfection levels. Anlysis of mtched smples from the sme infected sujects, however, reveled n unexpected pttern of CD8 þ Dy Dy 1 Dy ( 3) Tetrmer Tetrmer cells (% CD8 lymphocytes) CD8.5 A1-M-YLE. A-NS-FLV.5 B-N-NPK c d e 3.3 Tetrmer cells (% CD8 lymphocytes) 1 Tetrmer cells (% CD8 lymphocytes) Men tetrmer cells (% CD8 lymphocytes)..1 A1-M-YLE A-NS-FLV B-N-NPK Figure 5 RSV ntigen-specific CD8 þ T-cell kinetics diverge in compred with lood. Whole lood/pbmcs nd from RSV-infected individuls were co-stined with nti-cd3, CD8 nd tetrmers, nd then nlysed y flow cytometry. () Numers represent the percentge of A1-M-YLE þ CD8 þ T cells s proportion of CD3 þ lymphocytes t dy,, 1, 1 nd post inocultion. Representtive plots for one suject gted on CD3 þ lymphocytes re shown. () The frequencies of A1-M-YLE (n ¼ 9), A-NS-FLV (n ¼ 1) nd B-N-NPK (n ¼ 8) tetrmer-positive cells in lood s proportion of CD8 þ T cells re shown up to months follow-up. P vlues for Wilcoxon mtched pirs tests compred with dy re shown (A1-M-YLE dy 1, P ¼.39; dy 1, P ¼.39; dy, P ¼.39; A-NS-FLV dy 1, P ¼.88; B-N-NPK dy 1, P ¼.8; dy 1, P ¼.3; nd dy, P ¼.). (c) The frequencies of A1-YLE, A-NS-FLV nd B-N-NPK tetrmer-positive cells in s proportion of CD8 þ Tcells re shown. P vlues for Wilcoxon mtched pirs tests re shown compring dy 1 (P ¼.15) nd dy (P ¼.) frequencies with seline. Arevited P vlues re shown: NS (not significnt) ¼ P.5, Po.5, Po.1. Men±s.e.m. of epitope-specific CD8 þ T-cell responses re shown in (d) lood (n ¼ ) nd (e) (n ¼ 13). Tetrmer cells (% CD8 lymphocytes) Men tetrmer cells (% CD8 lymphocytes) 1 8 NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

8 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 T-cell kinetics tht diverged mrkedly from those in lood (Fig. 5c). While in this comprtment, tetrmer þ cells lso strted incresing in frequency fter dys, t oth dy 1 nd dy they were significntly more frequent thn in lood (P ¼.8 nd., respectively, Wilcoxon mtched pirs test), with sevenfold higher men frequency of A1-M-YLE þ cells in thn in lood t dy 1 (Fig. 5d,e). Furthermore, the frequency of tetrmer þ cells continued to rise in into the convlescent period despite the sence of symptoms or virus detection y qpcr, while contrction of these popultions hd occurred in lood. Therefore, t dy post infection, the men frequency of A1-M-YLE þ cells ws 11-fold greter in thn in lood. Phenotypiclly distinct RSV-specific CD8 þ T cells in irwys. In view of the strikingly divergent kinetics in nd lood, we went on to nlyse the phenotypic differences etween RSV-specific CD8 þ T cells in the two comprtments. In t rest, lmost ll (8 9%) tetrmer þ cells displyed the cnonicl CD9 þ CD13 þ Trm phenotype (Fig. ). In contrst, efore infection, no tetrmer þ cells in lood expressed either mrker. However, during infection, proportion of tetrmer þ cells in lood upregulted CD13, peking t dy 1 then decresing with resolution of infection. In, the CD13 single-positive popultion lso incresed in frequency t dy 1, trnsiently mking up greter proportion of tetrmer þ cells, which then returned to predominntly CD9 þ CD13 þ phenotype during convlescence. Anlysis of CD5RA nd CCR llowed further ctegoriztion of memory susets nd indicted tht the mjority of tetrmer þ cells exhiited effector memory (Tem) or effector memory re-expressing CD5RA (Temr) phenotypes, with predominntly Tem cells in (Fig. ). In lood, infection led to significnt increse in T-effector cells (expressing the CD5RA /CCR þ phenotype) t the expense of Temr cells, chnge tht ws not seen in the irwy. This occurred vi prolifertion strting etween dy 3 nd dy post infection, prlleling the polyclonl CD8 þ T-cell response nd peking t round dy 1, when B85% (IQR 9%) of epitope-specific CD8 þ T cells in lood showed ctivtion nd prolifertion y CD38 nd Ki- expression (Fig. c). This ws lso seen in tetrmer þ cells, lthough proportion of these remined quiescent. However, y dy, despite the continued ccumultion of tetrmer þ cells in the, there ws no evidence of on-going ctivtion or prolifertion. In lood, these cells upregulted perforin nd grnzyme B, which lso peked round dy 1 with round 33% (medin, IQR 1 %) expressing cytotoxicity molecules (Fig. ). However, few tetrmer þ cells expressed perforin. Furthermore, only minority upregulted grnzyme B, which gin peked round dy 1. During infection, the co-stimultory molecules CD nd CD were oth downregulted in proportion of RSV-specific CD8 þ T cells in the lood nd in the mjority of individuls hd not returned to seline frequencies y dy (Fig. ). In contrst, even efore infection, lrge proportion of RSV-specific CD8 þ T cells hd lredy downregulted CD nd CD þ CD þ doule-positive cells were in the minority. Furthermore, there were no significnt chnges in their expression following infection. Finlly, following ctivtion, RSV-specific CD8 þ T cells in lood upregulted CCR5 nd downregulted CDL, llowing homing to sites of inflmmtion nd wy from lymphoid tissues (Fig. c; Supplementry Fig. 3). In, there ws trend towrds tetrmer þ Trm cells upregulting CCR5 nd there ws no CDL expression t ny time. These dt suggest tht RSV infection induces trnsient increse of virus-specific CD8 þ T cells in the lood, with phenotype chrcteristic of cytotoxic effector cells, some of which chnges persist into the convlescent period. In contrst, Trm cells in the irwy lter less mrkedly following infection, with lrge proportion showing no prolifertive response nd overll reduced expression of cytotoxicity molecules. Limited functionlity of RSV-specific CD8 þ memory T cells. In studies of influenz, virus-specific CD8 þ memory T-cell frequencies in the lood hve een shown to correlte inversely with symptom severity during susequent infection 15,1. This hs not een demonstrted with other viruses, nd indeed we found no significnt difference in seline frequencies of RSV-specific CD8 þ T cells in peripherl lood etween those susequently deemed infected nd those who remined uninfected (Supplementry Fig. ). In ddition, there ws no sttisticl reltionship etween the mgnitude of pre-existing HLA-restricted responses nd the severity of disese, expressed throughout s cumultive virl lod or symptom score to smooth out dy-to-dy vriility in spite of the close correltion etween pek nd cumultive vlues (Supplementry Fig. ). Anlysis of CD8 þ T cells induced y highly protective systemic vccines, such s yellow fever, hve displyed polyfunctionlity tht my correlte with their efficcy 9. To investigte whether the functionlity of RSV-specific CD8 þ T cells might e contriuting to lck of protection, cytokine production ws nlysed y flow cytometry (Fig. 8). Overll, the frequencies of cytokine-producing CD8 þ T cells following short-term peptide stimultion were modest with verge medin frequencies of.35 (ginst YLE) nd.5% (ginst NPK). These were similr to those seen ginst the immunodominnt influenz epitope M1-GIL in A:1-expressing individuls from this cohort. However, most cytokine-expressing RSV-specific cells produced only single cytokine (IFN-g, tumour necrosis fctor or interleukin-) with n verge of only.3% (±s.e.m..3) producing three (Fig. 8,c; Supplementry Fig. 5). By comprison, influenz-specific CD8 þ T cells were more polyfunctionl, with 39% producing two cytokines nd significntly higher proportion (15.1%±s.e.m..3) producing three (P ¼.9 y Mnn Whitney test). Thus, RSV-specific memory CD8 þ T cells in lood hve reduced functionlity compred with those ginst influenz, potentilly contriuting to reduced protective cpcity. CD8 þ Trm cells in irwys correlte with reduced disese. These findings mirror our previous study tht showed RSV-specific nsl IgA correlting strongly with protection from PCR-confirmed infection with greter predictive power thn serum IgG in this regrd 9. However, where infection did occur, high nsl IgA titres did not reduce disese severity (Supplementry Fig. ). We hypothesized tht locl cell-medited immunity could provide the next lyer of defence y cting to directly eliminte cell-ssocited virus nd thus reduce symptoms. To test this, we nlysed the frequencies of tetrmer þ CD8 þ T cells ginst immunodominnt epitopes (representtive of the RSV-specific CD8 þ T-cell popultion efore infection) nd their reltionship with infection risk nd disese severity. At seline, RSV-specific CD8 þ Trm cells were lredy enriched in with significntly higher frequencies compred with lood irrespective of specificity (Fig. 9). However, in contrst to mucosl IgA, their frequency hd no impct on the likelihood of PCR-confirmed RSV infection (Fig. 9). Insted, the higher the frequency of RSV-specific CD8 þ T cells in seline, the lower the cumultive symptom score (Spermn s r ¼.91, P ¼.1; Fig. 9c) nd the lower the cumultive virl lod (Spermn s r ¼.8, 8 NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

9 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE c CD13 CD38 CCR Dy Dy 1 Dy CD9 Dy Dy 1 Dy CD5RA Dy Dy 1 Dy Ki CD13(% tetrmer CD8 lymphocytes) CD9CD13 (% tetrmercd8 lymphocytes) % tetrmercd8 lymphocytes % tetrmercd8 lymphocytes Ki-CD38(% tetrmer CD8 lymphocytes) Ki-CD38 (% tetrmer CD8 lymphocytes) Nive Tcm Tem/eff Temr Dy post-infection Figure RSV-specific CD8 þ T cells in disply distinctive resident memory phenotype. Tetrmer þ CD8 þ T cells in lood nd were costined for mrkers to ssess their differentition sttus. () CD9 nd CD13 s cnonicl mrkers of resident memory CD8 þ T cells re shown in lood (n ¼ 9) nd (n ¼ 5) from infected volunteers. Significnt P vlues for two-tiled Wilcoxon mtched pirs tests in lood compred with seline re shown (dy, P ¼.313; dy 1, P ¼.39; dy 1, P ¼.313; nd dy, P ¼.313). () Memory mrkers CD5RA nd CCR re shown in lood (n ¼ 19) nd (n ¼ 8). Men±significnt P vlues for two-tiled Wilcoxon mtched pirs tests compred with seline re shown in lood for T-effector/effector memory cells (dy, P ¼.3; dy 1, P ¼.; dy 1, P ¼.; dy, P ¼.) nd effector memory T cells re-expressing CD5RA (dy, P ¼.3; dy 1, P ¼.5; dy 1, P ¼.3; dy, P ¼.135). (c) Prolifertion nd ctivtion mrkers Ki- nd CD38 re shown in lood (n ¼ 19) nd (n ¼ 8). Significnt P vlues for two-tiled Wilcoxon mtched pirs tests re shown compred with seline in lood (dy, P ¼.5; dy 1, P ¼.1; nd dy 1, P ¼.5) nd (dy versus dy 1, P ¼.; nd dy 1 versus dy, P ¼. s no Ki- þ CD38 þ cells were found in ny seline smples). Throughout, representtive plots from single suject t dy, 1 nd post infection re shown with tetrmer þ cells s red dots nd totl CD8 þ T cells in grey contours. Summrized dt re shown for ll RSV-infected sujects (who could e nlysed using tetrmers) with medin frequencies shown in red. Arevited P vlues re shown: NS (not significnt) ¼ P.5, Po.5, Po.1. NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 9

10 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 c Grnzyme B CD CDL Dy Dy 1 Dy Dy Dy 1 Dy CD Dy Dy 1 Dy Perforin CCR Grnzyme Bperforin cells (% tetrmercd8 lymphocytes) Grnzyme Bperforin cells (% tetrmercd8 lymphocytes) % TetrmerCD8 lymphocytes % TetrmerCD8 lymphocytes CCR5 cells (% tetrmercd8 lymphocytes) CCR5 cells (% tetrmercd8 lymphocytes) CD CD CD CD CD CD CD CD Figure Reduced expression of cytotoxicity nd co-stimultory mrkers y RSV-specific CD8 þ T cells in the irwy. Tetrmer þ CD8 þ T cells in lood nd were co-stined for phenotypic mrkers. () Cytotoxicity molecules perforin nd grnzyme B re shown in lood (n ¼ 19) nd (n ¼ 5). Significnt P vlues for two-tiled Wilcoxon mtched pirs tests compred with seline re shown in lood (dy, P ¼.1; dy 1, P ¼.9; nd dy 1, P ¼.13). () Co-stimultory molecules CD nd CD re shown in lood (n ¼ 19) nd (n ¼ 1). Men±s.e.m. nd significnt P vlues for two-tiled Wilcoxon mtched pirs tests compred with seline re shown in lood for CD þ CD þ cells (dy 1, P ¼.5; dy, P ¼.8) nd CD CD cells (dy, P ¼.1). (c) Homing receptors CCR5 nd CDL re shown in lood (n ¼ 19) nd (n ¼ 5). Significnt P vlues for two-tiled Wilcoxon mtched pirs tests compred with seline re shown in lood (dy 1, P ¼.9; dy 1, P ¼.5). Throughout, representtive plots from single suject t dy, 1 nd post infection re shown with tetrmer þ cells s red dots nd totl CD8 þ T cells in grey contours. Summrized dt re shown for ll RSV-infected sujects (who could e nlysed using tetrmers) with medin frequencies shown in red. Arevited P vlues re shown: NS (not significnt) ¼ P.5, Po.5, Po.1. 1 NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

11 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE 1 5 Dy Dy 1 Dy c Dy Dy 1 Dy M-YLE IFN-γ % Responding CD8 T cells IFNγ TNF IL M-YLE TNF IL % Responding CD8 T cells N-NPK % Responding CD8 T cells Influenz M1-GIL 8 Influenz M1-GIL N-NPK Three functions Two functions One function Dy Dy 1 Dy Figure 8 RSV-specific CD8 þ T cells in lood show limited polyfunctionlity. PBMCs from individuls inoculted with RSV were stimulted with peptide epitopes (YLE, n ¼ 3; NPK, n ¼ ; nd influenz M1-GIL, n ¼ ) nd susequently intrcellulrly stined for IFN-g, tumour necrosis fctor (TNF) nd interleukin- (IL-) for nlysis y flow cytometry. () Flow cytometric dt gted on CD3 þ CD8 þ lymphocytes from one representtive donor re shown. Numers represent percentge of CD8 þ lymphocytes. () The men (±s.e.m.) frequencies of cytokine-producing CD8 þ Tcells s determined y Boolen gting re shown s percentges of totl responding cells. (c) The proportion of single, doule nd triple cytokine producers is shown. P ¼.31; Fig. 9d) in those who susequently developed PCR þ infection. These correltions held true on correltion with lower respirtory trct symptoms lone (Fig. 9e, Spermn s r ¼.3, P ¼.) ut less so with upper trct or systemic symptoms (Supplementry Fig. ). Similrly, higher frequencies of pre-existing CD8 þ Trm cells correlted with lower ronchil virl lod (Spermn s r ¼.5, P ¼.; Fig. 9f), suggesting tht these cells might hve direct role in virl clernce. In summry, while memory CD8 þ T cells in lood do not correlte with protection ginst RSV, higher frequencies of CD8 þ Trm cells in the irwy re ssocited with improved virl control nd reduced symptom severity, suggesting tht these re likely to hve role in meliorting disese if present in sufficiently high numers. Discussion Studies in niml models hve incresingly highlighted the distinctiveness of locl immunity nd the importnce of this comprtmentliztion to protective immune responses. Here we present the first interventionl study to trck virus-specific cell-medited immunity in the humn irwy. Longitudinl nlysis of the CD8 þ T-cell response showed the extent to which these cells in the humn irwy diverge from those in the lood during RSV infection in lmost every respect nd provides strong evidence for the role tht humn Trm cells ply in protection. RSV cused surprisingly extensive inflmmtion in most infected individuls despite miniml or no lower respirtory trct symptoms. Compred with rhinovirus (where neither mcroscopic inflmmtion nor cellulr infiltrtes re significntly seen on ronchoscopy in helthy dults 3 ), RSV led to sustntilly greter lower respirtory trct involvement. Infection cused CD8 þ T-cell infiltrtion of the respirtory mucos with n unexpected ccumultion of very lrge numers of RSV-specific cells well into convlescence, distinct from lood in the sme individuls. In oth lood nd irwy, ctivtion of CD8 þ T cells occurred cutely, coinciding with flling virl lod, suggesting their involvement in virus elimintion. Shortly fter pek virl shedding, prolifertion cesed, implying tht virl repliction ws required to drive this response. In immunodeficient children, the sence of T cells is ssocited with prolonged nd more severe infection, while RSV disese risk in older dults is elieved to e relted to fewer IFN-g-producing T cells 1,31. However, the low frequency, short-lived oosting nd possily limited functionlity of RSV-specific memory CD8 þ T cells in lood ment tht poor correltion with protection ws seen. In contrst, epitope-specific CD8 þ Trm cells in the irwys with their significntly higher frequencies nd locliztion ner the site of infection did indeed correlte with reduced disese severity. This strongly supports the hypothesis tht virus-specific CD8 þ Trm cells in the irwy ply direct role in erly clernce of respirtory viruses, while memory T cells in lood correlte indirectly (if t ll). Previous studies in mn, including those inferring the protective role of CD þ nd CD8 þ T cells from peripherl lood in influenz 15,1, mke the ssumption tht there is direct nd proportionl reltionship etween lood nd mucosl immunity. Our findings show tht this ssumption is fundmentlly incorrect nd tht these comprtmentlized popultions must e exmined directly. Recent phse-i clinicl trils hve shown tht virusvectored RSV vccine cndidtes cn induce T cells in lood 3.Our dt suggest tht their cpcity to induce Trm cells following intrnsl dministrtion should e explored further. These findings might lso explin why CD8 þ T-cell-inducing influenz vccines tht induce primrily systemic responses demonstrte suoptiml efficcy 5,33. In mice, expression of the receptors CD9 nd CD13 llows retention of CD8 þ Trm in tissues 3. Although they my not represent the entire Trm popultion 35, CD9 þ CD13 þ CD8 þ Trm cells hve een shown to loclize to the murine irwy NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 11

12 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 Tetrmer cells (% CD8 lymphocytes) Tetrmer cells pre-inocultion (% CD8 lymphocytes) Uninfected Infected A1-M-YLE A-NS-FLV B-N-NPK c Cumultive symptom score (trpezoidl AUC) r s =.91 P =.1 d Cumultive virl lod (AUC log 1 copies per ml) 3 1 r s =.8 P = Tetrmer cells in (% of CD8 lymphocytes) Tetrmer cells in (% of CD8 lymphocytes) e Cumultive LRT symptoms (trpezoidl AUC) r s =.3 P = Tetrmer cells in (% of CD8 lymphocytes) f Pek virl lod in ronchil rushings (log1 copies per ml) r s =. P = Tetrmer cells in (% of CD8 lymphocytes) Figure 9 Pre-existing RSV-specific memory CD8 þ T cells in the irwy correlte with reduced disese severity. RSV-specific CD8 þ T-cell responses to HLA-A1:1-, A:1- nd B:-restricted epitopes were nlysed y tetrmer stining efore inocultion. () Bseline frequencies of tetrmer þ cells in infected HLA-A1:1 þ (n ¼ 9), A:1 þ (n ¼ ) nd B: þ (n ¼ 11) in mtched lood nd smples were compred. The medins nd P vlues for Wilcoxon mtched pirs tests (A1-M-YLE, P ¼.3; A-NS-FLV, P ¼.313; B-N-NPK, P ¼.195) re shown (Po.5). () Bseline frequencies of tetrmer þ cells in from infected (n ¼ 1) nd uninfected (n ¼ 1) individuls were compred nd tested for significnt difference using the Mnn Whitney test (P ¼.95). Medins re shown. (c) Non-liner regression ws used to ssess the ssocition etween seline RSV-specific CD8 þ cell frequencies in nd disese severity in infected individuls s mesured y (c) cumultive symptom score, (d) nsl virl lod (trpezoidl re under the curve (AUC)), (e) cumultive lower respirtory trct symptoms nd (f) pek virl lod in ronchil rushings. Spermn s rnk correltion coefficient (r s ) nd P vlues re shown. following respirtory priming with influenz nd medite cross-protection ginst heterologous strins 1. Much less is known in humns, where previous studies hve een sed solely on uninfected lung tissue removed during cncer surgery or non-living orgn dontions nd limited to cross-sectionl nlysis of resting T cells,3. These estimtes of frequency showed tht Trm cells re undnt in the lung where, t rest, influenzspecific CD8 þ Trm cells re more frequent thn those for cytomeglovirus. Our longitudinl nlyses dvnce these findings y showing in vivo how Trm cells in the humn irwy rise following virl infection nd how they relte to disese severity. During cute infection, RSV-specific CD8 þ Trm cells were trnsiently joined y CD13 þ CD9 popultion tht could represent popultion tht migrte from lood to irwy, lthough the ontogeny of Trm cells hs yet to e fully elucidted 3,3. Interestingly, this my e the reverse of CD8 þ Trm cells generted ginst herpes simplex virus 1 (HSV-1) in murine skin, where sequentil induction of first CD9 nd then CD13 hs een oserved, suggesting the impct of tissue type, pthogen nd/or interspecies differences 38. RSV-specific Trm cells in the irwy showed phenotypic chnges suggestive of dvnced differentition, with downregultion of co-stimultory mrkers, ut lso reduced expression of cytotoxicity molecules. We therefore propose tht the very high Trm cell frequencies seen during erly convlescence cn confer solute protection ginst erly symptomtic reinfection ut my e functionlly regulted to prevent excessive dmge to the delicte lung rchitecture. Inflmmtory chnges seen in the irwy during convlescence my represent the effect of lrge numers of CD8 þ Trm cells intercting with residul RSV 1 NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1

13 NATURE COMMUNICATIONS DOI: 1.138/ncomms1 ARTICLE ntigen, ut these cused no symptoms in helthy dults. In contrst, in murine systems where lrge numers of RSV-specific CD8 þ T cells re present t the sme time s high virl titres, life-thretening immunopthology cn occur 39. In our study, it ws not deemed cceptle to suject volunteers to fourth ronchoscopy to determine the longevity of Trm cells, while other phenotypic nd functionl ssessments were likely to e qulittively similr to the preinfection time point. Nevertheless, it cn e inferred tht in these volunteers, CD8 þ Trm frequencies hd wned significntly, since the end of their lst nturl RSV infection (though less precipitously thn in lood). This wning in the lung contrsts with long-lived Trm cells in other tissues nd my represent nother mechnism y which immunopthology is reduced. Outstnding questions including how Trm cells differentite nd the conditions required for persistence remin mjor hurdles for generting protective cell-medited immunity in the mucos. Both residul ntigen nd cytokines such s TGF- hve een implicted in Trm cell mintennce. Blockde of ntigen recognition in the lungs of influenz-infected mice leds to loss of Trm cells, while TGF- is expressed in respirtory mucos nd in vitro cn induce upregultion of CD13 (ref. 1). This is supported y our findings of persistent RSV ntigen in the irwy ssocited with virus-specific CD8 þ Trm cells even dys fter inocultion. While there ws no pprent disese ssocited with high frequencies of RSV-specific CD8 þ Trm cells in our cohort, dmging immunopthology cused y oosting them in other ge groups nd settings remins risk. These dt show tht immune responses in lood cn fundmentlly misrepresent those t the site of infection. Our ex vivo nlyses of the respirtory comprtment show tht dptive immunity ginst respirtory viruses comprises multiple specilised nd non-redundnt protective mechnisms distinct in time nd sptil orgniztion. In RSV, loclly produced mucosl IgA constitutes n initil rrier to virus entry ut does not significntly modulte disese severity once the rrier is reched 9. When tht occurs, CD8 þ Trm cells provide erly recognition nd virus elimintion, thus reducing symptom severity nd virl lod. Only fter these does systemic immunity ct to prevent widespred disese. We therefore propose tht Trm cells represent one of severl immune mechnisms tht should e hrnessed together for optiml vccine-medited protection. Methods Ethics sttement. The study ws pproved y the UK Ntionl Ethics Service London Fulhm (study numers 1/H11/9 nd 11/LO/18). Written informed consent ws otined from ll volunteers. Study design. Helthy, non-smoking dults ged yers were recruited etween 1 nd 13. All sujects were genotyped for HLA clss-i loci using sequence-sed typing. A totl of 9 sujects were inoculted with 1 plqueforming units of RSV Memphis 3 (Meridin Lifesciences, Memphis, USA). Following the chllenge, prticipnts were qurntined for 1 dys. Cold symptoms were ssessed using symptoms diries completed dily y prticipnts. Reported symptoms with score of (sent), (moderte) or 3 (severe) included sneezing, nsl dischrge, nsl ostruction or sore throt (upper respirtory trct), hedche, mlise, fever (systemic) nd cough, wheeze, shortness of reth (lower respirtory trct symptoms). Cold ws confirmed if two out of the following three conditions were fulfilled: nsl dischrge lsting Z3 dys, sujective feeling of cold reported or cumultive 1-dy symptom score Z1. smples were otined t seline nd time points post inocultion s indicted in the text. Nsl lvge smples were collected dily nd used for virl lod quntifiction y qpcr s previously descried 9,. Briefly, totl RNA ws isolted using the QIAmp Virl RNA kit (Qigen) ccording to the mnufcturer s instructions. Reverse trnscription of 13 ml of totl isolted RNA ws chieved using the High Cpcity RNA-to-cDNA kit (Applied Biosystems) ccording to the mnufcturer s instructions. qpcr with reverse trncription rections for RSV N gene were chieved using primers shown in Supplementry Tle nd the TqMn Universl Mster Mix II (Applied Biosystems) nd 5 Fst Rel-Time PCR System (Applied Biosystems). Asolute quntifiction ws clculted using plsmid DNA stndrd curve. As shown previously, infection sttus s determined y qpcr positivity ccorded 1% with multiplex qulittive PCR nd plque ssy. Bronchoscopists were linded to the infection sttus of the volunteers. Bronchoscopies were undertken t 1 dys efore inocultion nd or 1 dys nd dys post infection. Mcroscopic ppernce of the irwys ws reported s norml, erythemtous or erythemtous with contct leeding. Bronchil rushings nd iopsies were otined t ech time point. Bronchil epithelil cells were wshed with RPMI/1% fetl clf serum (FCS; R1) nd stored in TRIzol regent (Invitrogen, Grnd Islnd, USA) for virl lod quntifiction. ws otined y instilltion of up to 1 ml of norml sline, filtered nd centrifuged efore flow cytometric nlysis. Immunohistochemistry. Endoronchil iopsies were fixed immeditely in % prformldehyde nd prffin emedded. RSV ws stined using polyvlent mouse nti-rsv ntiody (NCL-RSV3, Leic Biosystems, UK) t 1:5 dilution. CD8 þ T cells were identified y stining with mouse nti-cd8 (M, Dko) t 1:1 dilution using the EnVision peroxidse stining method (Dko, Denmrk) s previously descried 3. Briefly, 5-mm sections were stined ccording to the mnufcturer s instructions nd n irrelevnt mouse IgG1 kpp ntiody (MOPC1) ws used s negtive control for stining specificity of mouse monoclonl ntiodies. RSV-infected A59 cells were used s positive stining controls. Quntifiction ws chieved s previously descried with opertors linded to smple timing nd infection sttus 3. Briefly, slides were coded to void oserver is nd res of epithelium nd suepithelium ssessed using Leitz Dilux light microscope, Apple Mcintosh computer nd Imge 1.5 softwre. Totl epithelil nd suepithelil res of two to three ronchil iopsies were counted for ech ronchoscopy. Cell counts were expressed s the numer of cut cell profiles with visile nucleus per mm of suepithelium nd per.1 mm of epithlium. The coefficient of vrition for repet counts of positive cells y single oserver rnged from 5 to %. PBMC isoltion. PBMC isoltion ws performed y density centrifugtion using Histopque 1 (Sigm Aldrich, USA) ccording to the mnufcturer s protocol. Cells were either used immeditely or cryopreserved in FCS (Gico Life Technologies, USA) with 1% dimethyl sulfoxide (DMSO) in liquid nitrogen for future ssys. Ex vivo interferon-c ELISpot ssy. T-cell epitope mpping ws performed y INF-g ELISpot using 1 5 PBMCs in triplicte stimulted with peptide pools (1 mgml 1 of ech peptide) for initil screening followed y deconvolution with single peptides (1 mgml 1 ). Pltes were incuted for Z1 h t 3 C nd susequently developed (Mtech, Sweden). SFU were counted using AID ELISpot softwre (Autoimmun Dignostik GmH, Germny) nd nlysed with positive wells contining Z spots per 1 cells nd P vlue of r.5 using Student s t-test in t lest two experiments. Flow cytometry. HLA clss-i (A1:1 nd B:) tetrmers were gift from Rfi Ahmed (Emory University, Atlnt, USA). Custom-mde dextrmers (A:1) were purchsed from Immudex (Copenhgen, Denmrk). Whole lood or cell smples were stined y dding ml of tetrmer nd incuting for 1 min t room temperture efore stining with surfce ntiodies for min. Dextrmer stining ws performed in the sme wy on freshly isolted PBMCs or cells. Surfce stining of whole lood ws followed y erythrocytes lysis with BD Lysis uffer. Cells were then fixed with 1% prformldehyde solution or prepred for intrcellulr stining y fixing nd permeiliztion with BD Fixtion/Permeiliztion kit. The following surfce ntiodies (including clone nd ctlogue numer) were used in the study: CD3 PE-CF59 (UCHT1, BD#5), CD APC-H (SK3, BD#1398), CD38 PE Cy (HB, BD#33585), Ki- FITC (B5, BD#55), Perforin FITC (dg9, BD#555), Grnzyme B V5 (GB11, BD#51151), CD5RA FITC (HI1, BD#55588), CCR PE (1553, BD#55), CD V5 (M-T1, BD#58), CD PE Cy (CD., BD#58), CCR5 V5 (D/CCR5, BD#511), CDL PE (DREG-5, BD#5555; ll BD Biosciences); CD8 PerCP Cy 5.5 (RPA-T8, #5-88-), CD9 FITC (FN5, ) nd CD13 PE Cy (B-Ly, #5-138-; ll ebioscience). Dilutions t which ntiodies were used re shown in Supplementry Tle 8. FlowJo softwre (FlowJo LLC, USA) ws used for dt nlysis. In vitro peptide stimultion nd intrcellulr cytokine stining. Intrcellulr cytokine stining ws used to confirm epitopes identified y ELISpot. Thwed PBMCs were rested overnight nd stimulted the next dy for h in culture medium (RPMI 1, 1% FCS, mm glutmine, 1 IU ml 1 penicillin/ streptomycin) using selected peptides (1 mgml 1 ) plus nti-cd nd nti- CD9. After h, Brefeldin A (1 ml/ml) ws dded nd fter h cells were wshed nd stined with live/ded mrker (Invitrogen, USA); IFN-g APC (B, #55), TNF PE Cy (MA11, #55), interleukin- FITC (53.111, #38; ll BD Biosciences); nd relevnt surfce mrkers. Dilutions t which ntiodies were used re shown in Supplementry Tle 8. The frequencies of expression profiles for NATURE COMMUNICATIONS :1 DOI: 1.138/ncomms1 13