Responsiveness to Influenza Vaccination Correlates with NKG2C-Expression on NK Cells.

A icle Responsi eness o In luenza Vaccina ion Co ela es wi h NKG2C-Exp ession on NK Cells Peggy Riese 1,*,†, S ephanie T i el 1,†, Rishi D. Pa hi ana 2,3, F ank Klawonn 4,5, Rebecca J. Cox 2,3,6,†and Ca los A. Guzmán1,7,† 1Depa men o Vaccinology and Applied Mic obiology, Helmhol z Cen e o In ec ion Resea ch, 38124 B aunschweig, Ge many; [email p o ec ed] (S.T.); [email p o ec ed] (C.A.G.) 2Depa men o Clinical Science, The In luenza Cen e, Uni e si y o Be gen, 5007 Be gen, No way; [email p o ec ed] (R.D.P.); [email p o ec ed] (R.J.C.) 3K.G. Jebsen Cen e o In luenza Vaccine Resea ch, Uni e si y o Oslo, 0313 Oslo, No way 4Depa men o Bios a is ics, Helmhol z Cen e o In ec ion Resea ch, 38124 B aunschweig, Ge many; [email p o ec ed] 5Depa men o Compu e Science, Os alia Uni e si y o Applied Sciences, 38302 Wol enbue el, Ge many 6Depa men o Resea ch and De elopmen , Haukeland Uni e si y Hospi al, 5021 Be gen, No way 7Cen e o Indi idualized In ec ion Medicine, 30625 Hanno e , Ge many *Co espondence: peggy[email p o ec ed]; Tel.: +49-531-61814609; Fax: +49-531-61814699 †Au ho s con ibu ed equally. Recei ed: 30 Ap il 2020; Accep ed: 3 June 2020; Published: 5 June 2020   Abs ac : In luenza accina ion o en esul s in a la ge pe cen age o low esponde s, especially in high- isk g oups. As a i s line o de ense, na u al kille (NK) cells play a c ucial ole in he igh agains in ec ions. Howe e , hei implica ion wi h ega d o accine esponsi eness is insu icien ly assessed. The e o e, his s udy aimed a he alida ion o essen ial NK cell ea u es po en ially associa ed wi h di e en ial accine esponsi eness wi h a special ocus on NKG2C- and/o CD57-exp essing NK cells conside ed o ha bo memo y-like unc ions. To his end, 16 heal hy olun ee s we e accina ed wi h an adju an ed pandemic in luenza accine. Vaccine esponde s and low esponde s we e classi ied acco ding o hei hemagglu ina ion inhibi ion an ibody i e s. A majo i y o esponde s displayed enhanced equencies o NKG2C-exp essing NK cells 7- o 14-days pos - accina ion as compa ed o low esponde s, whe eas he exp ession o CD57 was no di e en ially modula ed. The NK cell cy o oxic po en ial was ound o be con ined o CD56 dim CD16 + NKG2C-exp essing NK cells in he esponde s bu no in he low esponde s, which was u he con i med by s ochas ic neighbo embedding analysis. The p esen ed s udy is he i s o i s kind ha asc ibes CD56 dim CD16 + NKG2C-exp essing NK cells a c ucial ole in biasing adap i e immune esponses upon in luenza accina ion and sugges s NKG2C as a po en ial bioma ke in p edic ing pandemic in luenza accine esponsi eness. Keywo ds: in luenza; accina ion; accine esponsi eness; NK cells; NKG2C 1. In oduc ion Acu e espi a o y in ec ions caused by he in luenza i us a e one o he majo public heal h p oblems leading o high mo ali y a es wo ldwide, and consequen ly, o a la ge socie al economic bu den [ 1 ]. Occasionally, a no el i us a ises, leading o wo ldwide sp ead and a pandemic. In he las decade, a numbe o pandemic in luenza accines ha e ecei ed ma ke ing au ho iza ion [ 2 ]. An inc ease in hemagglu ina ion inhibi ion (HAI) an ibody i e is commonly used o measu e he esponse o he accine. Howe e , an ibody esponses may be es ic ed in high- isk g oups (i.e., Vaccines 2020,8, 281; doi:10.3390/ accines8020281 www.mdpi.com/jou nal/ accines Vaccines 2020,8, 281 2 o 18 he e y young and he elde ly, indi iduals wi h co-mo bidi ies), esul ing in a la ge pe cen age o low esponde s [3,4]. The gene a ion o p o ec i e immuni y equi es he in e play be ween inna e and adap i e immune cells. NK cells a e desc ibed as c ucial inna e immune cells in he igh agains in luenza in ec ions [ 5 ]. Recen indings in NK cell biology p o ide u he e idence on speci ic unc ional ea u es o NK cells, which highligh a o me ly unde es ima ed ole du ing in ec ion. Se e al s udies e ealed unexpec ed NK cell cha ac e is ics, like hei con inuous di e en ia ion p ocess and he impac o he educa ion s a us on he magni ude o NK cell unc ionali y [ 6 – 9 ]. Likewise, i was disco e ed ha NK cells display adap i e immune ea u es simila o T cells, including he gene a ion o memo y-like NK cells [ 10 , 11 ]. Se e al epo s highligh ed he occu ence o mu ine memo y-like NK cells by (i) hap en-induced con ac hype sensi i i y, (ii) mu ine cy omegalo i us in ec ion (MCMV) and (iii) cy okine s imula ion [12–15]. Human memo y-like NK cells we e i s desc ibed in i o as a unique subse o NK cells exp essing CD57 and NKG2C de ec ed in a coho o indi iduals in ec ed wi h he human cy omegalo i us (HCMV) [ 16 – 18 ]. Subsequen ly, i was also demons a ed ha human memo y-like NK cells, cha ac e ized by he exp ession o NKG2C, could be induced in i o by cy okine s imula ion [ 19 , 20 ]. The CD94-NKG2C ac i a ing NK cell ecep o binds o HLA-E and ac s ia he ITAM-bea ing DAP12 signaling pa hway [ 21 ]. CD57 is mainly exp essed by e minally di e en ia ed NK cells, which a e sugges ed o ha e unde gone clonal expansion ollowing in ec ion. CD57 + NK cells a e desc ibed o ha bo a lowe p oli e a i e capaci y and o be less cy o oxic in esponse o cy okine s imula ion, bu show highe CD16-induced cy o oxici y [22]. Nex o hei newly disco e ed unc ional ea u es, NK cells a e well known o in e ac di ec ly and indi ec ly wi h adap i e immune cells. Thus, i can be hypo hesized ha NK cells migh be conside ed as ele an playe s in he ini ia ion o adap i e immuni y ollowing in luenza accina ion. Suppo ing e idence comes om a ecen s udy, in which NK cell esponsi eness ollowing in luenza accina ion was in es iga ed. The esul s o his s udy demons a ed ha NK cells wi h an in acellula immune memo y, cha ac e ized by enhanced IFN γ sec e ion ollowing an igen-speci ic e-s imula ion, a e gene a ed ollowing accina ion [ 23 ]. These NK cells displayed an inc eased in e naliza ion o he NKp46 ecep o , which is known o in e ac wi h he in luenza su ace p o ein hemagglu inin (HA). Howe e , despi e his agmen a y e idence, he e is s ill a conside able pauci y o knowledge in his ield. In his ega d, NK cell subse s exp essing CD57 and NKG2C ha e ye o be add essed. Thus, in he p esen s udy, he impac o he H1N1 accina ion on pheno ypic and unc ional changes o NK cells exp essing CD57 and NKG2C and hei ecip ocal in luence on he accina ion e icacy was in es iga ed. 2. Ma e ials and Me hods 2.1. S udy Design Six een heal hy olun ee s (heal h ca e wo ke s (HCWs)) we e accina ed wi h he pandemic in luenza accine Pandem ix ® (spli i ion, inac i a ed; A/Cali o nia/07/2009 (H1N1) -like s ain (X-179A), GlaxoSmi hKline, B en o d, UK), adju an ed wi h AS03 as pa o a la ge clinical ial. Fou een o he pa icipan s we e emale and wo we e male (one no mal and one low- esponde ), and hey we e bo n be ween 1951 and 1987 wi h a median bi h yea o 1974 and 1969 o no mal- and low- esponde s, espec i ely. O he han h ee pa icipan s (no mal esponde s), all pa icipan s ecei ed p e ious seasonal in luenza accines. All pa icipan s p o ided w i en in o med consen be o e inclusion in he s udy, which had e hical (Regional Commi ee o Medical Resea ch E hics (e hical app o al numbe is 2009/1224, issued by REC wes ), Wes e n No way (REK Ves )) and egula o y (No wegian Medicines Agency) app o al and is egis e ed a he Na ional Ins i u e o Heal h Da abase Clinical ials.go (NCT01003288). Human subjec igh s we e p o ec ed du ing he ial and he da a analysis. Blood (clo ed and Cell P epa a ion Tubes (CPTs)) was collec ed p io and 7-, 14-, 21- and 180-days pos - accina ion [ 24 ]. Pe iphe al blood mononuclea cells (PBMCs) we e Vaccines 2020,8, 281 3 o 18 isola ed om CPT ubes acco ding o he manu ac u e ’s ins uc ions and c yo-p ese ed in 90% e al bo ine se um (FBS)/10% dime hyl sul oxide (DMSO) un il u he analysis. 2.2. Humo al Immune Responses The HAI i e s in se um samples p e- accina ion and 7-, 14-, 21-, 90- and 180-days pos - accina ion we e de e mined by a HAI assay using he X179A i us. The assay was pe o med wi h 0.7% u key ed blood cells, as desc ibed p e iously [ 24 ]. The i e s analyzed a days 0 and 90 we e used o de ine esponde s and low esponde s. Vaccinees wi h a 4- old se ocon e sion o a i e inc ease >40 we e conside ed as esponde s. Human cy omegalo i us (CMV)-speci ic IgG an ibodies we e assessed using he Alini y i ins umen (Abbo ). 2.3. Cellula Immune Responses PBMCs we e hawed and 1 × 10 6 o 4 × 10 6 cells/sample we e e-s imula ed o 16 h in comple e RPMI 1640 (Gibco, supplemen ed wi h 10% FCS, 5% Penicillin/S ep omycin and 5% Glu amine) con aining he accine o mula ion wi h a inal concen a ion o 4 µ g hemagglu inin (HA)/mL spli i us accine (kindly p o ided by GlaxoSmi hKline, Belgium). Uns imula ed samples we e incuba ed o he same ime in comple e RPMI wi hou he accine o mula ion. B e eldin A and monensin we e added o all samples a e 5 h o incuba ion. Cells we e collec ed and s ained o low cy ome ic analysis. Su ace ma ke s aining was pe o med o 20 min a 4 ◦ C. The ollowing an ibodies we e used dilu ed in PBS: CD56 (PE-Cy7, clone B159, BD, F anklin Lakes, NJ, USA), CD3 (V450, clone UCHT1, BD), CD14 (Paci ic Blue, clone M5E2, BD), CD19 (V450, clone HIB19, BD Ho izon), CD16 (APC-H7, clone 3G8, BD Pha mingen), NKG2C (PE, clone 134591, R&D Sys ems, Minneapolis, MN, USA), CD57 (APC, clone HCD57, BioLegend, San Diego, CA, USA), Li e/Dead (Fixable Blue, In i ogen, Ca lsbad, CA, USA). The exp ession o CD107a was used as a co ela e o deg anula ion. To his end, he an i-CD107a an ibody (PE-Cy5, clone eBioH4A3, eBioscience, San Diego, CA, USA) was added o he cul u e. The sec e ion o IFN γ (Alexa Fluo 700, clone B27, BioLegend) was de ec ed by in acellula s aining using Cy o ix/Cy ope m solu ion (BD Biosciences). Samples we e acqui ed a a BD Fo essa low cy ome e and analyzed using FlowJo (FlowJo, LLC, Ashland, OR, USA). Uns ained, single s ained (one an ibody/sample) as well as luo escence-minus-one (FMO) samples we e used as con ols o he acquisi ion as well as he subsequen analysis. S a is ical di e ences we e de e mined by he G aphPad P ism so wa e. 2.4. S ochas ic Neighbo Embedding (SNE) Analysis Flow cy ome y da a o esponde s and low esponde s de i ed p e- and 7-days pos - accina ion we e impo ed in o FlowJo ( e sion 9) and compensa ion channel alues we e ex ac ed o he ollowing pa ame e s: CD56, CD16, NKG2C, CD57, CD107a and IFN γ . Up o 10,000 alues we e ex ac ed pe accinee and ime poin and hen pooled o esponde s and low esponde s. By using he R package “ sne”, a -dis ibu ed SNE analysis using Ba nes–Hu implemen a ion was pe o med and he esul ing da a we e plo ed wi h in ensi ies o he depic ed ma ke s (RS udio e sion 3.2.1, RS udio, Inc., Bos on, MA, USA), as desc ibed ea lie [25,26]. 2.5. S a is ical Analysis G aphPad P ism ( e sion 6.0 o Windows, G aphPad So wa e, La Jolla, CA, USA) was used o he s a is ical assessmen (unpai ed low-pa ame ic Mann–Whi ney o K uskal–Wallis es and Spea man co ela ion). Values o p≤0.05 we e conside ed signi ican . Vaccines 2020,8, 281 4 o 18 3. Resul s 3.1. In luenza Vaccina ion Leads o Enhanced F equencies o NKG2C-Exp essing NK Cells NK cells a e cha ac e ized by he in ensi y o CD56 exp ession and he co-exp ession o CD16 and can be he eby di ided in o di e en unc ional subse s (see ga ing s a egy, Figu e S1). The impac o pandemic accina ion on he dis ibu ion o blood NK cell subse s was assessed by low cy ome y a a ious ime poin s. A sligh ly educed equency o o al NK cells in accina ed indi iduals was obse ed du ing he i s 14 days a e accina ion ( ≈ 6% a day 0 o ≈ 5.4% a day 7 and 4.5% a day 14) (Figu e 1a). The ma ginally diminished equency emained ela i ely s able o e he obse a ion pe iod un il day 180 pos - accina ion ( ≈ 5% a day 21 and ≈ 4.8% a day 180). The di ision o NK cells in o p ima ily cy okine sec e ing CD56 b igh and highly cy o oxic CD56 dim subse s e ealed a dec ease in he CD56 dim NK cell equencies, especially o CD56 dim CD16 + cells ( ≈ 48% a day 0 o ≈ 34% a day 7), whe eas CD56 b igh NK cell equencies we e no a ec ed (Figu e 1b). These da a sugges ha accina ion-induced modula ion mainly a ec s CD56 dim CD16 + NK cells, which a e desc ibed o hold a highe cy o oxic bu lowe cy okine sec e ing capabili y as compa ed o CD56 b igh NK cells [ 27 ]. Human memo y-like NK cells based ei he on he exp ession o CD57 and NKG2C o NKG2C alone we e ecen ly desc ibed o exe ampli ied ecall esponses upon CMV in ec ion o o be induced a e cy okine s imula ion (IL-12, IL-15 and IL-18), espec i ely [ 13 , 17 , 20 ]. The analysis o CD56 dim CD16 + NK cells wi h ega d o CD57 and NKG2C exp ession a e pandemic accina ion e ealed ha he changes in NK cell equencies we e mainly es ic ed o he NKG2C-exp essing subse s. Inc eased equencies o bo h CD57 − NKG2C + ( om ≈ 2% a day 0–9% a day 7 and ≈ 8% a day 14) and CD57 + NKG2C + NK cell subse s ( om ≈ 1% a day 0–4% a day 7 and ≈ 7% a day 14) we e de ec ed a days 7 and 14 pos - accina ion (o ange and ed) in samples de i ed om 8 ou o 10 accine esponde s (ma ked wi h an a ow, Figu e 1c). The mos s iking di e ences we e obse ed a day 7 and 14 pos - accina ion, wi h signi ican ly ele a ed le els o bo h CD57 − NKG2C + and CD57 + NKG2C + NK cells, whe eas he equency o CD57 + NKG2C − NK cells was no a ec ed (Figu e 1d). Wi hin he NKG2C-exp essing subse s, high a ios compa ing pos - and p e- accina ion alues we e de ec ed ( ≥ 1.5, ed da a poin s) while he CD57 + NKG2C − NK cell subse showed a ios >1 (blue da a poin s), bu no ≥ 1.5. The analysis o NK cells ei he exp essing NKG2C o CD57 con i med he inc eased equency o NKG2C bu no CD57 exp ession (Figu e 1e). The da a sugges ha adju an ed pandemic accina ion induces changes in he subse o NKG2C-exp essing NK cells, which in u n can be po en ially in ol ed in de e mining he ou come o accina ion. While he equency o NKG2C-exp essing NK cells is desc ibed o be connec ed wi h CMV se o-posi i i y, no such co ela ion was ound he e (Figu e S2a) [ 16 , 17 ]. Fu he mo e, no di e ences be ween he mean CMV i e o low and no mal esponde s was obse ed (Figu e S2b). The co ela ion analysis o he CMV i e and he old change o he HAI i e o he age o he pa icipan s also did no yield any signi ican ela ion (Figu e S2c). Likewise, he old change o he HAI i e did no co ela e wi h he age o he accinees (Figu e S2d). Howe e , in no mal bu no in low esponde s, he equency o NKG2C + CD57 + NK cells a day 7 pos - accina ion showed a signi ican nega i e co ela ion wi h he age o he accinees (Figu e S2e). These indings highligh ha he hypo hesized ela ion be ween NKG2C+NK cells and he accina ion ou come is no dependen on he CMV se o-s a us, hus suppo ing publica ions s a ing ha CMV in ec ions do no a ec in luenza accine e icacy [28]. Vaccines 2020,8, 281 5 o 18 Vaccines 2020, 8, 281 5 o 18 Figu e 1. Con . Vaccines 2020,8, 281 6 o 18 Vaccines 2020, 8, 281 6 o 18 Figu e 1. In luenza accina ion a ec s he equency o NKG2C-exp essing na u al kille (NK) cells. Pe iphe al blood mononuclea cells (PBMCs) isola ed om accina ed indi iduals p io o accina ion and a he indica ed ime poin s pos - accina ion (dp = days pos - accina ion) we e s ained o he su ace ma ke s CD56, CD3, CD16, NKG2C and CD57. (a) F equencies o o al CD3−CD56+ NK cells and (b) o CD3-CD56b igh , CD3−CD56dim and CD3−CD56dimCD16+ NK cell subpopula ions. Diag ams show he connec ed column mean wi h 95% con idence in e al. (c) F equencies o NK cell popula ions cha ac e ized by he exp ession o CD57 and NKG2C. Columns ep esen indi idual da a poin s. (d) CD57- and NKG2C-exp essing CD56dimCD16+ subpopula ions depic ed as he a io o cell equencies de ec ed a he indica ed ime poin s pos - accina ion and he equencies de ec ed p io accina ion (day 0). Diag ams a e depic ed as sca e plo s wi h ba s o indi idual assigned da a poin s. (e) CD56dimCD16+NKG2C+ and CD56dimCD16+CD57+ NK cells displayed as equencies and as he a io o he day be o e accina ion. Columns ep esen indi idual da a poin s; diag ams a e depic ed as sca e plo s wi h ba s o indi idual assigned da a poin s. A ows indica e dono s wi h accine-induced immunological changes. As e isks deno e signi ican alues as calcula ed by unpai ed and non-pa ame ic K uskal–Wallis es . * p ≤ 0.05. Le e s in panels (d, e) indica e single esponde s. Figu e 1. In luenza accina ion a ec s he equency o NKG2C-exp essing na u al kille (NK) cells. Pe iphe al blood mononuclea cells (PBMCs) isola ed om accina ed indi iduals p io o accina ion and a he indica ed ime poin s pos - accina ion (dp =days pos - accina ion) we e s ained o he su ace ma ke s CD56, CD3, CD16, NKG2C and CD57. ( a ) F equencies o o al CD3 − CD56 + NK cells and ( b ) o CD3 − CD56b igh , CD3 − CD56 dim and CD3 − CD56 dim CD16 + NK cell subpopula ions. Diag ams show he connec ed column mean wi h 95% con idence in e al. ( c ) F equencies o NK cell popula ions cha ac e ized by he exp ession o CD57 and NKG2C. Columns ep esen indi idual da a poin s. ( d ) CD57- and NKG2C-exp essing CD56 dim CD16 + subpopula ions depic ed as he a io o cell equencies de ec ed a he indica ed ime poin s pos - accina ion and he equencies de ec ed p io accina ion (day 0). Diag ams a e depic ed as sca e plo s wi h ba s o indi idual assigned da a poin s. ( e ) CD56 dim CD16 + NKG2C + and CD56 dim CD16 + CD57 + NK cells displayed as equencies and as he a io o he day be o e accina ion. Columns ep esen indi idual da a poin s; diag ams a e depic ed as sca e plo s wi h ba s o indi idual assigned da a poin s. A ows indica e dono s wi h accine-induced immunological changes. As e isks deno e signi ican alues as calcula ed by unpai ed and non-pa ame ic K uskal–Wallis es . * p ≤ 0.05. Le e s in panels ( d , e ) indica e single esponde s. Vaccines 2020,8, 281 7 o 18 3.2. NK Cells o In luenza Vaccina ion Responde s and Low Responde s Display Di e ences in NKG2C and CD57 Exp ession The ine icacy o in luenza accines, cha ac e ized by he a ying occu ence o low esponde s, is a pe sis ing p oblem. To classi y no mal and low esponde s ollowing pandemic accina ion, he HAI i e o each accinee was e alua ed a days 0 and 90 pos - accina ion (Figu e S3). The o al equencies o NK cells de i ed om no mal and low esponde s we e compa ed, as well as he exp ession o CD57 and NKG2C. Reduced equencies o CD56 dim CD16 + NK cells we e obse ed in bo h esponse g oups a 7-days pos - accina ion, as compa ed o day 0. Howe e , he dec ease was less p o ound in no mal esponde s ( ≈ 30% educ ion o esponde s and ≈ 46% educ ion o low esponde s) (Figu e 2a). These indings we e consis en wi h he assessed a ios o NK cell equencies (day 7/day 0). The assessmen o CD57 exp ession p io o accina ion demons a ed di e ences in i s basal exp ession by NK cells de i ed om no mal and low esponde s (day 0). Howe e , he a io o CD56 dim CD16 + CD57 + NK cell equencies (day 7/day 0) e ealed no accine-induced e ec on CD57 exp ession ( a ios ≈ 1 o bo h esponde s and low esponde s (Figu e 2b). The analysis o CD56 dim CD16 + NKG2C + NK cells e ealed an inc eased equency a day 7 pos - accina ion in no mal esponde s ha was no obse ed in low esponde s (Figu e 2c). The a io o CD56 dim CD16 + NKG2C + NK cell equencies (day 7/day 0) suppo s his inding by displaying a highe a io o no mal esponde s ( ≈ 6) as compa ed o low esponde s ( ≈ 1). This di e ence is u he highligh ed by a ios mainly ≥ 1.5 ( ed do s) de ec ed o NKG2C-exp ession by CD16 + NK cells de i ed om no mal esponde s. This indica es ha esponde s show inc eased equencies o NKG2C + exp ession a day 7 pos - accina ion as compa ed o day 0, whe eas NKG2C exp ession in low esponde s emains la gely una ec ed. To add ess whe he he obse ed inc eased equency o NKG2C + NK cells is due o he speci ic an igen (HA) e-s imula ion, an indi idual analysis o single dono s was pe o med. These da a e ealed ha a signi ican numbe o esponde s displayed an HA-induced su ace exp ession o NKG2C ha was no obse ed in he g oup o low esponde s (Figu e 2d, a io (HA/uns imula ed (NS)) >1=blue, ≥1.5 = ed ). In e es ingly, he indi iduals esponding o HA e-s imula ion wi h enhanced exp ession o NKG2C al eady displayed a highe basal exp ession. Wi h ega d o he HA-induced su ace exp ession o CD57, nei he accine esponde s no low esponde s displayed a s ong modula ion, as indica ed by a ios (HA/uns imula ed) >1 bu no ≥ 1.5 (Figu e 2e). These indings sugges ha NKG2C-exp essing NK cells bias accine esponsi eness and migh se e as a de e minan o esponsi eness owa ds in luenza accina ion. Vaccines 2020,8, 281 8 o 18 Vaccines 2020, 8, 281 8 o 18 Figu e 2. Pheno ypic analysis o NK cells de i ed om no mal and low esponde s o he pandemic in luenza accina ion. Flow cy ome ic analysis o ozen PBMCs isola ed om accina ed indi iduals classi ied in o no mal esponde s (black do s) and low esponde s (whi e do s; a-c). F equencies and a ios o (a) CD56dimCD16+, (b) CD56dimCD16+CD57+ and (c) CD56dimCD16+NKG2C+ NK cells. Diag ams a e depic ed as sca e do plo s indica ing he mean by a ho izon al line. Ra ios we e de i ed om cell equencies de ec ed a day 7 pos - accina ion and he equencies de ec ed p io o accina ion (day 0) depic ed as sca e plo s wi h ba s. Ra ios o uns imula ed (NS) and HA- e-s imula ed (HA) (d) CD56dimCD16+NKG2C+ and (e) CD56dimCD16+CD57+ NK cells de i ed om no mal and low esponde s. Blue do s depic a ios >1 and ed do s depic alues ≥ 1.5. 3.3. CD107a Exp ession is Con ined o CD56dimCD16+NKG2C-Exp essing NK Cells in Responde s bu no in Low Responde s To dissec whe he , in addi ion o he pheno ypic al e a ions, he unc ionali y o CD56dim NK cells in no mal and low esponde s also di e s, CD16 exp ession, CD107a exp ession and IFNγ sec e ion we e add essed ex i o. Independen ly o he esponsi eness o accina ion, di e ences in he unc ionali y o CD16- and CD16+ NK cells wi hin he CD56dim subse we e de ec ed (Figu e 3a,b). The analysis o he NK cell deg anula ion capaci y e ealed ha wi hin he g oup o esponde s, CD56dimCD16− NK cells showed no changes in he equency o CD107a-exp essing cells a e accina ion (Figu e 3a). In con as , CD56dimCD16+ NK cells de i ed om esponde s exhibi ed an enhanced exp ession o CD107a peaking a day 7 pos - accina ion. Low esponde s showed simila unc ional di e ences be ween CD56dimCD16- and CD16+ NK cells (Figu e 3a). CD56dimCD16+ NK cells showed a highe equency o CD107a-exp essing cells a day 7 pos - accina ion as compa ed o day 0. The compa ison o no mal and low esponde s u he e ealed ha esponde s ha bo a lowe equency o CD56dimCD16+CD107a+ NK cells a day 7 pos - accina ion (≈4%) as compa ed o low esponde s (≈12%) (Figu e 3a). Figu e 2. Pheno ypic analysis o NK cells de i ed om no mal and low esponde s o he pandemic in luenza accina ion. Flow cy ome ic analysis o ozen PBMCs isola ed om accina ed indi iduals classi ied in o no mal esponde s (black do s) and low esponde s (whi e do s; a-c). F equencies and a ios o ( a ) CD56 dim CD16 + , ( b ) CD56 dim CD16 + CD57 + and ( c ) CD56 dim CD16 + NKG2C + NK cells. Diag ams a e depic ed as sca e do plo s indica ing he mean by a ho izon al line. Ra ios we e de i ed om cell equencies de ec ed a day 7 pos - accina ion and he equencies de ec ed p io o accina ion (day 0) depic ed as sca e plo s wi h ba s. Ra ios o uns imula ed (NS) and HA- e-s imula ed (HA) ( d ) CD56 dim CD16 + NKG2C + and ( e ) CD56 dim CD16 + CD57 + NK cells de i ed om no mal and low esponde s. Blue do s depic a ios >1 and ed do s depic alues ≥1.5. 3.3. CD107a Exp ession Is Con ined o CD56 dim CD16 + NKG2C-Exp essing NK Cells in Responde s bu No in Low Responde s To dissec whe he , in addi ion o he pheno ypic al e a ions, he unc ionali y o CD56 dim NK cells in no mal and low esponde s also di e s, CD16 exp ession, CD107a exp ession and IFN γ sec e ion we e add essed ex i o. Independen ly o he esponsi eness o accina ion, di e ences in he unc ionali y o CD16 − and CD16 + NK cells wi hin he CD56 dim subse we e de ec ed (Figu e 3a,b). The analysis o he NK cell deg anula ion capaci y e ealed ha wi hin he g oup o esponde s, CD56 dim CD16 − NK cells showed no changes in he equency o CD107a-exp essing cells a e accina ion (Figu e 3a). In con as , CD56 dim CD16 + NK cells de i ed om esponde s exhibi ed an enhanced exp ession o CD107a peaking a day 7 pos - accina ion. Low esponde s showed simila unc ional di e ences be ween CD56 dim CD16 − and CD16 + NK cells (Figu e 3a). CD56 dim CD16 + NK cells showed a highe equency o CD107a-exp essing cells a day 7 pos - accina ion as compa ed o day 0. The compa ison o no mal and low esponde s u he e ealed ha esponde s ha bo a lowe equency o CD56 dim CD16 + CD107a + NK cells a day 7 pos - accina ion ( ≈ 4%) as compa ed o low esponde s (≈12%) (Figu e 3a). Vaccines 2020,8, 281 9 o 18 Vaccines 2020, 8, 281 10 o 18 Figu e 3. Func ional di e ences cha ac e ize NK cells isola ed om no mal and low esponde s. Func ional low cy ome ic analysis o ozen PBMCs isola ed om accina ed indi iduals classi ied in o no mal and low esponde s. F equencies o (a) CD107a+CD56dim and (b) IFNγ+CD56dim NK cells wi h ega d o CD16 exp ession (CD16− whi e/CD16+ black). Ra io o CD107a-exp essing (c) CD57−NKG2C+, (d) CD57+NKG2C+ and (e) CD57+NKG2C− CD56dimCD16+ NK cells isola ed om esponde s and non- esponde s on he indica ed days pos - and p e- accina ion (day 0) depic ed as sca e plo s wi h ba s indica ing he mean wi h 95% con idence in e al. ( ) F equency o CD56dimCD16+ NK cells exp essing CD107a ( ed solid), NKG2C+CD57− (black solid), NKG2C+CD57+ (g een), NKG2C−CD57+ (black dashed) shown o indi idual esponde s and (g) non- esponde s. As e isks deno e signi ican alues as calcula ed by unpai ed and non-pa ame ic Mann–Whi ney es . * p ≤ 0.05, ** p ≤ 0.01. Figu e 3. Func ional di e ences cha ac e ize NK cells isola ed om no mal and low esponde s. Func ional low cy ome ic analysis o ozen PBMCs isola ed om accina ed indi iduals classi ied in o no mal and low esponde s. F equencies o ( a ) CD107a + CD56 dim and ( b ) IFN γ+ CD56 dim NK cells wi h ega d o CD16 exp ession (CD16 − whi e/CD16 + black). Ra io o CD107a-exp essing ( c ) CD57 − NKG2C + , ( d ) CD57 + NKG2C + and ( e ) CD57 + NKG2C − CD56 dim CD16 + NK cells isola ed om esponde s and non- esponde s on he indica ed days pos - and p e- accina ion (day 0) depic ed as sca e plo s wi h ba s indica ing he mean wi h 95% con idence in e al. ( ) F equency o CD56 dim CD16 + NK cells exp essing CD107a ( ed solid), NKG2C + CD57 − (black solid), NKG2C + CD57 + (g een), NKG2C − CD57 + (black dashed) shown o indi idual esponde s and ( g ) non- esponde s. As e isks deno e signi ican alues as calcula ed by unpai ed and non-pa ame ic Mann–Whi ney es . * p≤0.05, ** p≤0.01. Vaccines 2020,8, 281 16 o 18 24. Madhun, A.S.; Akselsen, P.E.; Sju sen, H.; Pede sen, G.; S indland, S.; Nos bakken, J.K.; Nilsen, M.; Mohn, K.; Jul-La sen, A.; Smi h, I.; e al. An adju an ed pandemic in luenza H1N1 accine p o ides ea ly and long e m p o ec ion in heal h ca e wo ke s. Vaccine 2010,29, 266–273. [C ossRe ] [PubMed] 25. 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Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h p://c ea i ecommons.o g/licenses/by/4.0/).