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
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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
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