A NKp80-based identification strategy reveals that CD56neg NK cells are not completely dysfunctional in health and disease.

iScience
A icle
A NKp80-Based Iden i ica ion S a egy Re eals
ha CD56
neg
NK Cells A e No Comple ely
Dys unc ional in Heal h and Disease
Ane O an ia,
In
˜igo Te e
´n, Alicia
Izquie do-
La uen e,...,Juan
C. Ga cı
´a-Ruiz,
Ola z
Zena uzabei ia,
F ancisco Bo ego
ancisco.bo ego abasco@
osakide za.eus
HIGHLIGHTS
NKp80 is a mo e p ecise
ma ke han CD16 in o de
o iden i y CD56
neg
NK
cells
CD16, bu no NKp80, is
downmodula ed a e
c yop ese a ion and cell
ac i a ion
CD56
neg
NK cells e ec o
unc ions a e no as
diminished as p e iously
desc ibed
O an ia e al., iScience 23,
101298
July 24, 2020 ª2020 The
Au ho s.
h ps://doi.o g/10.1016/
j.isci.2020.101298
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OPEN ACCESS
iScience
A icle
A NKp80-Based Iden i ica ion S a egy
Re eals ha CD56
neg
NK Cells A e No Comple ely
Dys unc ional in Heal h and Disease
Ane O an ia,
1
In
˜igo Te e
´n,
1
Alicia Izquie do-La uen e,
1
Juncal A. Alonso-Cab e a,
1
Vic o Sanda
´,
1
Joana Vi alle
´,
1
San iago Mo eno,
2
Ma ı
´a Tasias,
3
Alasne U anga,
4
Ca men Gonza
´lez,
4
Juan J. Ma eos,
5
Juan C. Ga cı
´a-Ruiz,
5
Ola z Zena uzabei ia,
1
and F ancisco Bo ego
1,6,7,
*
SUMMARY
Na u al kille (NK) cells a e usually iden i ied by he absence o o he lineage
ma ke s, due o he lack o cell-su ace-speci ic ecep o s. CD56
neg
NK cells, clas-
sically iden i ied as CD56
neg
CD16+, a e e y sca ce in he pe iphe al blood o
heal hy people bu hey expand in some pa hological condi ions. Howe e ,
s udies on CD56
neg
NK cells had e ealed di e en esul s ega ding he pheno-
ype and unc ionali y. This could be due o, among o he s, he uns able exp es-
sion o CD16, which hinde s CD56
neg
NK cells’ p ope iden i ica ion. Hence, we
aim o de e mine an al e na i e su ace ma ke o CD16 o be e iden i y
CD56
neg
NK cells. We ha e ound ha NKp80 is supe io o CD16. Fu he mo e,
we ound di e ences be ween he unc ionali y o CD56
neg
NKp80+ and
CD56
neg
CD16+, sugges ing ha he e ec o unc ions o CD56
neg
NK cells a e
no as diminished as p e iously hough . We p oposed NKp80 as a no ewo hy
ma ke o iden i y and accu a ely e-cha ac e ize human CD56
neg
NK cells.
INTRODUCTION
Na u al kille (NK) cells a e la ge g anula lymphocy es ha ha e he abili y o ecognize and kill ans-
o med and i us-in ec ed cells wi hou p io sensi iza ion (Caligiu i, 2008;F eud e al., 2017). In addi ion,
hey also p oduce and sec e e a a ie y o cy okines and chemokines ha modula e he immune esponse
(Banc o , 1993;Bi on e al., 1999;Coope e al., 2001;Robe son and Ri z, 1990). In human heal hy adul s,
hey comp ise 5%–15% o ci cula ing lymphocy es, being oge he wi h T cells and B cells, one o he
h ee majo lymphoid linages. Howe e , in con as o T and B cells, NK cells a e a membe o he inna e
lymphoid cells (ILCs) amily, o which he main cha ac e is ic is he absence o ea anged an igen ecep-
o s encoded by he ecombina ion ac i a ing genes (RAG) (A is and Spi s, 2015). Cu en ly, ILCs a e clas-
si ied in o i e di e en subse s, depending on hei e ec o unc ions, he cy okine pa e n hey sec e e,
and he ansc ip ion ac o s hey need o de elop and di e en ia e. The i e subse s a e NK cells, G oup
1 ILC (ILC1), ILC2, ILC3, and lymphoid issue-induce (LTi) cells (Colonna, 2018;Vi ie e al., 2018). NK cells
and ILC1 a e cells ha p oduce in e e on-g(IFN-g) as hei signa u e cy okine and need he T-be an-
sc ip ion ac o o de elop. On he o he hand, ILC1 exhibi e y li le o no cy o oxic ac i i y due o he
low o ze o le els o pe o in and g anzymes hey exp ess (Fuchs, 2016;Spi s e al., 2016). Ye , he dis inc-
ion be ween ILC1 and NK cells could be p oblema ic because hey exp ess simila cell su ace ma ke s
(Mjo
¨sbe g and Spi s, 2016;Spi s e al., 2013;T abanelli e al., 2018;Vi ie e al., 2018). Ne e heless, in
humans he NKp80 cell su ace ecep o is exp essed on NK cells and seems o be an NK-cell-speci ic
ma ke among human ILCs (F eud e al., 2016,2017). Fu he mo e, NK cells exp ess bo h T-be and Eo-
mesode min (Eomes) ansc ip ion ac o s, whe eas ILC1 only exp ess T-be (A is and Spi s, 2015;Bal e
al., 2020;Colonna, 2018;Mjo
¨sbe g and Spi s, 2016;Spi s e al., 2013;Vi ie e al., 2018).
Commonly, as he e is no known speci ic su ace ecep o ha leads o NK cell iden i ica ion wi hin pe iph-
e al blood mononuclea cells (PBMCs), hey a e pheno ypically de ined as CD56+ cells ha do no exp ess
o he lineage ma ke s, such as hose ha a e speci ic o T cells (CD3), B cells (CD19), and myeloid cells
(CD14). Fu he mo e, CD56 in combina ion wi h CD16, he low-a ini y Fc gamma ecep o IIIa, is gene ally
used o dis inguish di e en NK cell subse s ha a e p esen in heal hy human pe iphe al blood (F eud
1
Bioc uces Bizkaia Heal h
Resea ch Ins i u e,
Immunopa hology G oup,
Ba akaldo 48903, Spain
2
Ramo
´n y Cajal Heal h
Resea ch Ins i u e (IRYCIS),
Ramo
´n y Cajal Uni e si y
Hospi al, Mad id 28034,
Spain
3
Hospi al Uni e si a i i
Poli ecnic La Fe, Valencia
46026, Spain
4
Biodonos ia Heal h
Resea ch Ins i u e, Donos ia
Uni e si y Hospi al,
Donos ia-San Sebas ia
´n
20014, Spain
5
Bioc uces Bizkaia Heal h
Resea ch Ins i u e,
Hema ological Cance
G oup, C uces Uni e si y
Hospi al, Ba akaldo 48903,
Spain
6
Ike basque, Basque
Founda ion o Science,
Bilbao 48013, Spain
7
Lead Con ac
*Co espondence:
ancisco.bo ego abasco@
osakide za.eus
h ps://doi.o g/10.1016/j.isci.
2020.101298
iScience 23, 101298, July 24, 2020 ª2020 The Au ho s.
This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
1
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e al., 2017;Mon aldo e al., 2013). In his way, and based on he exp ession o hese ma ke s, wo majo
subse s a e iden i ied: CD56
b igh
CD16+/and CD56
dim
CD16+. In addi ion, wo mo e subse s ha e
been desc ibed: CD56
neg
(CD56
neg
CD16+) and uncon en ional CD56
dim
(CD56+CD16) NK cells (Hu
e al., 1995;Lugli e al., 2014;Robe o e al., 2018;Di Vi o e al., 2019). Howe e , none o hese ma ke s
a e speci ic o NK cells. Fo ins ance, al hough all ILCs a e also nega i e o he abo emen ioned linage
ma ke s (Spi s e al., 2013;T abanelli e al., 2018;Vi ie e al., 2018), CD56 is exp essed by a subse o
ILC3, known as NCR+ ILC3 and which, as NK cells, exp esses na u al cy o oxici y ecep o s (NCR)
NKp30, NKp44, and NKp46 (Spi s e al., 2013;T abanelli e al., 2018;Vallen in e al., 2015;Vi ie e al.,
2018). On he o he hand, al hough CD16 is widely accep ed o be an NK-cell-speci ic ma ke among
ILCs (Spi s e al., 2016;T abanelli e al., 2018;Vi ie e al., 2018), i is known ha CD16 could be down egu-
la ed ollowing a ge cell ac i a ion (Bo ego e al., 1994;G zywacz e al., 2007;Pe uzzi e al., 2013;Romee
e al., 2013;Zhou e al., 2013) and c yop ese a ion (Lug ha e al., 2015). The e o e, he lack o ma ke
speci ici y makes he p ocess o iden i ying NK cells qui e challenging and highligh s he need o s a using
o he se o cell su ace ma ke s.
The CD56
neg
subse exp esses NK-cell-associa ed su ace ma ke s, such as CD16, CD94, and NKp46, in
addi ion o he ansc ip ion ac o s Eomes and T-be (Ta azona e al., 2002;Voig e al.,2018). In
heal hy indi iduals, he p esence o CD56
neg
NK cells in blood is e y a e (Bjo
¨ ks o
¨me al.,2010;
Campos e al., 2014;Mu
¨lle -Du o ic e al., 2019). Howe e , yea s ago, in pa ien s wi h ch onic human
immunode iciency i us (HIV)-1 in ec ion, a signi ican expansion o CD56
neg
NK cells was epo ed (Hu
e al., 1995;Lugli e al., 2014), which was associa ed wi h high HIV-1 i al load (Al e e al., 2005;Ba ke
e al., 2007;Ma ilio e al., 2005). Indeed, long- e m non-p og esso s and pa ien s who success ully sup-
p ess i al load a e highly ac i e an i e o i al he apy ha e CD56
neg
NK cells le els compa able o
he ones ound in non-in ec ed subjec s (Al e e al., 2005;B une a e al., 2009). Howe e , pa ien s
who ail o supp ess i al load upon ea men ha e simila CD56
neg
numbe s o he ones wi h pe sis-
en i emia (Al e e al.,2005). In addi ion, in ch onically HIV-1-in ec ed indi iduals who de eloped
b oadly neu alizing an ibodies (bnAbs), a high p opo ion o NK cells ha e a CD56
neg
pheno ype,
whe eas in pa ien s who do no ha e bnAbs he p opo ion o CD56
neg
NK cells was lowe , al hough
s ill high when compa ed wi h HIV-1 se onega i e subjec s (B adley e al., 2018). Ele a ed equencies
o he CD56
neg
subse has also been desc ibed in hepa i is C i us (HCV)-monoin ec ed and HCV/HIV-
1-coin ec ed people (Gonzalez e al., 2008,2009). Fu he mo e, he abno mal expansion o hese cells
co ela ed wi h monoin ec ed pa ien ’s abili y o espond o pegyla ed-IFNaand iba i in ea men
(Gonzalez e al., 2009). Mo eo e , ea men s ha supp ess HCV eplica ion dec eases he numbe
o CD56
neg
NK cells in HCV-/HIV-1-coin ec ed pa ien s (Gonzalez e al., 2008). On he o he hand, i
has been desc ibed ha aging and human cy omegalo i us s a us has an e ec on he equency
and dis ibu ion o NK cell subse s, inc easing he pe cen age o CD56
neg
NK cells (Campos e al.,
2014;Mu
¨lle -Du o ic e al., 2019).
S udies wi h simila coho s o pa ien s di e in he equency, unc ionali y, and pheno ype o he
CD56
neg
NK cell subse , which could be due o di e en ga ing s a egies used o he iden i ica ion
o CD56
neg
NK cells (Bjo
¨ ks o
¨m e al., 2010;Elle e al., 2009;Ma ilio e al., 2005;Milush e al., 2013).
I is e y impo an o no e ha he abo emen ioned s udies, and many o he s, ha e iden i ied his
NK cell subse as CD56
neg
CD16+, and depending on he s udies, hey ha e o ha e no included in
he ga ing s a egy an exclusion channel o he exclusion o T cells, B cells, and/o monocy es wi hin
he cells o in e es , i.e. CD56
neg
NK cells. In addi ion, i is e y well known ha CD16 is down egula ed
by c yop ese a ion (Lug ha e al., 2015), a e cy okine ac i a ion and a ge cell s imula ion (Bo ego
e al., 1994;G zywacz e al., 2007;Pe uzzi e al., 2013;Romee e al., 2013;Zhou e al., 2013), and he e o e
he usage o his ma ke could lead o an inaccu a e iden i ica ion o he CD56
neg
NK cells and inconsis-
en esul s. In his wo k, we ha e explo ed he possibili y o using NKp80 as a ma ke o be e iden i y
CD56
neg
NK cells. NKp80, he p oduc o he KLRF1 gene, is an ac i a ing ecep o exp essed by i ually
all ma u e human NK cells (Vi ale e al., 2001).NKp80ma ksac i icals epinNKcellde elopmen ,asi
de ines unc ionally ma u e NK cells (F eud e al., 2016), and is an NK-cell-speci ic ma ke among human
inna e lymphoid cells (ILCs) (Vi ie e al., 2018). We show ha NKp80 is a mo e p ecise ma ke han CD16
in o de o iden i y CD56
neg
NK cells and ha i is no down egula ed a e sample c yop ese a ion o
cell ac i a ion. Impo an ly, using he NKp80 ma ke o he iden i ica ion, we ha e demons a ed ha he
e ec o unc ions o CD56
neg
NK cells a e no as diminished as p e iously hough , bo h in heal h and in
disease.
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2iScience 23, 101298, July 24, 2020
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RESULTS
The NKp80 Recep o Is Supe io o CD16 o he Iden i ica ion o Ci cula ing CD56
neg
NK Cell
Subse in Heal hy People
The CD16 ecep o has adi ionally been used, in combina ion wi h CD56, o iden i y he ci cula ing NK cell
subse s, wi h CD56
neg
NK cells de ined as CD56
neg
CD16+ (Bjo
¨ ks o
¨m e al., 2010). Howe e , CD16 is well
known o be down egula ed in some si ua ions, such as, c yop ese a ion, a e a ge cell s imula ion and
compounds, cell su ace ecep o , and cy okine ac i a ion (Bo ego e al., 1994;G zywacz e al., 2007;
Lug ha e al., 2015;Pe uzzi e al., 2013;Romee e al., 2013;Zhou e al., 2013). CD16 is shed om he
cell su ace as a consequence o ma ix me allop o einases ac i a ion, such as MT6 (also known as
MMP25) and ADAM17 (G zywacz e al., 2007;Pe uzzi e al., 2013;Romee e al., 2013). Wi h he aim o iden-
i y a mo e accu a e ma ke wi h a mo e s able exp ession, we i s compa ed CD16 wi h NKp80 ecep o o
iden i y CD56
neg
NK cells in heal hy dono s. Ou ga ing s a egy included an exclusion channel ( iabili y,
CD3, CD14, and CD19) ha allowed us o speci ically s udy non-T, non-B, non-monocy es iable cells (Fig-
u e S1A). As p e iously desc ibed (Lug ha e al., 2015), CD16 exp ession was down egula ed in c yop e-
se ed samples; howe e , he exp ession o NKp80 was no signi ican ly al e ed a e cell eezing (Fig-
u e S2), sugges ing ha his ecep o is mo e sui able o he de ec ion o CD56
neg
NK cells when i
conce ns o ozen cells. Ve y impo an ly, al hough no di e ences we e seen ega ding he pe cen age
o CD56
neg
NK cells selec ed using bo h ma ke s (Figu e S1B), he e was a signi ican ly highe equency
o Eomes+ cells in he CD56
neg
NKp80+ subpopula ion han in CD56
neg
CD16+ cells (Figu e 1A). Eomes
is a speci ic in acellula ma ke o he de ec ion o NK cells wi hin he ILCs, gi en ha i is a T-box an-
sc ip ion ac o needed o he de elopmen and unc ion o NK cells, whe eas o example, ILC1 do no
Figu e 1. NKp80 Be e Iden i ies CD56
neg
NKCells hanCD16inHeal hyIndi iduals
(A) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD56
neg
CD16+ and CD56
neg
NKp80+ popula ions.
(B) Le pa , ep esen a i e con ou plo showing he Eomes exp ession e sus he size (FSC-A) o CD56
neg
CD16+ cells.
Da a om a ep esen a i e heal hy dono is shown. Righ pa , ba g aph showing he median o FSC-A pa ame e wi hin
CD56
neg
CD16+Eomesand CD56
neg
CD16+Eomes+ popula ions.
(C) Ba g aph showing he pe cen age o CD123
+
cells wi hin CD56
neg
CD16+Eomes+, CD56
neg
CD16+Eomes,
CD56
neg
NKp80+Eomes+, and CD56
neg
NKp80+Eomespopula ions.
(D) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD56
neg
CD16+ and CD56
neg
NKp80+ popula ions wi h o
wi hou he addi ion o an i-CD123 mAb o he exclusion channel (Exclusion C.). The mean wi h he s anda d e o o he
mean (SEM) is ep esen ed, excep o (B) in which he median is ep esen ed. Each do ep esen s a dono . *p < 0.05,
**p < 0.01, ****p < 0.0001, ns: no signi ican .
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iScience 23, 101298, July 24, 2020 3
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exp ess Eomes (A is and Spi s, 2015;Bal e al., 2020;Colonna, 2018;Mjo
¨sbe g and Spi s, 2016;Spi s e al.,
2013;Vi ie e al., 2018). As he pe cen age o Eomes+ cells wi hin he CD56
neg
CD16+ subse was low, we
conside ed he possibili y ha o he CD16+ non-NK cells could ha e been selec ed using his ga ing s a -
egy. This hypo hesis was s eng hened by he ac ha wi hin he CD56
neg
CD16+ popula ion, he Eomes

cells had la ge size han Eomes+ cells (Figu e 1B). Thus, we s udied he exp ession o CD123 ecep o
(a-chain o he in e leukin 3 ecep o ) exp essed, among o he s, in plasmacy oid dend i ic cells (pDCs)
and basophils, which a e cha ac e ized by a la ge size and g anula i y (Collin e al., 2013;Han e al.,
2008;McKenna e al., 2005;Vi alle
´e al., 2019a;Zena uzabei ia e al., 2019). Resul s showed ha
CD56
neg
CD16+Eomescells exp essed CD123, in con as o CD56
neg
NKp80+Eomescells ha ba ely
did (Figu e 1C). Fu he mo e, he addi ion o an an i-CD123 mAb o he exclusion channel e ealed ha
he equency o CD56
neg
CD16+Eomes+ cells signi ican ly inc eased bu s ill ended o be lowe compa ed
wi h CD56
neg
NKp80+Eomes+ cells (Figu e 1D). These esul s sugges ed ha he inaccu acy in he iden i-
ica ion o he CD56
neg
NK cell subse using he CD16 ma ke in he ga ing s a egy is due o he selec ion
o Eomescells ha , a leas pa ially, could be pDCs and/o basophils, which a e cha ac e ized by he
exp ession o CD123.
Gi en ha he e a e no signi ican di e ences in he equency o CD56
neg
CD16+ and CD56
neg
NKp80+
cells (Figu e S1B), bu he la e exp essed signi ican ly highe le els o Eomes (Figu e 1A), we analyzed i
NKp80 is inclusi e o he CD56
neg
CD16+ subse . Resul s showed no signi ican di e ences in he e-
quency o CD16+NKp80,CD16NKp80+, and CD16+NKp80+ subse s wi hin he CD56
neg
cells
and ha hal o he CD56
neg
NKp80+ NK cells also co-exp ess CD16 (Figu e 2A).Impo an ly,when
he exp ession o Eomes was analyzed wi hin hese h ee subse s, we ound ha he equency o
Eomes+ cells was e y low in CD16+NKp80cells and signi ican ly highe in bo h CD16NKp80+
and CD16+NKp80+ cells (Figu e 2B). Mo eo e , al hough CD16+NKp80cells included a signi ican e-
quency o CD123+ cells (50%), bo h CD16NKp80+ and CD16+NKp80+ subse s comp ised negligible
le els o CD123+ cells (Figu e 2C). Al oge he , hese esul s sugges ha al hough CD16 and NKp80
do no comple ely iden i y he same CD56
neg
cells, NKp80 is mo e p ecise o he iden i ica ion o
CD56
neg
NK cells.
Mo e ecen ly, i was shown ha including CD7 as an addi ional ma ke o he CD56
neg
CD16+ cell subse
was an e ec i e me hod o accu a ely iden i y CD56
neg
NK cells (Milush e al., 2009,2013). The e o e, we
compa ed he equency o Eomes+ cells using CD7 o NKp80 ma ke s o iden i y he CD56
neg
NK cell
subpopula ion. The e we e no signi ican di e ences be ween CD7+CD56
neg
CD16+ and
CD56
neg
NKp80+ cells in e ms o Eomes exp ession, indica ing ha bo h s a egies we e equally e ec-
i e o he iden i ica ion o CD56
neg
cells in hese speci ic expe imen al se ings. Howe e , when we only
used he CD7 ma ke ins ead o NKp80 he equency o Eomes+ cells in he CD7+CD56
neg
popula ion
was much lowe han in bo h CD7+CD56
neg
CD16+ and CD56
neg
NKp80+ cells (Figu e 3A). On he o he
hand, combining he CD7 and CD16 ma ke s o iden i y he CD56
neg
NK cell subse could be an obs acle
in ce ain si ua ions, especially due o he uns able exp ession o he CD16 ecep o a e c yop ese a-
ion and cell s imula ion, and also he need o an addi ional monoclonal an ibody o he panel and an
ex a low cy ome e de ec o , which could be o e come by only using NKp80 as a ma ke o he iden-
i ica ion o CD56
neg
NK cells.
Nex , we s udied he CD300a (Dimi o a e al., 2016;Vi alle
´e al., 2019b;Zena uzabei ia e al., 2016)and
2B4 (CD244) (End e al., 2007) ecep o s ha a e also exp essed in NK cells, al hough no exclusi ely, as
ma ke s o he iden i ica ion o he CD56
neg
NK cells. Resul s showed a lowe equency o Eomes+ cells
bo h in CD56
neg
CD300a+ and in CD56
neg
2B4+ cells compa ed wi h CD56
neg
NKp80+ cells (Figu es 3Band
3C). Mo eo e , he addi ion o an an i-CD123 mAb o he exclusion channel minimally inc eased he e-
quency o Eomes+ cells (Figu e 3D). Al oge he , ou esul s demons a e ha NKp80 is he bes cell su ace
ma ke o iden i y he CD56
neg
NK cell subse wi h a e y high ce ain y and accu acy.
CD56
neg
NKp80+ Cells A e Expanded in HIV-In ec ed People and Pa ien s wi h Mul iple
Myeloma
As CD56
neg
NK cells a e in equen in he pe iphe al blood o heal hy dono s, we nex e alua ed he accu-
acy o he NKp80 ecep o o iden i y he expanded CD56
neg
NK cells in pa hological condi ions, such as
HIV in ec ion and mul iple myeloma (Figu e 4A; Table S1). No signi ican di e ences we e no iced in he
equency o Eomes+ cells be ween CD56
neg
CD16+ and CD56
neg
NKp80+ subpopula ions in un ea ed
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4iScience 23, 101298, July 24, 2020
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HIV-1 in ec ed subjec s. Howe e , he equency o Eomes+ cells was signi ican ly highe in
CD56
neg
NKp80+ han in CD56
neg
CD16+ cells in HIV-1 in ec ed subjec s unde combined an i e o i al
he apy (cART) (Figu e 4B). The di e ences be ween pa ien g oups could be explained because he ela-
i e equency o CD56
neg
CD16+ non-NK cells (Eomes) is lowe in un ea ed pa ien s due o a highe
expansion o he CD56
neg
CD16+ NK cells (Eomes+) (Al e e al., 2005;Ba ke e al., 2007;Ma ilio e al.,
2005). Thus, CD16 could only se e o iden i y CD56
neg
NK cells in ce ain pa hological condi ions in which
his subse is e y highly expanded.In addi ion, a highe equency o Eomes+ cells wi hin CD56
neg
NKp80+
cellsincompa isonwi hCD56
neg
CD16+ cells was also no iceable in mul iple myeloma pa ien s (Figu e 4C),
in which CD56
neg
NK cell expansion is mo e simila o he one o HIV-1 in ec ed subjec s unde cART (Fig-
u e 4A). These indings sugges ha NKp80, as demons a ed in heal hy dono s, is a no ewo hy al e na i e
o CD16 as a ma ke o iden i y CD56
neg
NK cells also in disease.
CD56
neg
NKp80+ Cells A e Mo e Func ional han CD56
neg
CD16+ Cells
Al hough su ace ecep o p o iling and p o eomic analyses indica e ha CD56
neg
ha e a pheno ypic ela-
ionship o CD56
dim
(Voig e al., 2018), CD56
neg
NK cells ha e been desc ibed as unc ionally impai ed
compa ed wi h CD56
dim
NK cells (Al e e al., 2005;Bjo
¨ ks o
¨m e al., 2010;Ma ilio e al., 2005;Milush
e al., 2013). Howe e , o he s ha e p oposed ha hese cells a e skewed a he han dys unc ional (Elle
Figu e 2. CD56
neg
CD16+NKp80Subse Mos ly Include Non-NK Cells
(A) Ba g aph showing he pe cen age o CD56
neg
CD16+NKp80,CD56
neg
CD16NKp80+ and CD56
neg
CD16+NKp80+
subse s wi hin he CD56
neg
cells.
(B) Ba g aph showing he pe cen age o Eomes+ cells wi hin he CD56
neg
CD16+NKp80,CD56
neg
CD16NKp80+, and
CD56
neg
CD16+NKp80+ popula ions.
(C) Ba g aphs showing he pe cen age o CD123+ cells wi hin he CD56
neg
CD16+NKp80,CD56
neg
CD16NKp80+, and
CD56
neg
CD16+NKp80+ popula ions. The mean wi h he s anda d e o o he mean (SEM) is ep esen ed. Each do
ep esen s a dono . ***p < 0.001, ****p < 0.0001, ns: no signi ican .
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e al., 2009). These di e ences may be due o inaccu a e iden i ica ion o CD56
neg
NK cells using he CD16
ma ke . The e o e, we s udied he e ec o unc ions o CD56
neg
CD16+ and CD56
neg
NKp80+ cells om
heal hy dono s and HIV-1 in ec ed subjec s, by measu ing deg anula ion (CD107a) (Al e e al., 2004)
and p oduc ion o TNF and IFNga e cy okines and K562 a ge cell s imula ions (Te e
´n e al., 2018).
Fi s , we wan ed o compa e CD16 and NKp80 exp ession down egula ion a e NK cell s imula ion. Resul s
showed ha NKp80 was no signi ican ly down egula ed a e K562 cell line and cy okine s imula ion,
whe eas CD16 exp ession signi ican ly dec eased a e s imula ion wi h bo h s imuli (Figu es 5Aand5B).
In e ms o unc ionali y, we obse ed ha CD56
neg
NKp80+ cells exhibi ed highe p oduc ion o TNF
and IFNg han CD56
neg
CD16+ cells in ea ed HIV-1 in ec ed subjec s and ha hey showed a endency
o a highe p oduc ion o bo h cy okines in un ea ed subjec s (Figu e 6A). Fu he mo e, in heal hy dono s,
bo h cy okine p oduc ion and he deg anula ioncapabili y ended obehighe in heCD56
neg
NKp80+
cells haninCD56
neg
CD16+ cells (Figu e 6B). Finally, we compa ed he unc ionali y o he CD56
dim
and
CD56
neg
NK cells. Ve y impo an ly, ou esul s showed ha , al hough CD56
neg
NK cells ha e lowe e ec o
unc ions han CD56
dim
NK cells (Figu e S3) in heal hy dono s and HIV-in ec ed people, hei unc ionali y is
much lowe when we used he CD16-based ga ing s a egy o iden i y CD56
neg
NK cells han when we used
Figu e 3. NKp80 Be e Iden i ies CD56
neg
NK Cells han CD7, CD300a, and 2B4 (CD244) in Heal hy Indi iduals
(A) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD7+CD56
neg
CD16+, CD56
neg
CD7+, CD56
neg
CD16+, and
CD56
neg
NKp80+ popula ions.
(B) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD56
neg
CD300a+, CD56
neg
CD16+, and CD56
neg
NKp80+
popula ions.
(C) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD56
neg
2B4+, CD56
neg
CD16+, and CD56
neg
NKp80+
popula ions.
(D) Ba g aphs showing he pe cen age o Eomes+ cells wi hin CD56
neg
CD300a+ and CD56
neg
2B4+ popula ions wi h o
wi hou he addi ion o an i-CD123 mAb o he exclusion channel (exclusion C.). The mean wi h he s anda d e o o he
mean (SEM) is ep esen ed. Each do ep esen s a dono . *p < 0.05, **p < 0.01, ***p < 0.001, ns: no signi ican .
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he NKp80-based ga ing s a egy. Al oge he , ou esul s indica e ha CD56
neg
NK cells, de ined as iable
CD3-CD19-CD14-CD56
neg
NKp80+ cells, a e signi ican ly less dys unc ional han p e iously hough .
DISCUSSION
I is o u mos impo ance o co ec ly pheno ype he di e en subpopula ions o immune cells no only in
heal hy people bu also in disease si ua ions. In he la e , a ia ions in he equency o cells subse s and in
hei e ec o unc ions, in compa ison o heal hy s a e, a e equen ly obse ed. These a ia ions can help
o unde s and he pa hogenesis o diseases, and mo eo e , hese changes can se e as bioma ke s o he
diagnosis, p ognosis, and/o o de e mine he e icacy o he ea men .
Some immune cell ypes a e cha ac e ized by he exp ession o speci ic lineage cell su ace ma ke s. Fo
example, T cells a e CD3+, whe eas o he cell ypes do no exp ess CD3. Howe e , a speci ic NK cell su -
ace ma ke has no been desc ibed ye . In gene al e ms, he minimum equi emen o de ine ci cula ing
human NK cells is based on he exp ession o CD56 and he absence o he CD3 ma ke , because an impo -
an subpopula ion o T cells exp esses CD56 (O aldo e al., 1991). Howe e , he e a e o he cells, such as
ILC3, which can also exp ess CD56 (A is and Spi s, 2015;Spi s e al., 2013;Vallen in e al., 2015;Vi ie e al.,
2018). On he o he hand, acco ding o he exp ession o CD56 and CD16, NK cells ha e been classi ied in o
ou subpopula ions: CD56
b igh
(CD56
b igh
CD16+/), CD56
dim
(CD56
dim
CD16+), uncon en ional CD56
dim
Figu e 4. NKp80 Be e Iden i ies CD56
neg
NKCells hanCD16inPa hologicalCondi ions
(A) Ba g aphs showing he pe cen age o CD56
neg
CD16+ (le pa ) and CD56
neg
NKp80+ ( igh pa ) subse s wi hin o al
NK cells om heal hy dono s (HD), un ea ed HIV-1 in ec ed people (HIV), HIV-1-in ec ed pa ien s unde cART (cART) and
mul iple myeloma pa ien s.
(B) Ba g aphs showing he pe cen age o Eomes+ cells wi hin CD7+CD56
neg
CD16+, CD56
neg
CD7+, CD56
neg
CD16+,
and CD56
neg
NKp80+ popula ions in un ea ed HIV-1-in ec ed subjec s (HIV) and HIV-1-in ec ed pa ien s unde cART
(cART).
(C) Ba g aph showing he pe cen age o Eomes+ cells wi hin CD56
neg
CD16+ and CD56
neg
NKp80+ popula ions in
mul iple myeloma pa ien s. The mean wi h he s anda d e o o he mean (SEM) is ep esen ed. Each do ep esen s a
dono . *p < 0.05, **p < 0.01, ns: no signi ican .
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(CD56
dim
CD16), and CD56
neg
(CD56
neg
CD16+) (F eud e al., 2017;Hu e al., 1995;Mon aldo e al., 2013;
Robe o e al., 2018;Di Vi o e al., 2019). Finally, al hough he Eomes ansc ip ion ac o is also exp essed in
CD4+ and CD8+ T cells (Knox e al., 2014;Na ayanan e al., 2010), i is used as a speci ic in acellula ma ke
o NK cells wi hin he ILCs.
CD56
neg
cells ep esen a e y low pe cen age o NK cells in pe iphe al blood om heal hy people
(Bjo
¨ ks o
¨m e al., 2010;Campos e al., 2014;Mu
¨lle -Du o ic e al., 2019). Howe e , in ce ain diseases
he e is a e y signi ican expansion o his cell subpopula ion (Al e e al., 2005;Ba ke e al., 2007;Fo -
coni e al., 2018;Hu e al., 1995;Lugli e al., 2014;Ma ilio e al., 2005). To ou knowledge, in p e ious
publica ions, he exp ession o he CD16 ma ke ha e so a been used in all he ga ing s a egies o
iden i y he CD56
neg
NK cell subse . Some au ho s ha e made use o a s a egy based on only h ee
ma ke s (CD3, CD56, and CD16) (Al e e al., 2005,2006;Ba ke e al., 2007;Campos e al., 2014;F ias
Figu e 5. CD16, Bu no NKp80, Is Down egula ed a e K562 Cell Line and IL-12+IL-18 Cy okine S imula ion
(A) Rep esen a i e pseudocolo plo g aphs compa ing he exp ession o CD16 and NKp80 in non-s imula ed condi ion,
and a e K562 cell line and IL-12+IL-18 cy okine s imula ion. Da a om a ep esen a i e heal hy dono is shown.
(B) His og ams showing he median luo escence in ensi y (MFI) o CD16 and NKp80 on NK cells in non-s imulus condi ion
and a e K562 cell line and IL-12+IL-18 cy okine s imula ion. Da a om a ep esen a i e heal hy dono is shown.
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iScience, Volume 23
Supplemen al In o ma ion
A NKp80-Based Iden ifica ion S a egy
Re eals ha CD56
neg
NK Cells A e No Comple ely
Dys unc ional in Heal h and Disease
Ane O an ia, Iñigo Te én, Alicia Izquie do-La uen e, Juncal A. Alonso-Cab e a, Vic o
Sandá, Joana Vi allé, San iago Mo eno, Ma ía Tasias, Alasne U anga, Ca men
González, Juan J. Ma eos, Juan C. Ga cía-Ruiz, Ola z Zena uzabei ia, and F ancisco
Bo ego

Viable/CD3/14/19-
3.96
NK cells CD56neg
NK cells
A
B
Figu e S1. Iden i ica ion o CD56neg NK cells. Rela ed o Figu e 1. (A) Pseudocolo and con ou plo
g aphs ep esen ing he ga ing s a egy u ilized o he iden i ica ion o CD56neg NK cells. Da a om a
ep esen a i e c yop ese ed sample om a heal hy dono is shown. Lymphocy es we e elec onically
ga ed based on hei o wa d and side sca e pa ame e s and hen single cells we e selec ed. To iden i y
NK cells, he popula ion nega i e o he exclusion channel ( iabili y, CD3, CD14 and CD19) was
selec ed. Then CD56neg NK cells we e iden i ied using di e en ga ing s a egies. (B) Pe cen age o
CD56negCD16+ and CD56negNKp80+ cells in heal hy dono s. Ba g aph showing he pe cen age o
CD56negCD16+ and CD56negNKp80+ cells in heal hy dono s. The mean wi h he s anda d e o o he
mean (SEM) is ep esen ed. Each do ep esen s a dono . ns: no signi ican .
A
CD56
CD16
NKp80
B
Fig. S2. CD16 bu no NKp80 is down egula ed a e cell c yop ese a ion. Rela ed o Figu e 1. (A)
Rep esen a i e pseudocolo plo g aphs compa ing he exp ession o CD16 and NKp80 in esh and
c yop ese ed samples. Da a om a ep esen a i e heal hy dono is shown. (B) His og ams showing he median
luo escence in ensi y (MFI) o CD16 and NKp80 on NK cells in esh and c yop ese ed samples. Da a om a
ep esen a i e heal hy dono is shown.
CD56dim
CD56neg
K562 cell line
CD16 based ga ing
IL-12 + IL-18
NKp80 based ga ing
HIV cART HD HIV cART HD
HIV cART HD HIV cART HD
HIV cART HD HIV cART HD
Fig. S3. Deg anula ion (CD107a) and cy okine p oduc ion by NK cells in esponse o he K562 cell line
and IL-12+IL-18 cy okine s imula ion. Rela ed o Figu e 6. Ba g aphs showing he pe cen age o CD56dim
and CD56neg NK cells posi i e o CD107a and TNF a e K562 cell line s imula ion and IFNγ a e IL-12+IL-18
cy okine s imula ion om HIV-1 in ec ed subjec s (HIV), HIV-1 in ec ed pa ien s unde cART (cART) and
heal hy dono s (HD). The mean wi h he s anda d e o o he mean (SEM) is ep esen ed. Each do
ep esen s a dono .
Table S1. Clinical da a o un ea ed HIV-1 in ec ed subjec s, unde cART HIV-1
in ec ed pa ien s and mul iple myeloma pa ien s. Rela ed o Figu e 4 and Figu e 6.
Un ea ed HIV-1
subjec s
HIV-1 pa ien s on
cART
Mul iple myeloma
pa ien s
Median
Range
(min-
max)
Median
Range
(min-
max)
Median
Range
(min-
max)
Sex
Female: n=0
Male: n=9
-
Female: n=1
Male: n=7
-
Female: n=6
Male: n=3
-
Age
(yea s)
39
(32-59)
49
(34-56)
63
(53-74)
cART
(mon hs)
-
-
13
(10-27)
-
-
TRANSPARENT METHODS
Con ac o eagen s and esou ce sha ing
Fu he in o ma ion and eques o esou ces and eagen s should be di ec ed o and will be
ul illed by he Lead Con ac , F ancisco Bo ego ( ancisco.bo ego abasco@osakide za.eus).
This s udy did no gene a e new unique eagen s.
Expe imen al Model and Subjec De ails
Fo his s udy, bu y coa s om 24 heal hy adul dono s and c yop ese ed pe iphe al blood
mononuclea cells (PBMCs) om 9 mul iple myeloma pa ien s we e collec ed h ough he
Basque Biobank o Resea ch (h p://www.biobanco asco.o g), which complies wi h he quali y
managemen , aceabili y and biosecu i y, se ou in he Spanish Law 14/2007 o Biomedical
Resea ch and in he Royal Dec ee 1716/2011. The s udy was app o ed by he Basque E hics
Commi ee o Clinical Resea ch (PI2014017 and PI+CES+INC-BIOEF 2017-03). All subjec s
p o ided w i en and signed in o med consen in acco dance wi h he Decla a ion o Helsinki. In
addi ion, c yop ese ed PBMCs om heal hy dono s (n=5), un ea ed HIV-1 in ec ed subjec s
(n=9) and pa ien s unde cART (n=8) we e p o ided by he HIV BioBank in eg a ed in he
Spanish AIDS Resea ch Ne wo k (RIS) (Supplemen a y In o ma ion, Appendix I). Samples we e
p ocessed ollowing cu en p ocedu es and ozen immedia ely a e hei ecep ion. All pa ien s
pa icipa ing in he s udy ga e hei in o med consen and p o ocols we e app o ed by
ins i u ional e hical commi ees.
All HIV-1 in ec ed pa ien s we e asymp oma ic when he sample was collec ed, we e no co-
in ec ed wi h hepa i is C i us (HCV), had mo e han 200 CD4+ T cells/mm3 and hey had ne e
been diagnosed wi h AIDS. Un ea ed HIV-1 in ec ed subjec s had de ec able i emia (>10,000
HIV-RNA copies/ml) and hey had ne e been ea ed wi h cART, while pa ien s unde cART
had unde ec able i emia and had been ea ed wi h cART a leas o 6 mon hs. Clinical da a o
HIV-1 in ec ed pa ien s we e ob ained om he RIS da abase. Clinical da a a e shown in
Table S1.
An ibodies and eagen s
Fo low cy ome y-based p ocedu es, he ollowing luo och ome-conjuga ed an i-human
monoclonal an ibodies (mAbs) we e used: B illian Viole (BV)421 an i-CD56 (NCAM 16.2),
BV510 an i-CD3 (UCHT1), BV510 an i-CD14 (MФP9), BV510 an i-CD19 (SJ25C1), BV510 an i-
CD123 (9F5), PE an i-CD123 (9F5), PE an i-CD7 (M-T701) and Pe CP-Cy5.5 an i-IFNγ (B27)
om BD Biosciences; FITC an i-CD16 (B73.1) and APC an i-TNF (MAb11) om BioLegend; PE
an i-CD300a (E59.126) and PE an i-2B4 (clone C1.7) om Beckman Coul e ; PE an i-CD107a
(REA792) and PE-Vio770 an i-NKp80 (4A4.D10) om Mil enyi Bio ec; eFluo 660 an i-Eomes
(WD1928) om eBioscience. Dead cells we e de ec ed wi h he LIVE/DEAD™ Fixable Aqua
Dead Cell S ain Ki o 405nm exci a ion om In i ogen, ollowing manu ac u e ’s p o ocol.
The ollowing eagen s we e also used: Foxp3/T ansc ip ion Fac o S aining Bu e Se om
eBioscience; B illian S ain Bu e , BD GolgiS op™ P o ein T anspo Inhibi o (monensin), BD
GolgiPlug™ P o ein T anspo Inhibi o (b e eldin A) and BD Pe m/Wash™ Bu e om BD
Bioscience; and pa a o maldehyde (PFA) om Sigma-Ald ich/Me ck.
Me hods De ails
Pe iphe al blood mononuclea cell isola ion.
F esh PBMCs om heal hy dono s we e ob ained om bu y coa s by Ficoll (GE Heal hca e)
densi y g adien cen i uga ion and c yop ese ed in Fe al Bo ine Se um (FBS) (GE Heal hca e
Hyclone) wi h 10% Dime hylsul oxide (DMSO) (The mo Scien i ic Scien i ic).

Flow cy ome y: Pheno ypical s udies.
Fo pheno ypical s udies, c yop ese ed PBMCs om heal hy dono s, HIV-1-in ec ed subjec s
and mul iple myeloma pa ien s we e hawed a 37ºC and washed wice wi h RPMI 1640 medium
wi h L-Glu amine (Lonza). Then, cells we e incuba ed o 1 hou a 37ºC wi h 10U DNase
(Roche) in R10 medium (RPMI 1640 medium con aining Glu aMAX om The mo Fishe
Scien i ic, 10% FBS and 1% Penicillin-S ep omycin om The mo Fishe Scien i ic). A e wa ds,
cells we e coun ed and washed wi h Phospha e Bu e ed Saline (PBS) (Gibco, The mo Fishe
Scien i ic). Then, dead cells we e excluded by using he LIVE/DEAD eagen (In i ogen,
The mo Fishe Scien i ic). Fo he s aining o NK cell su ace ma ke s, cells we e i s washed
wi h PBS con aining 2.5% o Bo ine Se um Albumin (BSA) (Millipo e) and hen incuba ed o 30
minu es a 4ºC wi h luo och ome-conjuga ed mAbs. To iden i y NK cells, i s iable cells ha
we e nega i e o CD3, CD14 and CD19 we e elec onically ga ed, and hen, by using he an i-
CD56 mAb in combina ion wi h mAbs agains CD16, NKp80, CD300a, 2B4 and/o CD7, NK
cells we e classi ied in h ee subse s: CD56b igh , CD56dim and CD56neg. A e his, cells we e
washed again wi h 2.5% BSA in PBS and ixed and pe meabilized wi h Foxp3/T ansc ip ion
Fac o S aining Bu e Se (eBioscience, The mo Fishe Scien i ic) ollowing manu ac u e ’s
ecommenda ions. Finally, cells we e s ained using an i-Eomes mAb o 30 minu es a oom
empe a u e (RT) and washed wi h Pe meabiliza ion Bu e 1x (eBioscience). Sample
acquisi ion was ca ied ou in a MACSQuan Analyze 10 low cy ome e (Mil enyi Bio ec).
Flow cy ome y: Func ional assays.
Fo unc ional assays, a e DNase ea men , PBMCs om HIV-1-in ec ed subjec s and heal hy
dono s we e coun ed and pla ed a 0.5 x 106 cells/well in 48 well pla es in NK cell cul u e
medium (RPMI 1640 medium wi h Glu aMAX, 10% FBS, 1% penicillin s ep omycin, 1% non-
essen ial amino acids and 1% Sodium-Py u a e). PBMCs we e hen p imed wi h in e leukin (IL)-
15 (10ng/mL) and cul u ed o 20 hou s. Fo cy okine s imula ion, IL-12 (10ng/mL) and IL-18
(50ng/mL) we e also added o pla ed PBMCs. Fo a ge cell s imula ion, K562 cells we e added
a e he 20 hou s o cul u e in IL-15 a E ec o :Ta ge (E:T) 1:1 a io (0.5x106 PBMCs and
0.5x106 K562 cells). Then, IL-12+IL-18 and K562 s imula ed PBMCs we e cul u ed o 6 hou s.
CD107a was added a he s a o he co-cul u e pe iod and p o ein anspo inhibi o s we e
added a e 1 hou o he es o he incuba ion ime ollowing manu ac u e ’s p o ocol.
A e wa ds, iabili y and su ace ma ke s aining was pe o med as explained abo e. Fo
in acellula s aining, cells we e ixed wi h 4% PFA o 15 minu es on ice and hen washed wice
wi h 2.5% BSA in PBS. A e his, cells we e pe meabilized wi h BD Pe m/Wash Bu e 1X o 15
minu es a RT. Finally, he co esponding mAbs we e added o 30 minu e and cells we e
washed wi h BD Pe m/Wash Bu e 1X be o e acquisi ion in he MACSQuan Analyze 10 low
cy ome e (Mil enyi Bio ec). The pe cen age o posi i e cells o CD107a, IFNγ and TNF was
calcula ed a e sub ac ing he non-s imulus condi ion.
Quan i ica ion and S a is ical Analysis.
Da a we e analysed using FlowJo™ 10.4.1. G aphPad P ism 8.01 so wa e was used o
g aphical ep esen a ion and s a is ical analysis. As speci ied in all igu e legends, each do in
he g aphs ep esen s a dono . Da a we e ep esen ed showing means ± s anda d e o o he
mean (SEM) o median as indica ed in he igu e legend. P io o s a is ical analyses, da a we e
es ed o no mal dis ibu ion wi h Kolmogó o -Smi no no mali y es . In he case o mul iple
myeloma pa ien s, an ou lie was iden i ied and emo ed using G ubb es (alpha=0.05). I da a
we e no mally dis ibu ed, es o pai ed alues was used o de e mine signi ican di e ences.
Non-no mal dis ibu ed da a we e compa ed wi h Wilcoxon ma ched-pai s signed ank es .
K uskal-Wallis es was used o mul iple compa isons o non-no mal da a (Figu e 4A). *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001.
Appendix I: CoRIS Membe s
Execu i e commi ee
San iago Mo eno, Inma Ja ín, Da id Dalmau, Ma ia Luisa Na a o, Ma ia Isabel González,
Fede ico Ga cia, E a Po eda, Jose An onio I iba en, Félix Gu ié ez, Ra ael Rubio, F ancesc
Vidal, Juan Be engue , Juan González, M Ángeles Muñoz-Fe nández.
Fieldwo k da a managemen and analysis
Inmaculada Ja in, Belén Alejos, C is ina Mo eno, Ca los Inies a, Luis Miguel Ga cia Sousa,
Nie es Sanz Pe ez, Ma a Ra a
BioBanK HIV Hospi al Gene al Uni e si a io G ego io Ma añón
M Ángeles Muñoz-Fe nández, I ene Consueg a Fe nández
Hospi al Gene al Uni e si a io de Alican e (Alican e)
Espe anza Me ino, Gema Ga cía, I ene Po illa, I án Agea, Joaquín Po illa, José Sánchez-
Payá., Juan Ca los Rod íguez, Lina Gimeno, Li ia Gine , Ma cos Díez, Melissa Ca e es,
Se gio Reus, Vicen e Boix, Diego To ús
Hospi al Uni e si a io Cen al de As u ias (O iedo)
Víc o Asensi, Eulalia Valle, Ma ía Eugenia Ri as Ca menado, Tomas Sua ez-Za acina
Secades, Lau a Pé ez Is
Hospi al Uni e si a io 12 de Oc ub e (Mad id)
Ra ael Rubio, Fede ico Pulido, O ilia Bisbal, Asunción He nando, Lou des Domínguez, Da id
Rial C es elo, Lau a Be mejo, Mi eia San ac eu
Hospi al Uni e si a io de Donos ia (Donos ia-San Sebas ián)
José An onio I iba en, Julio A izabalaga, Ma ía José A ambu u, Xabie Camino, F ancisco
Rod íguez-A ondo, Miguel Ángel on Wichmann, Lidia Pascual Tomé, Miguel Ángel Goenaga,
Mª Jesús Bus induy, Ha kai z Azkune, Maialen Iba gu en, Ai zibe Liza di, Xabie Ko aja ena.,
Mª Pila Ca mona Oyaga, Mai ane Ume ez Iga ua
Hospi al Gene al Uni e si a io De Elche (Elche)
Félix Gu ié ez, Ma Masiá, Se gio Padilla, Ca alina Robledano, Joan G ego i Colomé, A aceli
Adsua , Ra ael Pascual, Ma a Fe nández, José Albe o Ga cía, Xa ie Ba be , Vanessa Agullo
Re, Ja ie Ga cia Abellan, Reyes Pascual Pé ez, Ma ía Roca
Hospi al Gene al Uni e si a io G ego io Ma añón (Mad id)
Juan Be engue , Juan Ca los López Be naldo de Qui ós, Isabel Gu ié ez, Ma ga i a Ramí ez,
Belén Padilla, Paloma Gijón, Te esa Aldamiz-Eche a ía, F ancisco Teje ina, F ancisco José
Pa as, Pascual Balsalob e, C is ina Diez, Lei e Pé ez La o e., Chia a Fanciulli
Hospi al Uni e si a i de Ta agona Joan XXIII (Ta agona)
F ancesc Vidal, Joaquín Pe ai e, Consuelo Viladés, Se gio Veloso, Mon se a Va gas,
Mon se a Olona, Anna Rull, Es he Rod íguez-Gallego, Ve ónica Alba., Al onso Ja ie
Cas ellanos, Miguel López-Dupla
Hospi al Uni e si a io y Poli écnico de La Fe (Valencia)
Ma a Mon e o Alonso, José López Aldegue , Ma ino Blanes Juliá, Ma ía Tasias Pi a ch, I án
Cas o He nández, E a Calabuig Muñoz, Sand a Cuélla To a , Miguel Sala e Lle í, Juan
Fe nández Na a o.
Hospi al Uni e si a io La Paz/IdiPAZ
Juan González-Ga cia, F ancisco A nalich, José Ramón A ibas, Jose Ignacio Be na dino de la
Se na, Juan Miguel Cas o, Ana Delgado Hie o, Luis Escosa, Ped o He anz, Víc o
Hon añón, Sil ia Ga cía-Bujalance, Milag os Ga cía López-Ho elano, Alicia González-Baeza,
Ma ia Luz Ma ín-Ca bone o, Ma io Mayo al, Ma ia Jose Mellado, Ra ael Es eban Micán, Rocio
Mon ejano, Ma ía Luisa Mon es, Vic o ia Mo eno, Ignacio Pé ez-Vale o, Guadalupe Rúa
Ceb ián, Be a Rodés, Talia Sainz, Elena Sendago a, Na alia S ella Alcá iz, Eulalia Valencia.
Hospi al Uni e si a i Mu uaTe assa (Te asa)
Da id Dalmau, Angels Jaén, Mon se Sanma í, Mi eia Cai ó, Ja ie Ma inez-Lacasa, Pablo
Velli, Rose Fon , Ma ina Ma inez, F ancesco Aiello
Hospi al Uni e si a io de La P incesa (Mad id)
Ignacio de los San os, Jesus Sanz Sanz, Ana Salas Apa icio, C is ina Sa ia Cepeda, Lucio
Ga cia-F aile F aile, En ique Ma ín Gayo.
Hospi al Uni e si a io Ramón y Cajal (Mad id)
San iago Mo eno, José Luis Casado Oso io, Fe nando D onda Nuñez, Ana Mo eno Zamo a,
Ma ia Jesús Pé ez Elías, Ca olina Gu ié ez, Nadia Mad id, San os del Campo Te ón, Se gio
Se ano Villa , Ma ia Jesús Vi ancos Gallego, Ja ie Ma ínez Sanz, Usua Anxa U oz, Tama a
Velasco
Hospi al Gene al Uni e si a io Reina So ía (Mu cia)
En ique Be nal, Al edo Cano Sanchez, An onia Alca az Ga cía, Joaquín B a o U bie a,
Angeles Muñoz Pe ez, Ma ia Jose Alca az, Ma ia del Ca men Villalba.
Hospi al Nue o San Cecilio (G anada)
Fede ico Ga cía, José He nández Que o, Leopoldo Muñoz Medina , Ma a Al a ez, Na alia
Chueca, Da id Vinuesa Ga cía , Cla a Ma inez-Mon es., Ca los Gue e o Bel an, Adol o de
Salaza Gonzale z, Ana Fuen es Lopez
Cen o Sani a io Sando al (Mad id)
Mon se a Raposo U illa, Jo ge Del Rome o, Ca men Rod íguez, Te esa Pue a, Juan Ca los
Ca ió, Ma Ve a, Juan Balles e os, Oska Aye di.
Hospi al Uni e si a io Son Espases (Palma de Mallo ca)
Melcho Rie a, Ma ía Peña anda, Mª Angels Ribas, An oni A Campins, Ca men Vidal, F ancisco
Fanjul, Ja ie Mu illas, F ancisco Homa ., Helem H Vilchez, Ma ia Luisa Ma in, An oni Paye as.
Hospi al Uni e si a io Vi gen de la Vic o ia (Málaga)
Jesús San os, C isi ina Gómez Aye be, Isabel Viciana, Rosa io Palacios, Ca men Pé ez López,
Ca men Ma ia Gonzalez-Domenec
Hospi al Uni e si a io Vi gen del Rocío (Se illa)
Pompeyo Viciana, Nu ia Espinosa, Luis Fe nando López-Co és.
Hospi al Uni e si a io de Bell i ge (Hospi ale de Llob ega )
Daniel Podzamcze , A kai z Imaz, Juan Ti aboschi, Ana Sil a, Ma ía Saumoy, Paula P ie o
Hospi al Cos a del Sol (Ma bella)
Julián Olalla Sie a, Ja ie Pé ez S achowski., Al onso del A co, Ja ie de la To e, José Luis
P ada, José Ma ía Ga cía de Lomas Gue e o
Hospi al Gene al Uni e si a io San a Lucía (Ca agena)
Ono e Juan Ma ínez, F ancisco Jesús Ve a, Lo ena Ma ínez, Jose ina Ga cía, Begoña
Alca az, Amaya Jimeno.
Complejo Hospi ala io Uni e si a io a Co uña (Chuac) (A Co uña)
Angeles Cas o Iglesias, Be a Pe nas Sou o, Al a o Mena de Cea.
Hospi al Uni e si a io Vi gen de la A ixaca (El Palma )
Ca los Gale a, Helena Albendin, Au o a Pé ez, Asunción Ibo a, An onio Mo eno, Ma ia
Angus ias Me los, Asunción Vidal, Ma isa Meca
Hospi al Uni e si a io In an a So ia (San Sebas ian de los Reyes)
Inés Suá ez-Ga cía, Edua do Malmie ca, Pa icia González-Ruano, Dolo es Ma ín Rod igo, Mª
Pila Ruiz Seco.
Hospi al Uni e si a io P íncipe de As u ias (Alcalá de Hena es)
José Sanz Mo eno, Albe o A anz Caso, C is ina He nández Gu ié ez, Ma ía No ella Mena.
Hospi al Clínico Uni e si a io de Valencia (València)
Ma ía José Galindo Pue o, Ramón Fe nando Vilal a, Ana Fe e Ribe a.
Hospi al Reina So ía (Có doba)
An onio Ri e o Román, An onio Ri e o Juá ez, Ped o López López, Isabel Machuca Sánchez,
Ma io F ias Casas, Angela Camacho Espejo
Hospi al Uni e si a io Se e o Ochoa (Leganés)
Miguel Ce e o Jiménez, Ra ael To es Pe ea
Nues a Seño a de Valme (Se illa)
Juan A Pineda, Pila Rincón Mayo, Juan Macias Sanchez, Nicolas Me chan e Gu ie ez, Luis
Miguel Real, Anais Co ma Gomez, Ma a Fe nandez Fue es, Alejand o Gonzalez-Se na