An igen P esen ing Cell-Media ed Expansion o Human
Umbilical Co d Blood Yields Log-Scale Expansion o
Na u al Kille Cells wi h An i-Myeloma Ac i i y
Nina Shah
1
*, Bea iz Ma in-An onio
1
, Hong Yang
1
, S ephanie Ku
2
, Dean A. Lee
3
, Lau ence J. N. Coope
3
,
William K. Decke
2,4
, Su ang Li
1
, Simon N. Robinson
1
, Takuya Sekine
1
, Sim i Pa ma
1
, John G ibben
5
,
Michael Wang
6
, Ka y Rez ani
1
, E ic Y on
1
, Ame Najja
7
, Ja ed Bu ks
8
, Ind eshpal Kau
1
,
Richa d E. Champlin
1
, Ca he ine M. Bolla d
2
, Elizabe h J. Shpall
1
1Depa men o S em Cell T ansplan a ion and Cellula The apy, The Uni e si y o Texas M.D. Ande son Cance Cen e , Hous on, Texas, Uni ed S a es o Ame ica, 2Cen e
o Cell and Gene The apy, Baylo College o Medicine, Hous on, Texas, Uni ed S a es o Ame ica, 3Depa men o Pedia ics, The Uni e si y o Texas M.D. Ande son
Cance Cen e , Hous on, Texas, Uni ed S a es o Ame ica, 4Depa men o Pa hology and Immunology, Baylo College o Medicine, Hous on, Texas, Uni ed S a es o
Ame ica, 5Ins i u e o Cance , Queen Ma y Uni e si y o London, Cen e o Medical Oncology, Ba s and The London School o Medicine, London, Uni ed Kingdom,
6Depa men o Lymphoma, The Uni e si y o Texas M.D. Ande son Cance Cen e , Hous on, Texas, Uni ed S a es o Ame ica, 7Depa men o Expe imen al Diagnos ic
Imaging, The Uni e si y o Texas M.D. Ande son Cance Cen e , Hous on, Texas, Uni ed S a es o Ame ica, 8Depa men o Leukemia Resea ch, The Uni e si y o Texas
M.D. Ande son Cance Cen e , Hous on, Texas, Uni ed S a es o Ame ica
Abs ac
Na u al kille (NK) cells a e impo an media o s o an i- umo immuni y and a e ac i e agains se e al hema ologic
malignancies, including mul iple myeloma (MM). Umbilical co d blood (CB) is a p omising sou ce o allogeneic NK cells bu
la ge scale ex i o expansion is equi ed o gene a ion o clinically ele an CB-de i ed NK (CB-NK) cell doses. He e we
desc ibe a no el s a egy o expanding NK cells om c yop ese ed CB uni s using a i icial an igen p esen ing eede cells
(aAPC) in a gas pe meable cul u e sys em. A e 14 days, mean old expansion o CB-NK cells was 1848- old om esh and
2389- old om c yop ese ed CB wi h .95% pu i y o NK cells (CD56
+
/CD3
2
) and less han 1% CD3
+
cells. Though su ace
exp ession o some cy o oxici y ecep o s was dec eased, aAPC-expanded CB-NK cells exhibi ed a pheno ype simila o CB-
NK cells expanded wi h IL-2 alone wi h espec o a ious inhibi o y ecep o s, NKG2C and CD94 and main ained s ong
exp ession o ansc ip ion ac o s Eomesode min and T-be . Fu he mo e, CB-NK cells o med unc ional immune synapses
wi h and demons a ed cy o oxici y agains a ious MM a ge s. Finally, aAPC-expanded CB-NK cells showed signi ican
in i o ac i i y agains MM in a xenogenic mouse model. Ou indings in oduce a clinically applicable s a egy o he
gene a ion o highly unc ional CB-NK cells which can be used o e adica e MM.
Ci a ion: Shah N, Ma in-An onio B, Yang H, Ku S, Lee DA, e al. (2013) An igen P esen ing Cell-Media ed Expansion o Human Umbilical Co d Blood Yields Log-
Scale Expansion o Na u al Kille Cells wi h An i-Myeloma Ac i i y. PLoS ONE 8(10): e76781. doi:10.1371/jou nal.pone.0076781
Edi o : E en Alici, Ka olinska Ins i u e , Sweden
Recei ed Ap il 16, 2013; Accep ed Augus 29, 2013; Published Oc obe 18, 2013
Copy igh : ß2013 Shah e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s
un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed.
Funding: This wo k was suppo ed by he Na ional Ins i u es o Heal h K12 CA088084 (Shah) and Cance P e en ion and Resea ch Ins i u e o Texas RP#100430
(Shpall). The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip .
Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis .
* E-mail: nshah@mdande son.o g
In oduc ion
Mul iple myeloma (MM) is he second mos common hema o-
logic malignancy in adul s [1]. I is cu en ly conside ed incu able,
e en a e high dose chemo he apy and au ologous hema opoie ic
s em cell ansplan a ion (HSCT) [2]. Na u al kille (NK) cells a e
CD56
+
/CD3
2
cy o oxic lymphocy es ha a e inc easingly ecog-
nized as a po en cellula he apy. NK cells ha e been shown o be
ac i e agains MM in se e al p eclinical s udies [3,4]. In addi ion,
a ela i e dec ease in NK cell equency o unc ion in MM
pa ien s has been shown o co ela e wi h mo e ad anced disease
o poo e ou come [5,6].
NK cell cy o oxic ac i i y can be igge ed by cy okines,
an ibodies o a shi in he balance be ween hei ac i a ing and
inhibi o y ecep o s. Speci ically, NK cells a e cy o oxic o cells
lacking app op ia e sel -majo his ocompa ibili y complex (MHC)
class I molecules ia disinhibi ion o he kille immunoglobulin-like
ecep o (KIR). This o ms he basis o he ‘‘missing sel ’’
hypo hesis [7] and is hough o media e dono NK cell
allo eac i i y in he se ing o allogeneic HSCT. Howe e he
p ecise ole o KIR-ligand misma ch in HSCT is no known. In
some pa ien s ea ed wi h allogeneic-HSCT, PB-NK cell allo -
eac i i y as de e mined by missing KIR ligands appea s o p edic
educed a es o elapse and g a e sus hos disease (GVHD)
[8,9]. Addi ionally, in MM pa ien s unde going ma ched alloge-
neic-HSCT, an ac i a ed dono KIR haplo ype (Bx) has been
associa ed wi h a signi ican ly lowe isk o elapse and be e PFS
[10]. In con as , o he s udies ha e sugges ed ha he e ec o
KIR-ligand incompa ibili y is no consis en , pa icula ly as i
ela es o condi ioning egimen, dono sou ce and GVHD
ou comes [11,12,13,14].
Al hough allogeneic NK cells appea p omising in MM,
au ologous PB-NK cells om MM pa ien s appea o be
hypo unc ional [15]. This may be due o inhibi o y cy okines
PLOS ONE | www.plosone.o g 1 Oc obe 2013 | Volume 8 | Issue 10 | e76781
such as TGF-b, IL-6 and IL-10 p esen in he MM mic oen i-
onmen [16,17,18] o dys egula ion o IL-15 signaling in a o o
MM cells o e ac i a ion o NK cells [19,20]. While some p e-
clinical s udies sugges ha his NK cell dys unc ion can be
e e sed ia ex i o expansion/ac i a ion [4,21,22], he po en ially
unp edic able na u e o au ologous NK cells om hea ily p e-
ea ed pa ien s wa an s u he op imiza ion o echniques o
allogeneic adop i e NK cell he apy. Fu he mo e, in ad anced
disease s a es, MM cells may up egula e Class I exp ession [23].
This sugges s ha KIR-MHC class I misma ched, allogeneic NK
cell he apy would be ad an ageous o e au ologous NK cell
he apy, as allogeneic NK cells would be less inhibi ed by cogna e
MHC class I in con as o au ologous NK cells.
To da e, he majo i y o clinical ials o NK cell he apy o
a ious malignancies ha e used allogeneic PB as a sou ce o NK
cells. We a e in e es ed in NK cells de i ed om human umbilical
co d blood (CB) as an al e na i e and mo e eadily a ailable
sou ce o NK cells. Ou g oup has p e iously demons a ed ha ex
i o expansion wi h IL-2 ac i a es o he wise quiescen CB-NK
cells. These CB-NK cells exhibi a ma u e pheno ype, simila o
PB-NK cells, and a e as ac i e as PB-NK cells agains leukemia
a ge s [24].
The limi ed numbe o NK cells in an unmanipula ed CB uni
equi es an e icien and obus NK cell ex i o expansion s a egy.
Se e al g oups ha e ecen ly epo ed expansion o PB-NK cells
using gene ically enginee ed a i icial an igen p esen ing cells
(aAPCs) de i ed om he K562 cell line [25,26]. In his s udy, we
build upon ecen ly de eloped echnology wi h aAPCs [26] and
desc ibe a no el echnique o expanding CB-NK cells o use in
MM. This good manu ac u ing p ac ice (GMP)-complian me hod
yields clinical scale expansion o pheno ypically ma u e CB-NK
cells which a e cy o oxic o MM cells in i o and demons a e
in i o an i-MM ac i i y in a xenogenic model. Taken oge he , ou
esul s p o ide he basis o u he explo a ion o CB-NK cell
he apy o pa ien s wi h MM.
Ma e ials and Me hods
E hics S a emen
All esea ch in ol ing human ma e ials was app o ed by he
MD Ande son (MDACC) Ins i u ional Re iew Boa d (IRB). Co d
blood uni s we e ob ained om heal hy dono s who ga e w i en
in o med consen . All animal wo k was pe o med unde an
MDACC Ins i u ional Animal Ca e and Use Commi ee (IA-
CUC)-app o ed p o ocol speci ic o his s udy.
Cells and Cell Lines
K562-based aAPCs exp essing memb ane bound IL-21 ‘‘Clone
9.mbIL21’’ we e gene ously p o ided by D . Lau ence Coope
(MDACC, Hous on TX). Clone 9.mbIL21 cells exp ess mem-
b ane-bound IL-21, 41BB ligand, CD64 (FccRI) and CD86. This
cell line has ecen ly been shown o p omo e PB NK cell
expansion [26].and is GMP-g ade o clinical use. Ta ge s o NK
cell unc ional assays consis ed o K562 cells (Ame ican Type
Cul u e Collec ion (ATCC), Rock ille, MD) and MM cell lines
RPMI 8226 (ATCC), ARP-1 (Mul iple Myeloma Resea ch
Cen e , Li le Rock AK), and U266 (ATCC). Au ologous,
unselec ed CB cells ( om he same CB uni as he NK cells) we e
used as a nega i e con ol o
51
ch omium (C ) expe imen s.
Gene a ion o eGFP-FFLuc-exp essing ARP-1 Cell Line o
in i o Expe imen s
The gene a ion o e o i us ec o s encoding g een luo escen
p o ein (eGFP)-Fi e ly Luci e ase (eGFP-FFLuc) and p oduc ion o
ansien e o i al supe na an ha e been p e iously desc ibed
[27,28]. B ie ly, he usion p o ein eGFP-FFLuc was cloned in o
an SFG e o i al ec o and e o i al supe na an was p oduced
using 293-T cells co- ans ec ed wi h he ollowing e o i al
ec o s: eGFP-FFLuc SFG plasmid, he Peg-Pam-e plasmid
con aining he sequence o he MoMLV gag-pol and he RDF
plasmid encoding o he RD114 en elope. Re o i al supe na an
was collec ed a 48 and 72 hou s a e ans ec ion and s o ed a
-80uC o u he use. Fo he gene a ion o eGFP-FFLuc-
exp essing ARP-1 umo cells, 50,000 cells we e pla ed in p esence
o e o i al supe na an encoding eGFP-FFLuc in one well o a
24-well pla e p e-coa ed wi h ecombinan ib onec in agmen
(CH-296; Taka a Shuzo, O su, Japan). T ansduced ARP-1 cells
we e expanded and eGFP exp ession e alua ed by luo escence-
ac i a ed cell so e (FACSCalibu ; Bec on-Dickinson (BD), San
Jose, CA) analysis, whe eas exp ession o FFLuc was de ec ed
using D-luci e in (P omega, Madison, WI) and bioluminescence
measu ed wi h a luminome e (Modulus; Tu ne BioSys ems,
Sunny ale, CA). Because o he absence o selec ion gene in he
eGFP-FFLuc e o i al cons uc , single cell cloning o he ARP-1-
ansduced cells was pe o med o isola e and expand an ARP-1
clone (clone #24) wi h high le el o eGFP and FFLuc exp ession.
As ARP-1 exp esses bo h CD138 and kappa ligh chain [29,30],
Clone 24 was u he alida ed by FACS analysis o CD138 and
Kappa ligh chain exp ession and ELISA o Kappa ligh chain
sec e ion.
Isola ion and Expansion o Umbilical Co d Blood-de i ed
NK Cells
CB uni s we e ob ained om heal hy dono s who ga e in o med
consen unde MDACC IRB-app o ed p o ocols. Cul u e media
was comp ised o 45% RPMI-1640 (Cellg o, Manassas, VA) and
45% Click’s media (I ine Scien i ic, San a Ana, CA) supplemen -
ed wi h 10% AB human se um (A lan a Biologicals, Law ence ille,
GA) and 100 IU/mL IL-2 (P oleukin; Chi on, Eme y ille, CA).
CB mononuclea cells (MNCs) we e isola ed om esh o
ozen CB uni s by icoll densi y g adien cen i uga ion. Twen y
million MNCs we e pla ed in 400 mL media in a GP500 gas
pe meable bio eac o (Wilson Wol Co po a ion, New B igh on,
MN) wi h i adia ed (100 Gy) aAPC eede cells (2:1 eede
cell:MNC a io) a 37uC. IL-2 was eplenished e e y 2–3 days. On
day 7, cul u ed cells we e CD3-deple ed ia immunomagne ic
deple ion acco ding o manu ac u e ’s ins uc ions (Mil enyi
Bio ech, Aubu n, CA). Remaining cells we e hen e-pla ed in
he same condi ions, e-s imula ed wi h aAPC eede cells and
cul u ed o an addi ional 7 days (Figu e 1). Flow cy ome ic
analysis was pe o med on Days 0, 7 and 14 du ing he expansion.
NK cell numbe was de e mined by mul iplying he li e o al
nuclea ed cell coun by he pe cen age o CD56
+
/CD3
2
cells.
Di e ences in cell g ow h we e calcula ed using a 2- ailed s uden ’s
- es (Mic oso Excel 2010, Redmond, WA).
O iginal Expansion Techniques
Fo compa ison, CB-NK cells we e also expanded by a me hod
al eady known o be success ul in ou labo a o y [24]. F esh CB
MNCs we e isola ed as abo e and hen subjec ed o CD56
+
immunomagne ic selec ion. These cells we e hen suspended a
1610
6
cells/mL cul u e media wi h IL-2 a 500 IU/mL. The cells
we e cul u ed o 14 days a 37uC; IL-2 was eplenished e e y 2–3
days.
Expanded Co d Blood NK Cells Kill Myeloma
PLOS ONE | www.plosone.o g 2 Oc obe 2013 | Volume 8 | Issue 10 | e76781
NK Cell Pheno yping ia Flow Cy ome y
The ollowing an ibodies we e used: FITC-conjuga ed CD45,
CD158a, CD158b, CD94; PE-conjuga ed CD16, CD56, NKp30,
NKp46, NKp44, NKG2C; Pe CP-conjuga ed CD3; APC-conju-
ga ed CD56, NKG2A; Alexa Fluo 647- conjuga ed Eomesode min,
T-be (BD Biosciences); FITC-conjuga ed CD158e1 (BioLegend,
San Diego, CA); aAPC-conjuga ed NKG2A (Beckman Coul e ,
B ea, CA). In acellula s aining o Eomes and T-be was
pe o med pe manu ac u e ’s guidelines (BD Cy o ix/Cy ope m,
BD Biosciences). Da a we e acqui ed by he BD FACSCalibu
de ice using BD CellQues -P o so wa e. Flow cy ome y analysis
was pe o med using CellQues and FlowJo (T ee S a , Ashland,
OR) so wa e. Di e ences in MFI we e calcula ed using a wo-sided
pai ed - es (Mic oso Excel 2010).
Immuno luo escence and Con ocal Mic oscopy Image
Acquisi ion
Immuno luo escen labeling was pe o med as p e iously
desc ibed [31]. Ta ge cells we e labeled wi h CellT acke Blue
CMAC (7-amino-4-chlo ome hylcouma in, Molecula P obes,
Eugene, OR). NK cell- a ge cell conjuga es we e o med by
suspending equal olumes and cell numbe s o NK e ec o cells
and a ge cells (5610
6
/mL) in cul u e media o 15 min a 37uC.
Cells we e hen ans e ed on o mic oscope slides using a cell
concen a o (Cy o uge 2, IRIS In e na ional, and Cha swo h,
CA), ixed wi h 3% me hanol- ee o maldehyde and hen
pe meabilized. NK e ec o cell F-ac in was s ained wi h hoda-
mine-phalloidin (Molecula P obes, In i ogen, Ca lsbad, CA).
Images we e acqui ed using an Olympus IX81 mic oscope (Cen e
Valley, PA).
NK Cell
51
C Cy o oxici y Assay
Se ial dilu ions o NK cells we e co-incuba ed in iplica e o 4
hou s wi h 5000
51
C -labeled a ge cells (Ame sham Pha macia
Bio ech, Pisca away, NJ), in a o al olume o 100 mlinaV-
bo om 96-well pla e (Co ning, Co ning, NY). The ea e ,
supe na an s (50 ml) we e ha es ed and ans e ed o a Luma-
Pla e-96 (Pe kin-Elme , Wal ham, MA). A e d ying o e nigh ,
51
C elease was measu ed on a TOPCoun NXT mic opla e
scin illa ion and luminescence coun e (Pe kin-Elme ). Cy o oxic-
i y was de e mined by he o mula: cy o oxici y = (sample alue-
spon aneous lysis)/(max-lysis-spon aneous lysis) 6100%.
ARP-1 Myeloma Mu ine Model
NOD/SCID IL-2Rc
null
(NSG) mice (Jackson Labo a o ies, Ba
Ha bo , ME) we e i adia ed wi h 300 cGy and inocula ed wi h
160
6
eGFP-FFLuc - ansduced ARP-1 cells (Clone 24) in a e-
nously on day 21. Whe e indica ed, 10610
6
ex i o, esh, aAPC-
expanded CB NK cells we e gi en e o-o bi ally on days 0, 12
and 19 wi h IL-2 (2000 IU in ape ioneally (IP) h ee imes pe
week). Mice we e subjec ed o wice weekly bioluminescence
imaging (BLI) and weekly se um kappa ligh chain measu emen s.
P io o image acquisi ion mice we e anes he ized wi h 2%
iso lu ane in 98% oxygen. BLI was pe o med using a Xenogen
IVIS 200 sys em (Calipe , Wal ham, MA) 10 minu es ollowing a
100 mL IP injec ion o D-luci e in (20 mg/mL PBS). BLI images
we e acqui ed a 5-minu e exposu es and supe imposed on b igh
ield pho og aphs o he animals. Signal quan i a ion in pho ons/
second (p/s) was pe o med by de e mining he pho on lux a e
wi hin s anda dized egions o in e es (ROI) using Li ing Image
so wa e (Calipe ). Se um kappa le els we e measu ed by a
comme cially a ailable enzyme-linked immunoso ben assay
(ELISA) ki (Be hyl Labo a o ies, Mon gome y, TX) acco ding
o manu ac u e ’s ins uc ions. Resul s epo ed a e a ep esen a-
i e expe imen wi h 5 mice in each g oup. Di e ences in BLI and
se um kappa le els we e calcula ed using a 2- ailed s uden ’s - es
(Mic oso Excel 2010). Su i al was calcula ed using he Kaplan-
Meie me hod (SAS s a is ical so wa e, e sion 9.2, Ca y, NC).
Resul s
aAPC-media ed CB-NK Expansion om F esh o
C yop ese ed CB Uni s yields Signi ican ly G ea e Fold
Expansion o NK Cells han Expansion o CD56
+
Cells wi h
IL-2 Alone
In compa ison wi h ou o iginal expansion app oach o CD56-
selec ed cells cul u ed wi h IL2 alone, cul u e o ei he esh o
ozen CB MNCs wi h aAPC eede cells esul ed in g ea e
Figu e 1. Cul u e o CB-NK cells. Unselec ed CB MNCs we e cul u ed o 7 days in a GP500 bio eac o wi h IL-2 (100 IU/mL) and aAPCs a 2:1
aAPC:MNC a io. Cells we e immunomagne ically CD3-deple ed on Day 7 and e-cul u ed in same condi ions o an addi ional 7 days. On day 7 cells
we e again CD3-deple ed and subjec o pheno ypic and unc ional s udies.
doi:10.1371/jou nal.pone.0076781.g001
Expanded Co d Blood NK Cells Kill Myeloma
PLOS ONE | www.plosone.o g 3 Oc obe 2013 | Volume 8 | Issue 10 | e76781
expansion o NK cells a e cul u e o 14 days (p,0.05 o bo h
esh o ozen condi ions, Figu es 2A and 2B). Cul u ing o esh
CB MNCs (n = 8) wi h aAPC eede cells yielded a mean old
expansion o 1848 old (609 old –4778 old) while cul u ing o
ozen CB MNCs (n = 6) wi h eede cells yielded a mean old
expansion o 2389 old (103 old –4931 old). This was in
compa ison o 20 old (11 old -27 old) expansion om cul u e o
esh CD56
+
-selec ed cells wi h IL-2 alone (n = 3). The di e ence
in NK cell yield was appa en by day 7 o he esh CB cul u e
wi h aAPC eede s (p,0.05) bu did no each s a is ical
signi icance o he ozen CB condi ion un il day 14 (p = 0.06 a
day 7). As seen in Figu e 2C, he inal cul u e con ained e y ew
(#1%) CD3
+
cells and his was no signi ican ly di e en be ween
he 3 cul u e condi ions: mean alue o 0.44% CD3
+
cells om he
cul u e wi h IL-2 alone, 0.74% CD3
+
cells om esh CB MNCs
wi h aAPC eede s and 0.66% CD3
+
cells om ozen CB MNCs
wi h aAPC eede s (p.0.5 o all compa isons).
aAPC-media ed Expansion Yields a Pu e Popula ion o NK
Cells wi h a Ma u e Pheno ype
As seen in Figu e 3A, co-cul u e o CB MNCs wi h IL-2 and
aAPC eede cells yielded a popula ion ha was pu e o NK cells
a he end o he 2 week expansion pe iod. A e CD3-deple ion,
96% o cells we e CD56
+
/CD3
2
and less han 1% we e CD3
+
.
CB-NK cells expanded wi h aAPCs demons a ed a CD56
hi
pheno ype simila o CB-NK cells expanded wi h IL-2 alone. O
no e, cul u e o unselec ed CB MNCs wi h IL-2 and soluble IL-21
yielded a ela i ely pu e CD56
+
/CD3
2
NK cell popula ion bu
wi h limi ed expansion o cells (mean expansion o 14 old, da a
no shown). In addi ion, a e log- old expansion, aAPC-expanded
CB-NK cells did no appea exhaus ed; a he , CB-NK cells
con inued o s ongly exp ess Eomesode min and T-be , an-
sc ip ion ac o s ecen ly ecognized as necessa y o NK cell
ma u a ion and ac i a ion [32,33] (Figu e 3B). In e es ingly, he
su ace exp ession o NK cy o oxici y ecep o s (NCRs) NKp30,
NKp46 and NKp44 was signi ican ly lowe o aAPC-expanded
CB-NK cells e sus IL-2-expanded CB-NK cells (p#0.05 o all
h ee NCRs). Howe e , he exp ession o KIR an igens, NKG2A,
co- ecep o CD94 and he ac i a ing ecep o NKG2C was simila
be ween he wo expansion me hods (Figu e 3C).
CB-NK Cells Cul u ed wi h aAPCs Demons a e in i o
An i-myeloma Ac i i y
In o de o kill a ge s, NK cells mus di ec ly con ac he cell o
in e es and o m he ‘‘NK immune synapse’’ (NKIS) [34,35]. Ou
lab has p e iously demons a ed ha expansion o CB-NK cells is
necessa y o epai he de ec i e NKIS exhibi ed by naı
¨ e CB-NK
cells [24]. To demons a e ha his synapse abili y is main ained in
CB-NK cells expanded wi h aAPC eede cells, we pe o med a
se ies o synapse assays wi h a ious MM a ge s. As shown in
Figu e 4A, NK cells cul u ed wi h aAPC eede cells o med a
unc ional NKIS (demons a ed by F-ac in pola iza ion) wi h he
classic NK cell a ge K562, MM cell lines RPMI 8226, aARP-1
and U266.
To demons a e he unc ionali y o CB-NK cells expanded wi h
aAPC eede s imula ion, we pe o med a s anda d
51
C
cy o oxici y assay. aAPC-expanded CB-NK cells we e cy o oxic
o all o he MM cell line a ge s (Figu e 4B). Fu he mo e, despi e
he di e ences in pheno ype wi h ega d o he NCRs, in
compa ison wi h CB-NK cells expanded wi h IL-2 alone, he
aAPC-media ed expanded CB-NK cells demons a ed equal o
g ea e cy o oxici y agains K562 (Figu e 4C). This inding was
consis en ac oss he MM cell lines as well (Figu e S1). Nei he o
he CB-NK p epa a ions demons a ed au ologous cy o oxici y.
T ea men wi h Expanded CB-NK Cells Delays
De elopmen o Myeloma in a Mu ine Model
To in es iga e whe he ex i o expanded CB-NK cells can inhibi
he g ow h o MM cells in i o, we s udied NSG mice ea ed wi h
GFP i e ly luci e ase- ansduced ARP-1 cells (Clone 24). Using
he bioluminescen signal in ensi y as a su oga e o umo cell
densi y, se ial images demons a ed ha mice ea ed wi h CB-NK
cells had a delay in he onse o MM (Figu e 5A). A e 1 week, he
signal in ensi y (p/s) was signi ican ly g ea e in hose mice who
Figu e 2. Co-cul u e o CB MNCs wi h IL-2 and aAPCs yields
signi ican ly g ea e expansion o NK cells han cul u e wi h IL-
2 alone. A. Mean old g ow h o CD56
+
/CD3
2
NK cells om 8 esh and
6 ozen co d blood expansions wi h aAPCs and IL-2 e sus 3
expansions wi h IL-2 alone (14 day cul u e). B. Time cou se o NK cell
g ow h o e 14 day cul u e be ween all 3 condi ions. By day 7, he esh
CB aAPC-con aining cul u e demons a ed g ea e NK cell g ow h han
cul u e wi h IL-2 alone (p,0.05). The ozen CB showed a simila end
a day 7, which did no each s a is ical signi icance (p = 0.06). C. All
h ee cul u e condi ions yielded compa able, low pe cen ages o CD3
+
cells:. 0.44%, 0.74% and 0.66% CD3
+
cells om he cul u e wi h IL-2
alone, esh CB MNCs wi h aAPC eede s o ozen CB MNCs wi h aAPC
eede s espec i ely (p.0.5 o all compa isons). Mean +/2SD is shown
o each igu e. P,0.05 whe e indica ed (*).
doi:10.1371/jou nal.pone.0076781.g002
Expanded Co d Blood NK Cells Kill Myeloma
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ecei ed Clone 24 ARP-1 cells alone e sus hose who ecei ed
Clone 24 ARP-1 cells and CB-NK cells (Figu e 5B, p,0.05 om
Day 8–22) This was consis en wi h he ELISA analysis o se um
kappa ligh chains; mice ecei ing Clone 24 ARP-1 cells alone had
signi ican ly mo e measu able se um kappa han mice who
ecei ed Clone 24 ARP-1 cells and CB-NK cells, (Figu e 5C,
p,0.01 a each ime poin ). Finally, he e was also a di e ence in
su i al be ween he 2 g oups wi h a median su i al o 31 days in
he mice who ecei ed Clone 24 ARP-1 cells alone e sus 38 days
o he mice who ecei ed Clone 24 ARP-1 cells and CB-NK cells,
(Figu e 5D, p = 0.003).
Discussion
To ou knowledge, his is he i s s udy explo ing ex i o
expanded CB-NK cells o he ea men o MM. Clinical ials
wi h allogeneic HSCT o MM consis en ly show an enhanced
comple e emission a e in compa ison wi h au ologous HSCT
egimens [36,37,38], sugges ing a ue g a e sus MM e ec .
Howe e , his bene i is o -se by inc eased ea men - ela ed
mo ali y associa ed wi h GVHD [39]. MM is hus an ideal disease
candida e o NK cell he apy: in compa ison wi h a T cell eple e
allog a , NK cells exe an allogeneic g a e sus umo e ec bu
do no appea o inc ease he isk o GVHD [40,41]. Indeed a
clinical ial wi h allogeneic PB-de i ed NK cells o MM has
demons a ed sa e y and no inc ease in GVHD [42], hough he
ole o KIR-HLA I incompa ibili y on NK cell allo eac i i y
emains o be de ined.
The in i o and in i o da a p esen ed he e suppo he use o
CB-NK cells agains MM. Expanded CB-NK cells exhibi ed
imp essi e cy o oxici y and immune synapse o ma ion agains
MM a ge s. In addi ion, CB-NK cells we e able o signi ican ly
delay es ablishmen o disease in a mu ine MM model. The
e en ual umo bu den in ou in i o model sugges s ha cellula
he apy would likely ha e g ea es success i adminis e ed in
combina ion wi h o he con en ional he apies, which could
include alkyla ing o immunomodula o y agen s. In addi ion, he
iming o se ial NK cell doses may be u he op imized o exe
g ea e an i- umo ac i i y, as has been done in a simila in i o
assay [4].
In compa ison o expansion wi h IL-2 alone, CB-NK cells
expanded wi h aAPCs demons a ed a dec eased su ace exp es-
sion o he ac i a ing NCRs NKp30, NKp46 and NKp44.
Howe e he exp ession o KIR an igens, inhibi o y ecep o
NKG2a, co- ecep o CD94 and ac i a ing ecep o NKG2C was
simila be ween he 2 condi ions. The eason o he dec ease in
NCR exp ession is no comple ely clea . I is possible ha he
in e ac ion be ween he CB-NK cells and he K562-based aAPCs
du ing co-cul u e media ed a ans e o he ecep o s o he a ge
cells, as has been seen wi h o he NK cell ecep o s and a ge cell
lines [43]. In e es ingly, he di e ences in NCR su ace exp ession
did no appea o impai he unc ional cy o oxici y o he aAPC-
expanded CB NK cells, sugges ing ha he gain in cell numbe is
no accompanied by a comp omise in unc ion. In addi ion,
aAPC-expanded CB-NK cells showed p ese a ion o Eomeso-
de min and T-be exp ession, wo ansc ip ion ac o s which ha e
ecen ly been ecognized as in eg al o NK cell unc ion
[32,44,45]. Recen mu ine s udies ha e epo ed ha down-
egula ion o hese wo ansc ip ion ac o s in NK cells ollowing
adop i e NK cell ans e and homeos a ic p oli e a ion is
accompanied by an exhaus ed pheno ype and limi ed NK cell
an i- umo ac i i y [32]. While one migh expec a simila
educ ion o Eomesode min and T-be exp ession a e he log-
old expansion o ou CB-NK, his was no he case. Addi ional
in i o s udies a e in p og ess o in es iga e i expanded CB NK
cells a e in insically less suscep ible o exhaus ion and mo e likely
o main ain he exp ession o hese ansc ip ion ac o s ollowing
adop i e ans e .
The challenge o expanding allogeneic NK cells o a clinically
ele an dose emains, as does inding he app op ia e dono , i
Figu e 3. Pheno ype o CB-NK cells cul u ed wi h aAPCs. A. O e he 14-day expansion, CB-NK cells cul u ed wi h aAPC eede cells
demons a ed a p og essi ely pu e, CD56
+
/CD3
2
popula ion, ( ep esen a i e do plo s o 17 expansions). B. aAPC-expanded CB-NK cells main ained
Eomesode min
hi
and T-be
hi
pheno ype a e expansion. Rep esen a i e his og ams om 3 di e en CB-NK expansions; cells a e ga ed on he li e
CD56
+
popula ion. C. CB MNCs om he same CB uni we e expanded wi h aAPCs +IL-2 o IL-2 alone (n = 3 sepa a e CB uni s). Rep esen a i e do
plo s o NK cell su ace ecep o exp ession on day 14 a e shown. D. By median luo escence in ensi y (MFI), aAPC-expanded CB-NK demons a ed a
dec eased su ace exp ession o he NCRs NKp30, NKp46 and NKp44. Howe e he e was a simila exp ession be ween he condi ions o he KIR
an igens, inhibi o y ecep o NKG2A, co- ecep o CD94 and ac i a ing ecep o NKG2C) (n = 3 pai ed expansions, mean +/2SD is shown, p#0.05
whe e indica ed).
doi:10.1371/jou nal.pone.0076781.g003
Expanded Co d Blood NK Cells Kill Myeloma
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indeed he ac i i y o hese cells depends on misma ch be ween
dono KIR and ecipien HLA I. He e we demons a e ha CB
can se e as a eliable sou ce o NK cells o adop i e cellula
immuno he apy. In ansla ing ou indings o he clinic, i should
be no ed ha , om 20610
6
MNCs (app oxima ely 10% o a
clinical CB uni ), ou cul u e sys em would allow o he
gene a ion o app oxima ely 1.4610
9
NK cells o in usion, o
1.9610
7
NK cells/kg o an a e age 70 kg adul . This is o e 18
old highe han he g ow h seen wi h CD56
+
selec ed cells
expanded wi h IL-2 alone. Addi ionally, his NK p oduc is
ela i ely pu e, wi h only 6610
4
CD3
+
cells/kg, hus educing he
po en ial o GVHD. In compa ison wi h o he c yop ese ed CB-
NK cul u e sys ems [46,47,48], he me hod desc ibed in his pape
has se e al ad an ages. Fi s , i equi es only wo weeks o cul u e,
which could minimize bo h he cos and po en ial o mic obial
con amina ion seen wi h longe du a ion cul u es. In addi ion, his
sys em equi es only a ac ion o he CB uni . A minimum o
2610
8
CB MNCs a e ypically ob ained om a ozen CB uni ;
hus he NK dose could po en ially be inc eased by a leas 10- old,
o a o al o 1.9610
8
NK cells/kg. As CB uni s can be hawed in
ac ions, his would allow o conside a ion o se ial doses o NK
cell he apy o enhance an i- umo e icacy.
CB-NK cells could be conside ed a easonable al e na i e o
PB-NK cells o adop i e ans e . The po en ial bene i s o
expanded NK cells om CB o e PB include he lowe a es o
acu e GVHD seen in he allogeneic HSCT se ing [49,50,51] as
well as apid a ailabili y, wi h o e 600,000 banked uni s
wo ldwide [52]. In addi ion, CB-NK cells do no equi e collec ion
om a li e dono . Finally, o hose pa ien s who do no ha e a
eadily a ailable amily dono , he CB pool p o ides a unique
oppo uni y o ind a sui ably ma ched allog a .
Figu e 4. aAPC-expanded CB-NK cells o m immunological synapses wi h and a e cy o oxic agains myeloma a ge s. A. CMAC-
labeled umo a ge s (blue) we e incuba ed a a 1:1 a io wi h aAPC-expanded CB-NK cells o 15 minu es. Conjuga es we e hen ixed, pe meabilized
and s ained o NK e ec o cell F-ac in wi h hodamine-phalloidin ( ed). Con ocal and b igh ield images we e acqui ed; ep esen a i e images om
each slide a e shown. aAPC-expanded CB-NK cells o m immune synapses wi h he classic NK a ge K562 as well as a a ie y o MM cell lines. B. aAPC-
expanded CB-NK cells we e co-incuba ed in iplica e o 4 hou s wi h
51
C -labeled a ge cells a a ios as shown. Supe na an s we e hen ha es ed
and analyzed he nex day o
51
C con en . % Cy o oxici y =(sample alue-spon aneous lysis)/(max-lysis-spon aneous lysis) x 100%. CB-NK cells
demons a e dose-dependen cy o oxici y agains K562 (classic NK cell a ge ) and MM cells lines RPMI 8266, ARP-1 and U266 ( ep esen a i e o n.3
assays o each cell line). C. aAPC-Expanded CB-NK cells displayed equal o mo e cy o oxici y agains K562 cells e sus CB-NK cells expanded wi h IL-2
alone ( ep esen a i e om n = 4 assays).
doi:10.1371/jou nal.pone.0076781.g004
Expanded Co d Blood NK Cells Kill Myeloma
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Taken oge he , ou esul s sugges ha CB-NK cells a e ac i e
agains MM and can be eliably gene a ed by a GMP-complian
me hod o ob ain clinically ele an doses. S udies a e in p og ess
o be e de e mine he ole, i any, o KIR-HLA misma ch on NK
cell cy o oxici y agains p ima y CD138
+
MM cells. Finally, a
clinical ial using aAPC-expanded CB-NK cells in conjunc ion
wi h high dose chemo he apy and au ologous HSCT o MM is
being de eloped.
Suppo ing In o ma ion
Figu e S1 aAPC-Expanded CB-NK cells displayed equal
o mo e cy o oxici y agains MM cells e sus CB-NK
Figu e 5. aAPC-expanded CB-NK cells delay de elopmen o myeloma in a NSG mu ine model. 1610
6
GFP i e ly luci e ase- ansduced
ARP-1 cells (Clone 24) we e gi en IV on day -1. In he CB-NK ea ed g oup, 10610
6
ex i o, aAPC-expanded CB NK cells we e gi en e o-o bi ally on
days 0, 12 and 19 wi h IL-2, 2000 IU (IP) h ee imes pe week. Se ial BLI and kappa ELISA measu emen s we e acqui ed un il day 18. Resul s ep esen
mean alues o n = 5 mice in each g oup un il day 18, by which ime 1 mouse in he ARP-1 alone g oup had died. A. Se ial BLI images demons a e
impai ed myeloma de elopmen in mice ecei ing CB-NK cells. B. Signal in ensi y (p/s) was signi ican ly g ea e in mice ecei ing Clone 24 ARP-1 cells
alone e sus hose ecei ing bo h Clone 24 ARP-1 cells and CB-NK cells. Region o in e es (ROI) is indica ed by ec angles supe imposed on each
mouse om Figu e 5A, p#0.05 a days 8–22. C. Se um kappa le els (ng/mL) we e signi ican ly highe in mice ea ed wi h Clone 24 ARP-1 cells e sus
hose ea ed wi h Clone 24 ARP-1 cells and CB-NK cells, p#0.01 a each ime poin . D. By Kalpan-Meie me hod, he e was a signi ican di e ence in
su i al o he mice, (p = 0.003) in a o o he NK- ea ed g oup. The mice who ecei ed Clone 24 ARP-1 cells alone had a median su i al o 31 days
e sus 38 days o he mice who ecei ed Clone 24 ARP-1 cells and CB-NK cells.
doi:10.1371/jou nal.pone.0076781.g005
Expanded Co d Blood NK Cells Kill Myeloma
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cells expanded wi h IL-2 alone. IL-2 expanded o aAPC-
expanded CB-NK cells we e co-incuba ed in iplica e o 4 hou s
wi h
51
C -labeled a ge cells as de ailed o Figu e 4. Cy o oxici y
o aAPC-expanded CB-NK cells was equal o o g ea e han ha
o CB-NK cells expanded wi hou aAPCs agains a ious MM cell
lines (A: RPMI 8226, B: U266, C: ARP-1; ep esen a i e da a
om n = 3 expe imen s).
(TIF)
Acknowledgmen s
The au ho s wish o hank D . Qing Yi and Jin He o assis ance wi h he
mul iple myeloma mu ine model and Wilson Wol Co po a ion o
p o iding GP500 bio eac o s. We would also like o hank he MD
Ande son Co d Blood Bank and Myeloma Tissue Bank o he no mal and
malignan cells used in hese s udies.
Au ho Con ibu ions
Concei ed and designed he expe imen s: NS BMA HY SK DL LC WD
SL TS SP JG KR EY AN JB IK CB EJS. Pe o med he expe imen s: NS
BMA HY SK WD SL TS EY AN JB IK. Analyzed he da a: NS BMA HY
SK WD SL SR TS SP JG MW KR EY AN JB IK CB EJS. Con ibu ed
eagen s/ma e ials/analysis ools: DL LC JG MW AN JB RC CB EJS.
W o e he pape : NS BMA HY LC WD KR EY AN IK RC CB EJS.
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