T ansmissible cy o oxici y o mul iple myeloma cells
by co d blood-de i ed NK cells is media ed by esicle
a icking
B Ma in-An onio
1,2
, A Najja
3
, SN Robinson
1
, C Chew
1
,SLi
1
, E Y on
1
, MW Thomas
1
, I Mc Niece
1
, R O lowski
4
, C Mun
˜oz-Pinedo
5
,
C Bueno
6
, P Menendez
6,7
, C Fe na
´ndez de La ea
2
, A U bano-Ispizua
2
, EJ Shpall
1
and N Shah
1
Na u al kille cells (NK) a e impo an e ec o s o an i- umo immuni y, ac i a ed ei he by he down egula ion o HLA-I molecules
on umo cells and/o he in e ac ion o NK-ac i a ing ecep o s wi h ligands ha a e o e exp essed on a ge cells upon umo
ans o ma ion (including NKG2D and NKP30). NK kill a ge cells by he esicula deli e y o cy oly ic molecules such as
G anzyme-B and G anulysin ac i a ing di e en cell dea h pa hways, which can be Caspase-3 dependen o Caspase-3
independen . Mul iple myeloma (MM) emains an incu able neoplas ic plasma-cell diso de . Howe e , we p e iously epo ed he
encou aging obse a ion ha co d blood-de i ed NK (CB-NK), a new sou ce o NK, showed an i- umo ac i i y in an in i o
mu ine model o MM and con i med a co ela ion be ween high le els o NKG2D exp ession by MM cells and inc eased e icacy o
CB-NK in educing umo bu den. We aimed o cha ac e ize he mechanism o CB-NK-media ed cy o oxici y agains MM cells. We
show a Caspase-3- and G anzyme-B-independen cell dea h, and we e eal a mechanism o ansmissible cell dea h be ween
cells, which in ol es lipid–p o ein esicle ans e om CB-NK o MM cells. These esicles a e seconda ily ans e ed om
ecipien MM cells o neighbo ing MM cells ampli ying he ini ial CB-NK cy o oxici y achie ed. This indi ec cy o oxici y in ol es
he ans e o NKG2D and NKP30 and leads o lysosomal cell dea h and dec eased le els o eac i e oxygen species in MM cells.
These indings sugges a no el and unique mechanism o CB-NK cy o oxici y agains MM cells and highligh he impo ance o
lipids and lipid ans e in his p ocess. Fu he , hese da a p o ide a a ionale o he de elopmen o CB-NK-based cellula
he apies in he ea men o MM.
Cell Dea h and Di e en ia ion (2015) 22, 96–107; doi:10.1038/cdd.2014.120; published online 29 Augus 2014
Na u al kille cells (NK) a e impo an e ec o s o an i- umo
immuni y o he immune sys em. They can be ac i a ed by
inhibi ion o kille cell immunoglobulin (Ig)-like ecep o (KIR)
ecep o s on NK due o down egula ion o HLA-I on umo
cells o by he in e ac ion o NK-ac i a ing ecep o s wi h
ligands ha a e o e exp essed on a ge cells. These
ecep o s include NKG2D and he amily o NK cy o oxici y
ecep o s (NKP30, NKP44, NKP46),
1
which can be on he cell
su ace and in he endocy ic compa men
2
om whe e hey
a ic o umo cells in exosomes o exe cy o oxici y.
3
NK deli e cy oly ic molecules such as G anzyme-B (G B) and
G anulysin o a ge cells. G B induces Caspase-3-dependen
apop o ic cell dea h wi h eac i e oxygen species (ROS)
gene a ion.
4
Al e na i ely, G anulysin ac i a es Caspase-3-
dependen cell dea h h ough ROS gene a ion
5,6
and Cas-
pase-3-independen cell dea h ia endoplasmic e iculum (ER)
s ess
7
o lysosomal cell dea h h ough elease o ca hepsins.
8
Mul iple myeloma (MM) is a plasma-cell diso de cha ac e -
ized by clonal p oli e a ion o malignan plasma-cells in he
bone ma ow (BM) and monoclonal p o ein in he blood o
u ine.
9,10
Plasma cells syn hesize la ge quan i ies o Igs,
which a e olded in he ER. An excess o Ig syn hesis causes a
ailu e in his olding p ocess leading o he elease o
un olded/mis olded Igs.
11
These Igs a e deg aded by in a-
cellula p o ein deg ada ion pa hways, including he ubiqui in–
p o easome sys em and au ophagy. P o easome inhibi o s
(PIs) a e po en an i-MM agen s,
12
which block he p o ein
deg ada ion p ocess in MM cells leading o ER s ess-
media ed cell dea h.
13,14
An excess o PI-induced ER s ess
can inc ease au ophagy
15,16
leading o e en ual e ac o y
disease in some pa ien s.
17–19
The e o e new an i-MM
s a egies a e needed.
P e iously, we ha e demons a ed ha co d blood-de i ed
NK (CB-NK) show an i- umo ac i i y in an in i o MM mu ine
model
20
and obse ed ha he exp ession o NKG2D by MM
umo cells co ela ed wi h lowe umo bu den ollowing
CB-NK cellula he apy. He e we cha ac e ize he CB-NK-
media ed cy o oxici y agains MM cells and obse e a
1
Depa men o S em Cell T ansplan a ion and Cellula The apy, The Uni e si y o Texs M.D. Ande son Cance Cen e , Hous on, TX, USA;
2
Depa men o Hema ology,
Hospi al Clinic, IDIBAPS, Josep Ca e as Leukaemia Resea ch Ins i u e/Uni e si y o Ba celona, Ba celona, Spain;
3
Depa men o Cance Sys ems Imaging, The
Uni e si y o Texs M.D. Ande son Cance Cen e , Hous on, TX, USA;
4
Depa men o Lymphoma/Myeloma, The Uni e si y o Texas M.D. Ande son Cance Cen e ,
Hous on, TX, USA;
5
Cell Dea h Regula ion G oup, Bell i ge Biomedical Resea ch Ins i u e (IDIBELL), Ba celona, Spain;
6
Josep Ca e as Leukemia Resea ch Ins i u e
and Cell The apy P og am o he School o Medicine, Uni e si y o Ba celona, Ba celona, Spain and
7
Ins i ucio
´Ca alana de Rece ca i Es udis A anc¸a s (ICREA),
Ba celona, Spain
*Co esponding au ho : B Ma in-An onio, Depa men o Hema ology, Hospi al Clinic, IDIBAPS, Josep Ca e as Leukaemia Resea ch Ins i u e, Facul a de Medicina,
Uni e si y o Ba celona, Ca e Casano a 143, Ba celona 08036, Spain. Tel: þ935572810; Fax: þ34 934035263; E-mail: [email p o ec ed]
Recei ed 05.2.14; e ised 02.7.14; accep ed 09.7.14; Edi ed by JP Medema; published online 29.8.14
Abb e ia ions: NK, na u al kille cells; CB-NK, co d blood-de i ed NK cells; MM, mul iple myeloma; ROS, eac i e oxygen species; ER, endoplasmic e iculum;
G B, G anzyme-B
Cell Dea h and Di e en ia ion (2015) 22, 96–107
&
2015 Macmillan Publishe s Limi ed All igh s ese ed 1350-9047/15
www.na u e.com/cdd
Caspase-3- and G -B-independen cell dea h and e eal a
mechanism o ansmissible cell dea h be ween cells ha
in ol es lipid–p o ein esicle ans e om CB-NK o MM
cells. These esicles a e seconda ily ans e ed om
ecipien MM cells o neighbo ing MM cells, he eby ampli ying
he ini ial CB-NK cy o oxici y achie ed. This indi ec cy o oxi-
ci y in ol es he ans e o NKG2D and NKP30 and leads o
lysosomal cell dea h and educed ROS le els in MM cells.
Resul s
NKG2D exp ession in MM cells a e CB-NK ea men
co ela es wi h lowe MM p og ession, and NKG2D and
NKP30 con ibu e mo e o he cy o oxici y o MM cells
han in K562 cells. We ha e p e iously shown ha CB-NK
exe an i-MM ac i i y in a mu ine MM model.
20
Immunode i-
cien mice we e injec ed wi h he ARP1 MM cell line. MM
cells we e iden i ied in he BM, spleen, lymph nodes (LN) and
o a ies and demons a ed highe MFI o NKG2D in he BM,
LN and o a ies om CB-NK- ea ed mice e sus un ea ed
mice (Supplemen a y Figu es S1A–D and F–I). CB-NK in
CB-NK ecipien mice we e CD56
þ
, CD3
, NKG2D
þ
and
CD16
þ
(Supplemen a y Figu e S1J). In ea ed mice, MFI o
NKG2D in MM cells in LN co ela ed in e sely wi h umo
bu den (R¼0.886, P¼0.04; Supplemen a y Figu e S1J),
sugges ing a ans e o NKG2D om CB-NK o MM cells
wi h a bene icial e ec .
As he p esence o NKG2D in MM cells was associa ed wi h
lowe umo bu den, we in es iga ed he cy o oxic ole o
NKG2D. We also examined NKP30, as i has an impo an ole
in he killing o MM cell lines,
21
MICA/B (NKG2D ligand) and
he syne gis ic e ec s o hese ecep o s. We compa ed ou
esul s om ARP1 cells wi h K562 cells, whe e, in he absence
o HLA-I, NK cy o oxici y is media ed by KIR ecep o s.
A e blocking hese ecep o s, we ound a highe con ibu-
ion o hese in he killing o ARP1 e sus K562 cells (Figu es
1a–d). The g ea es impac in ARP1 cells was media ed by
NKP30 ollowed by NKG2D (Figu es 1b and d). A a 20 : 1
e ec o : a ge cell a io, we obse ed a 33% (NKG2D
blocked), 49% (NKP30 blocked) and 73% (bo h blocked)
educ ion in ARP1 killing (Figu e 1d) e sus 4% (NKG2D
blocked), 3% (NKP30 blocked) and 19% (bo h blocked)
educ ion in K562 killing (Figu e 1c). This e ec was
ep oduced a high e ec o : a ge cell a ios (Figu es 1c
and d, Po0.0001). A simila pa e n was obse ed wi h
p ima y MM CD138
þ
cells showing a 20 : 1 e ec o : a ge
cell a io a 26.6% (NKG2D blocked) and 51% (NKP30
blocked) educ ion in CD138
þ
cell killing (Figu es 1e–g).
Mo eo e , in NK om MM pa ien s (MM-NK) ha showed
lowe cy o oxici y han CB-NK (Supplemen a y Figu e S2A),
NKG2D and NKP30 ecep o s did no impac NK-MM
cy o oxici y (Supplemen a y Figu e S2B), e ealing his
unique ea u e o CB-NK cy o oxici y and he supe io i y o
CB-NK o MM ea men .
NKG2D and NKP30 a e ans e ed in o MM cells ia lipid
a icking and he endocy ic pa hway. We epo ed an
in e se co ela ion be ween NKG2D exp ession in MM-ARP1
cells and disease p og ession and demons a ed ha
NKG2D and NKP30 we e in ol ed in CB-NK cy o oxici y.
The a icking o hese ecep o s o MM cells was subse-
quen ly ollowed using con ocal luo escence mic oscopy.
Cell acke s we e used o label CB-NK and umo cells.
Tumo cells we e labeled in blue using CMAC and CB-NK in
g een wi h bodipy-505/515. This has an a ini y o neu al
lipids and has a chlo ome hyl g oup ha allows binding o
memb ane p o eins, he eby labeling p o eins and lipids. We
obse ed ha bodipy s aining allowed us o ollow he
a icking o con en s om CB-NK o ARP1 cells (Figu e 2,
and Supplemen a y Mo ie S1).
Wi hin 10 min o umo cell/CB-NK co-cul u e, CB-NK
ans e ed hei con en s o MM cells. This was ia lipid-
p o ein esicles and la ge olume esicles (Figu e 2a).
Fu he , some o he lipid-p o ein esicles a icked be ween
ARP1 cells a e ans e om CB-NK (Figu e 2b). Such
ans e was no de ec ed in he absence o CB-NK
(Figu e 2c). This a icking also in ol ed he immunological
synapse, as demons a ed by ac in co-s aining (Figu es 2a
and b). O no e, MM-NK showed less in ense immunological
synapse e sus MM cells and lowe bodipy s aining
(Supplemen a y Figu e S2C) in compa ison o CB-NK
suppo ing he inding o lowe MM-NK cy o oxici y and
indica ing lowe le els o lipids/cy o oxic esicles in MM-NK.
The s onge immunological synapses and bodipy s aining,
indica i e o highe le els o lipids, we e cha ac e is ic o mo e
po en CB-NK.
Time-lapse in i o imaging e ealed ha he in ensi y o he
ma e ials ans e ed o ARP1 cells by CB-NK inc eased wi h
ime as indica ed by he inc ease in Bodipy ( egions o in e es
(ROIs) 1–3 in Figu es 2d and e). Also, a e CB-NK ans e ed
esicles o ARP1 cells, hese ARP1 cells could hen ans e
Bodipy-con aining esicles o neighbo ing ARP1 cells (ROIs 4
and 5 in Figu es 2d, and g and Supplemen a y Mo ie S1).
This ans e was associa ed wi h memb ane blebbing
appea ance (Figu e 2g) indica i e o apop o ic-like mechan-
isms.
22
Imaging e ealed ha , a e 20 min o co-cul u e,
NKG2D and NKP30 we e de ec ed in ARP1 cells (Figu es 3a–
c), whe eas hey could no be de ec ed in ARP1 cells ha had
no been wi h CB-NK (Figu e 3d). A e 3 days o co-cul u e,
low cy ome y con i med su ace and in acellula p esence
o NKG2D and NKP30 in ARP1 cells (Figu es 3e and ).
We in es iga ed he ole o he endocy ic pa hway in he
NKG2D and NKP30 ans e o ARP1 cells. Cells up ake
molecules om he ex acellula en i onmen by endocy osis.
NK-ac i a ing ecep o s a e con inuously ecycled and
deg aded by endocy ic pa hway.
2
Once p oduced, NK-
ac i a ing ecep o s a e deli e ed o ea ly endosomes
(de ined by EEA1). They a e hen ei he ecycled o he
plasma memb ane (de ined by Rab11) and exocy osed, o
deli e ed o la e endosomes (de ined by Rab7), o be a ge ed
o lysosomes o deg ada ion.
2,23,24
We he e o e analyzed
whe he NKG2D and NKP30 once ans e ed be ween cells
en e ed he endocy ic pa hway in MM cells.
We obse ed ha bodipy co-localized wi h NKG2D and
NKP30 (column 3, Figu e 4) con i ming concomi an lipid and
p o ein a icking be ween cells. Co-localiza ion o NKG2D
and NKP30 wi h EEA1 (column 4, Figu es 4a and d), Rab11
(column 4, Figu es 4b and e) and Rab7 (column 4, Figu es 4c
and ) indica ed ac i e a icking o NKG2D and NKP30
h ough endocy ic pa hway in ol ing lipid–p o ein a icking.
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
97
Cell Dea h and Di e en ia ion
To con i m he impo ance o lipid–p o ein a icking in he
ans e o NKG2D and NKP30 and in CB-NK cy o oxici y, we
analyzed he exp ession le els o NKG2D and NKP30 in
a ge cells ollowing p eincuba ion o CB-NK wi h Filipin III
(a lipid a inhibi o )
22,25
and pe o med cy o oxic y assays
adding U18666A (a choles e ol syn hesis and anspo
inhibi o
26
). Lipid a s inhibi ion educed he exp ession o
NKG2D and NKP30 in ARP1 cells a e CB-NK exposu e
(Figu e 4g), p o iding e idence o suppo a ole o lipid–
p o ein a icking in he ans e o NKG2D and NKP30.
Choles e ol syn hesis inhibi ion in CB-NK educed cy o oxici y
only e sus ARP1 (Figu es 4h and i); howe e , o K562 we
obse ed highe cy o oxici y a 10 : 1 e ec o : a ge a io,
sugges ing di e en oles o lipids in CB-NK cy o oxici y
depending on he a ge cell. We con i med in U266 and
KMM1 MM cells he cy o oxic ole o CB-NK-de i ed lipids
(Supplemen a y Figu e S3A). In e es ingly, pe iphe al blood
NK (PB-NK) o heal hy indi iduals ailed o show e idence o a
cy o oxic ole o lipids e sus MM (Supplemen a y Figu e
S4A), sugges ing ano he unique ea u e o CB-NK.
The mechanisms unde lying CB-NK-media ed cy o-
oxici y di e be ween a ge cells. As we had e idence
o di e en cy o oxic mechanisms depending on he a ge
cells (MM e sus K562) (Figu e 1), o he pa ame e s we e
assessed. Upon in e ac ion wi h a ge cells, NK deg anula e
eleasing sec e o y lysosomes wi h G B,
27
ac i a ing
Caspase-3 in a ge cells wi h ea u es o apop osis, such
as memb ane blebbing and ROS gene a ion.
4
CB-NK
showed less deg anula ion e sus ARP1 han e sus K562
cells (mean alue: 7.6 e sus 68%, P¼0.029) (Figu es 5a
and b), which was con i med o o he MM cell lines
(Supplemen a y Figu e S3B). Fu he , we no ed ha G B
was ans e ed o ARP1 cells a e con ac (Figu e 5c).
In e es ingly, inhibi ion o G B and Caspase-3 in NK-K562
and NK-ARP1 co-cul u es educed he killing o K562 cells by
CB-NK as expec ed bu inc eased he killing o ARP1 cells by
CB-NK (Figu es 5d and e). Inhibi ion o G B and Caspase-3
a a 20 : 1 e ec o : a ge a io yielded a educ ion o 13% in
CB-NK cy o oxici y agains K562 cells bu an inc ease o
12% in killing agains ARP1 cells (Figu es 5e, Po0.05).
Figu e 1 NKG2D and NKP30 con ibu e mo e o he cy o oxici y o MM cells han in K562 cells: (aand b) Cy oxici y assays o CB-NK agains K562 (a) and ARP1 (b) cells
a e blocking NKG2D, NKP30 in CB-NK and MICA/B in a ge cells. (cand d) Pe cen age o killing educ ion ob ained in panels aand ba e blocking he ecep o s. Ba s
ep esen mean±S.E.M. alues a a e ec o : a ge cell a io 20 : 1 (c) and 10 : 1 (d)(n¼8). (e) CB-NK cy o oxici y assay e sus p ima y CD138
þ
MM cells, adding CB-NK
be o e and a e blocking NKG2D, NKP30 and bo h oge he . ( ) Pe cen age o killing educ ion ob ained in panel ea e blocking he ecep o s. (g) Mean alues o expe imen
shown in panels eand wi h CD138
þ
cells om i e MM pa ien s. Ba s ep esen mean±S.E.M. *P 0.05. **P 0.0001
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
98
Cell Dea h and Di e en ia ion
G B- and Caspase-3-independen CB-NK cy o oxici y
was also con i med o o he MM cell lines (Supplemen a y
Figu e S3C). Howe e , cy o oxici y o PB-NK om heal hy
indi iduals e sus K562 and ARP1 was G B and Caspase-3
dependen (Supplemen a y Figu e S4B), p o iding ano he
unique cha ac e is ic o CB-NK. Because CB-NK deg anula-
ion indica es elease o lysosomes, we measu ed lysosomes
in K562 and ARP1 cells a e CB-NK. As con ols, CD19
þ
cells om heal hy indi iduals we e used. Whe eas in K562
and CD19
þ
cells he e was an inc ease in lysosomes a e
CB-NK (Figu e 5 , Po0.0001), in ARP1 he e was a
dec ease (Figu e 5 , Po0.0001), con i ming ha di e en
mechanisms o CB-NK cy o oxici y agains MM cells exis .
CB-NK induce lysosomal cell dea h in MM cells, and
NKG2D and NKP30 a e in ol ed in his mechanism. We
aimed o unde s and he mechanisms unde lying CB-NK
cy o oxici y e sus MM cells. We had obse ed lipid
a icking be ween cells, cy o oxici y ha was independen
G B and Caspase-3 and dec eased lysosomes a e CB-NK
exposu e (Figu e 5). G anulysin can cause Caspase-3-
independen cell dea h wi h ER s ess and lysosomal
pe meabiliza ion.
7,8
A e pe meabiliza ion, lysosomes
elease ca hepsins leading o a Caspase-3-independen cell
killing called ‘lysosomal cell dea h’.
8
We hypo hesized ha
CB-NK induce lysosomal cell dea h in MM cells. We
analyzed lysosome ma ke s (Lyso- acke and Rab7, which
also de ines lysosomes) and he impac o cys eine
ca hepsins in CB-NK cy o oxici y. A e 40 min wi h CB-NK,
ARP1 cells had educed lysosomes le els (Figu es 6a, b,
and d, Po0.0001). Fu he , ca hepsins inhibi ion educed
CB-NK cy o oxici y e sus ARP1 and no e sus K562
(Figu e 6e), con i ming a lysosomal cell dea h o MM cells;
which was con i med in o he MM cell lines, by de ec ing bo h
a dec ease in Rab7 and he impac o ca hepsins in CB-NK
cy o oxici y (Supplemen a y Figu es S3D and E). P ima y
MM CD138
þ
cells also showed dec eased lysosomes le els
a e CB-NK (Supplemen a y Figu e S3F). In e es ingly,
cy o oxici y o PB-NK om heal hy indi iduals e sus K562
o ARP1 cells was no a ec ed by ca hepsins inhibi ion
(Supplemen a y Figu e S4C), indica ing ano he unique
ea u e o CB-NK.
ROS we e also dec eased in ARP1 cells (Figu es 6c and ,
Po0.0001) and in p ima y MM CD138
þ
cells a e CB-NK
exposu e (Supplemen a y Figu e S3F). As G B leads o ROS
p oduc ion
4
and Caspase-3-dependen cell dea h, we
Figu e 2 CB-NK con en s a e ans e ed o ARP1 cells ia lipid esicles o lipid d ople s (LDs). (a) CB-NK and ARP1 cells we e co-cul u ed in cell pelle o 10 min. The
wo images a e he same showing ARP1 cells s ained in blue (CMAC) and CB-NK in g een (bodipy). Ac in is shown in ed. CMAC and ac in a e shown in he i s image and he
h ee colo s oge he in he second image. A ows indica e ans e o CB-NK con en in g een in o ARP1 cells, indica ing lipid a icking. (b) Same expe imen as panel a
showing one CB-NK ans e ing con en o one ARP1 cell and his ARP1 cell ans e ing con en o a seconda y ARP1 cell h ough he immunological synapse. (c) ARP1 cells
alone as con ol s ained wi h CMAC and ac in in ed. (d) Di e en ime poin s aken om a ime lapse in i o imaging o 2 h (Supplemen a y Mo ie S1) whe e ARP1 cells we e
s ained in blue (CMAC) and CB-NK in g een (bodipy). Fi e di e en ARP1 cells a e indica ed in egions o in e es (ROIs). ROIs 1–3 indica e h ee ARP1 cells ha had been
ouched by he same CB-NK. ROI 4 indica es one ARP1 cell ouched by a CB-NK and hen his ARP1 cell ans e s he CB-NK con en in o ano he ARP1 cell (ROI 5).
(e) Analysis o he inc ease du ing ime o g een s aining (bodipy) in ROIs 1–3 om panel d.( ) Analysis o he inc ease du ing ime o g een s aining (bodipy) in ROIs 4 and 5 in
panel d.(g) Ampli ied images om ROIs 4 and 5 in panel d o show ARP1 cells showing memb ane blebbing. See also Supplemen a y Mo ie S1
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
99
Cell Dea h and Di e en ia ion
in es iga ed whe he his pa hway was esponsible o he
dec eased ROS le els obse ed. Inhibi ion o G B and
Caspase-3 did no ab oga e he dec ease in ROS
(Figu e 6 ), con i ming ha G B and Caspase-3 in MM cells
nei he ac i a e cell dea h no ROS p oduc ion. We in es i-
ga ed whe he he dec eased ROS a e CB-NK ea men
was only cha ac e is ic o MM cells. In o he umo cells
analyzed (K562), he e was also a dec ease in ROS a e CB-
NK ea men (Figu e 6g, Po0.0001); howe e , CD19
þ
cells
om heal hy indi iduals showed an inc ease in ROS a e
CB-NK (Figu e 6g, Po0.0001).
To de e mine whe he NKG2D and NKP30 we e in ol ed in
he dec eased le els o lysosomes and ROS in MM cells a e
CB-NK exposu e, hese ecep o s we e blocked, and lyso-
somes and ROS le els analyzed. Blocking ei he NKG2D
(Figu e 6h, Po0.0001) o NKP30 (Figu e 6i, Po0.0001)
ab oga ed he CB-NK e ec on he dec eased lysosome and
ROS le els in MM cells a e CB-NK exposu e, indica ing ha
Figu e 3 NKG2D and NKP30 a e ans e ed in o MM cells: (a) NKG2D and (b) NKP30 in ARP1 cells a e co-cul u ing wi h CB-NK o 20 min. ARP1 cells we e s ained in
blue (CMAC) and CB-NK in g een (bodipy). NKG2D and NKP30 a e indica ed in ed. All he images ep esen he same showing di e en combina ions o he h ee colo s.
A ows indica e ARP1 cells wi h NKG2D o NKP30. (c) Fluo escence alues ob ained om images in panels aand b. Ba s ep esen mean±S.E.M. (d) As con ol, ARP1 cells
alone we e s ained o NKG2D and NKP30 wi h he same se ings han in images aand b. Resul s om panels a o dwe e con i med in h ee di e en expe imen s.
(eand ) De ec ion by low cy ome y o NKG2D and NKP30 in ARP1 cells a e 24, 48 and 72 h o co-cul u e wi h CB-NK. ARP1 cells we e ga ed based on CMAC s aining.
(e) Rep esen a i e plo o he expe imen a 24 h o co-cul u e. ARP1 cells uns ained alone we e used as con ol. ( ) Median luo escence in ensi y (MFI) alues o NKG2D and
NKP30 in ARP1 cells a he di e en imes o co-cul u e, pe o ming bo h su ace and in acellula s aining
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
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Cell Dea h and Di e en ia ion
bo h ecep o s we e in ol ed in he cell dea h media ed by
CB-NK.
MM s oma dec eases CB-NK cy o oxici y. Because o
hep o ec i e oleo hes omainMM,weanalyzed he
impac o MM s oma in CB-NK cy o oxici y. MM s oma
educed CB-NK cy o oxici y o MM cells (ARP1, RPMI and
KMM1, Figu e 7a, Po0.05). We obse ed ha bo h MM
cells and CB-NK adhe ed s ongly o he s oma making
di icul he o ma ion o he immunological synapse
be ween CB-NK and MM cells. Howe e , we s ill obse ed
esicle ans e om CB-NK o MM cells and also
seconda y ans e be ween MM cells (Figu e 7b). Because
CXCL12 e he s hemopoie ic cells in o he s oma,
28
we
measu ed CXCL12 sec e ion by he s oma and obse ed a
educed CXCL12 sec e ion a e he addi ion o MM cells o
MM cells and CB-NK (Figu e 7c, Po0.05), sugges ing a
possible ole o CXCL12 in educing he an i-MM cy o oxi-
ci y o CB-NK. The e o e we pe o med cy o oxici y assay
adding AMD3100, which inhibi s binding o CXCL12 o
CXCR4 in hemopoie ic cells. A sligh eco e y in CB-NK
cy o oxici y e sus RPMI MM cell line was obse ed
(P¼0.09, Figu e 7d), p o iding e idence o suppo his
possibili y.
CB-NK cy o oxici y is ansmissible be ween MM cells.
We demons a ed lipid–p o ein a icking be ween MM cells
a e CB-NK con ac . Recipien MM cells appea ed o
become apop o ic wi h memb ane blebbing (Figu e 2g and
Supplemen a y Mo ie S1). We examined whe he NKG2D
and NKP30 we e seconda ily ans e ed om ARP1 cells
p e iously exposed o CB-NK (‘p ima y (11) ARP1 cells’) o
adjacen ARP1 cells (‘seconda y (21) ARP1 cells’). Bodipy-
labeled CB-NK and CMAC-labeled ARP1 cells we e co-
cul u ed o 40 min; hese ARP1 cells we e de ined as
11ARP1 cells. Ali e 11ARP1 cells we e hen FACS-so ed.
These so ed 11ARP1 cells we e hen ei he le alone
(11ARP1 alone) o co-cul u ed (11ARP1 wi h 21ARP1) in
cell pelle o ano he 40 min wi h uns ained esh 21ARP1
(21ARP1 a e 11ARP1) cells allowing di e en ia ion
be ween he wo ARP1 cell popula ions (CMAC
þ
11ARP1
cells e sus uns ained 21ARP1 cells). Then, in hese wo
se s o ARP1 cells, we analyzed he exp ession o NKG2D
and NKP30 (Figu e 8a).
Figu e 4 The ans e o NKG2D and NKP30 in o MM cells in ol es lipid a icking and he endocy ic pa hway: Co-localiza ion o NKG2D wi h (a) EEA1, (b) Rab11 and (c)
Rab7 and o NKP30 wi h (d) EEA1, (e) Rab11 and ( ) Rab7 in ARP1 cells a e being co-cul u ed wi h CB-NK o 20 min. ARP1 cells we e s ained in blue (CMAC) and CB-NK in
g een (bodipy), NKG2D and NKP30 a e indica ed in ed. All he images in one ow a e he same bu showing di e en combina ions o colo s. In column 1, CMAC and bodipy
a e shown o indica e ARP1 cells and CB-NK. In column 2, NKG2D o NKP30 and CMAC a e shown indica ing ARP1 cells wi h NKG2D o NKP30. In column 3, bodipy in g een
and NKG2D and NKP30 in ed indica e co-localiza ion (yellow) o NKG2D and NKP30 wi h lipids. In column 4, NKG2D and NKP30 a e indica ed in ed and he endocy ic
ma ke in g een. In column 5, bodipy in g een and he endocy ic ma ke in ed indica e co-localiza ion o lipid esicles wi h endocy ic pa hway. A ows indica e ARP1 cells.
(g) NKG2D and NKP30 exp ession in ARP1 cells alone (ARP1 alone), a e being co-cul u ed wi h CB-NK o 40 min (ARP1 a e NK) and a e being co-cul u ed o 40 min
wi h CB-NK p e iously incuba ed wi h Filipin III (ARP1 a e NK þFilipin). (h) CB-NK cy o oxici y educ ion e sus ARP1 and K562 cells a e incuba ing p e iously CB-NK wi h
U18666A o 2 h. (i) CB-NK cy o oxici y educ ion o esul s shown in panel h. Ba s ep esen mean±S.E.M. *P 0.05*. **P 0.0001
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
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Cell Dea h and Di e en ia ion
NKG2D (Figu es 8b and d, Po0.0001) and NKP30 (Figu es
8c and e, Po0.0001) we e ans e ed seconda ily o
neighbo ing 21ARP1 cells (21ARP1 a e 11ARP1 in
Figu e 8) om 11ARP1 cells, which had p ima ily synapsed
wi h NK cells (11ARP1 wi h 21ARP1 in Figu e 8). Co-s aining
o ac in indica ed ha his ans e occu ed h ough an
immunological synapse be ween ARP1 cells, especially o
NKG2D (Figu es 8d and e).
As NKG2D and NKP30 we e ans e ed o 21ARP1 cells,
we also analyzed he downs eam e ec s o his ans e on
lysosomes and ROS le els and whe he his e ec was
ansla ed in some indi ec cy o oxici y o CB-NK agains
hese 21ARP1 cells. These 21ARP1 cells demons a ed
dec eased lysosome and ROS le els a e con ac wi h 11
ARP1 cells (21ARP1 a e 11ARP1: in Figu es 8 and g,
Po0.0001), in compa ison o he con ol so ed ARP1 cells
(ARP1 c l: in Figu es 8 and g). We also no iced a
compensa ing e ec be ween he wo popula ions o ARP1
cells, as a e 11ARP1 cells we e exposed o CB-NK and hen
incuba ed wi h 21ARP1 cells, he 11ARP1 cells began o
no malize hei loss in lysosomes and ROS (11ARP1 alone
e sus 11ARP1 wi h 21ARP1: in Figu es 8 and g,
Po0.0001).
To de e mine whe he CB-NK cy o oxici y was also
ansmi ed be ween ARP1 cells, we pe o med cy o oxici y
assay by adding 11ARP1-so ed cells as e ec o s and
21ARP1 cells as a ge s. 11ARP1 cells, which p e iously
synapsed wi h CB-NK, demons a ed 7% cy o oxici y agains
21ARP1 cells a a 40 : 1 e ec o : a ge a io. Al hough his
cy o oxici y dec eased a lowe e ec o : a ge cell a ios, i
was g ea e han he cy o oxici y o he con ol ARP1 cells
un ouched by CB-NK (Figu e 8h), indica ing a ansmissible
cell dea h om CB-NK o MM cells and hen om hese MM
cells o neighbo ing MM cells. Finally, we es ed whe he he
MM s oma impac ed on his indi ec ‘domino-e ec ’ cy o oxi-
ci y. Cy o oxici y assay con aining MM s oma educed
indi ec cy o oxici y o 11ARP1 e sus 21ARP1 a 20 : 1
e ec o : a ge cell a io (Figu e 8i), sugges ing ha MM
s oma may educe ‘domino-e ec ’ cy o oxici y.
Discussion
We show a no el CB-NK-media ed cy o oxici y agains MM
cells ha is G B and Caspase-3 independen , in ol es
ansmissible cy o oxici y be ween CB-NK and MM cells ia
lipid–p o ein esicle a icking and seconda ily ans e ed
om hese MM cells o neighbo ing MM cells. This lipid–
p o ein ans e in ol es NKG2D and NKP30 ecep o s,
causes lysosomal cell dea h and educed ROS le els. This
cy o oxici y appea s o be unique o CB-NK and di e en om
Figu e 5 The mechanisms unde lying CB-NK-media ed cy o oxici y di e be ween a ge cells: (a) Deg anula ion (CD107a exp ession) o CB-NK a e 4 h o incuba ion
wi h K562 o ARP1 cells. (b) Mean±S.E.M. alues om a ep esen a i e expe imen shown in a(n¼4). (c) G B exp ession in ARP1 cells a e being co-cul u ed wi h CB-NK
o 20 min. ARP1 cells we e s ained in blue (CMAC) and CB-NK in g een (bodipy). G B is indica ed in ed. (d) CB-NK cy o oxici y educ ion a e inhibi ing G B and Caspase-3.
Killing is shown o con ol CB-NK, a e inhibi ing G B in CB-NK, Caspase-3 in a ge cells and bo h, G B in CB-NK and Caspase-3 in a ge cells. (e) Pe cen age o killing
educ ion a e inhibi ing G B and Capase-3 in ou di e en CB uni s o he ep esen a i e expe imen shown in d.( ) Lysosome le els be o e and a e CB-NK exposu e o
40 min o K562 cells, CD19
þ
cells om a heal hy dono and ARP1 cells. Ba s ep esen mean±S.E.M. *P 0.05. **P 0.0001
CB-NK cy o oxici y agains mul iple myeloma cells
B Ma in-An onio e al
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Cell Dea h and Di e en ia ion
ha seen agains K562 cells whe e NKG2D and NKP30 do no
ha e such a ele an ole.
Lysosomal cell dea h happens a e up u ed lysosomes
elease ca hepsins leading o Caspase-3-independen cell
dea h, which can show memb ane blebbing, and which,
depending on he magni ude o lysosomal pe meabiliza ion,
will ac i a e an apop o ic-like p og ammed cell dea h o
nec osis cell dea h.
29
Lipids and G anulysin a e in ol ed in
his ype o cell dea h as lipids des abilize lysosomes
30
and
G anulysin media es a Caspase-3-independen lysosomal cell
dea hwi hmemb aneblebbing.
8
These s udies suppo ou
indings whe e we show a ansmissible MM cell dea h
media ed by CB-NK, in ol ing lipid–p o ein ans e wi h a
dec ease in lysosomes, cell dea h dependen on Ca hepsins
and showing memb ane blebbing in MM cells. NKG2D and
NKP30, associa ed wi h he dec eased lysosomes le els, we e
also indi ec ly ans e ed o new MM cells indica ing hei
possible ole in his indi ec cy o oxici y. Al hough no measu e-
men o G anulysin was made, ou indings do no disca d a
possible ole o G anulysin in his lysosomal cell dea h.
Cells exchange in o ma ion ia exosomes/lipid esicles,
and NK elease cy o oxic exosomes/ esicles exp essing
NK-ac i a ing ecep o s.
3,31
Lipids inhibi au ophagy
32
and
can ac i a e ER s ess-associa ed cell dea h.
33
He e we show
lipid a icking be ween cells and ha his exchange o
‘in o ma ion’ in lipid s uc u es, which con ain NKG2D and
NKP30, occu s be ween CB-NK and MM cells and a e wa ds
be ween adjacen MM cells being associa ed wi h cy o oxici y
agains MM cells. E en he bone ma ow s oma educed his
CB-NK di ec and indi ec cy o oxici y, i was no an impedi-
men o his lipid ans e om CB-NK o MM cells and
seconda ily o neighbo ing MM cells.
G B-induced NK dea h is usually accompanied by
inc eased ROS p oduc ion, a p ocess which is Caspase-3
dependen .
4
We ound ha e en hough G B is ans e ed
in o ARP1 cells by CB-NK, he G B and Caspase-3 pa hway
was no in ol ed nei he in he killing o ARP1 cells no in he
dec eased ROS le els de ec ed. By compa ison, NKG2D-
and NKP30-associa ed MM cell killing was associa ed wi h
dec eased ROS, p o iding e idence o a ole o hese
ecep o s. As ROS is associa ed wi h su i al in umo cells
and wi h a lowe ing o he in e ac ion be ween cance cells
and he immune sys em,
34,35
his CB-NK-associa ed ROS
educ ion migh make umo cells mo e ulne able o cell-
based immuno he apeu ic s a egies.
MM cells, which a e suscep ible o ER s ess cell dea h,
ha e high au ophagy le els.
18,36
Au ophagy uses lysosomes
o deg ada ion,
37
associa es o inc eased ROS
38
and
con ibu es o chemo he apeu ic esis ance.
17
We obse ed
dec eased lysosomes and ROS in MM cells a e CB-NK
Figu e 6 CB-NK induce lysosomal cell dea h in MM cells, and NKG2D and NKP30 a e in ol ed in his mechanism: (a–d) Analysis o lysosomes (Rab7 and Lyso- acke :
Lys) and ROS p oduc ion in ARP1 cells be o e and a e co-cul u e in cell pelle wi h CB-NK o 40 min. ARP1 cells we e labeled in blue (CMAC), CB-NK in g een (bodipy) and
each ma ke is indica ed in ed. (a) La e endocy ic and lysosome ma ke Rab7. (b) Lysosome ma ke (Lyso- acke ). (c) ROS p oduc ion. (d) Mean±S.E.M. alues o slides
analyzed om a o d. Resul s om a o cwe e con i med in h ee di e en expe imen s. (e) CB-NK cy o oxici y educ ion a e incuba ing a ge cells (K562 and ARP1) wi h E64
(a cys eine ca hepsin inhibi o ). ( ) ROS a ia ion a e inhibi ing G B in CB-NK and Caspase-3 in ARP1 cells and co-cul u ing o 40min. (g) ROS le els be o e and a e CB-
NK exposu e o 40 min o K562 cells and CD19
þ
cells om heal hy indi iduals. (hand i) Va ia ion o lysosome (Lys) and ROS le els a e CB-NK exposu e o 40 min (ARP1
a e NK) and a e blocking (h) NKG2D and (i) NKP30 in CB-NK (NKG2D block and NKP30 block). Lysosomes we e de e mined wi h he Lyso- acke . Ba s ep esen
mean±S.E.M. **P 0.0001
CB-NK cy o oxici y agains mul iple myeloma cells
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Cell Dea h and Di e en ia ion
exposu e, which could sugges dec eased au ophagy le els.
Al hough an ex ensi e s udy o au ophagy ma ke s was no
unde aken he e, he possible au ophagy educ ion in MM
cells by CB-NK could educe he e ac o iness, which
happens in some MM pa ien s a e PI ea men .
17,19
In summa y, we show a unique mechanism o CB-NK
cy o oxici y ha in ol es ansmissible cy o oxici y be ween
cells, whe e lipids appea as ‘ ehicles’ o pe o m his
mechanism. This opens new pa hways o he use o CB-NK
as a cellula immuno he apy op ion and o de eloping
he apeu ic s a egies using CB-NK-de i ed lipids.
Ma e ial 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 Cance Cen e (MDACC) Ins i u ional Re iew Boa d (IRB). CB
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 (IACUC)-app o ed p o ocol speci ic o his s udy.
Figu e 7 MM s oma dec eases CB-NK cy o oxici y: (a) CB-NK cy o oxici y educ ion a e adding MM s omal cells in o cy o oxici y assays. Cy o oxici y assays o MM
cells we e done in pa allel ei he adding (Ta ge cell þCB þNK þS oma) o no adding (Ta ge cell þCB-NK) MM s omal cells o he assay. (b) Con ocal luo escence
image o s omal MM cells wi h CB-NK (in g een wi h bodipy) and KMM1 MM cells (in blue wi h CMAC). Ac in is shown in ed. S omal cells can be isualized by he ac in
ilamen s. A ows in he lowe pa show CB-NK adhe ed o s omal cells. A ows in he uppe pa show ans e o CB-NK con en o MM cells and also seconda ily be ween
MM cells. The whi e squa e is shown ampli ied in he igh side showing ei he wo colo s (CMAC and ac in) o he h ee colo s oge he (CMAC, bodipy and ac in). Same
images we e con i med o o he MM cell lines. (c) CXCL12 le els in he supe na an o cy o oxici y assays wi h MM cells adding ei he s oma wi h a ge MM cells and CB-NK
(S oma þ a ge þCB-NK) o s oma wi h a ge cells alone (S oma þ a ge ). Se um MM pa ien was added as posi i e con ol. P- alues ep esen analysis o s oma alone
e sus s oma þ a ge . (d) Cy o oxici y CB-NK educ ion a 20 : 1 o 10 : 1 e ec o : a ge a io, a e adding (S oma þAMD3100) o no adding (S oma) AMD3100 o he
cy o oxici y assay wi h s oma, CB-NK and MM cells. *P 0.05
CB-NK cy o oxici y agains mul iple myeloma cells
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Cell Dea h and Di e en ia ion
