Ci a ion: Ploege , L.; Kaleja, P.;
Tholey, A.; Le au, M.; Janssen, O.
Analysis o Cy o oxic G anules and
Cons i u i ely P oduced Ex acellula
Vesicles om La ge G anula
Lymphocy ic Leukemia Cell Lines.
Cells 2024,13, 1310. h ps://doi.o g/
10.3390/cells13161310
Academic Edi o : Fuguo Liu
Recei ed: 15 July 2024
Re ised: 30 July 2024
Accep ed: 2 Augus 2024
Published: 6 Augus 2024
Copy igh : © 2024 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
cells
A icle
Analysis o Cy o oxic G anules and Cons i u i ely P oduced
Ex acellula Vesicles om La ge G anula Lymphocy ic
Leukemia Cell Lines
La a Ploege 1,† , Pa ick Kaleja 2,†, And eas Tholey 2, Ma cus Le au 3,* and O ma Janssen 1,*
1Molecula Immunology—Ins i u e o Immunology, Uni e si y Hospi al Schleswig-Hols ein, Campus Kiel,
24105 Kiel, Ge many
2Sys ema ic P o eomics & Bioanaly ics—Ins i u e o Expe imen al Medicine, Uni e si y o Kiel,
24105 Kiel, Ge many
3S em Cell T ansplan a ion and Immuno he apy—In e nal Medicine II, Uni e si y Hospi al
Schleswig-Hols ein, Campus Kiel, 24105 Kiel, Ge many
*
Co espondence: ma cus.le [email protected] (M.L.); o ma [email protected] (O.J.); Tel.: +49-(0)431-500-22775 (M.L.);
+49-(0)431-500-31031 (O.J.)
†These au ho s con ibu ed equally o his wo k.
Abs ac : Backg ound: La ge g anula lymphocy e leukemias (LGLLs) a e a e lymphop oli e a i e
malignancies caused by clonal expansion o g anula lymphocy es. T-cell LGLL and na u al kille
(NK) cell LGLL a e de ined based on hei cellula o igin. Thei clinical mani es a ion and pa hophysi-
ology a y depending on he sub ype and include, e.g., neu openia, anemia, ecu en in ec ions, and
au oimmuni y. A limi ed numbe o a ailable pa ien -de i ed cell lines a e conside ed aluable ools
o s udy he biology o hese malignancies. They di e in he exp ession o lineage-speci ic su ace
ma ke s, bu gene ally con ain cy o oxic e ec o molecules in cha ac e is ic g anules. Me hods: We in-
es iga ed he p esence and elease o lysosome-associa ed e ec o p o eins in pa ien -de i ed LGLL
cell lines by low and imaging cy ome y, by Wes e n blo ing and by bo om–up p o eomics p o iling.
Resul s: The es ed cell lines did no exp ess FasL (CD178), bu did exp ess CD26/DPP4
+
. In acel-
lula ly, we de ec ed majo di e ences in he abundance and subcellula dis ibu ion o g anzymes,
pe o in, and g anulysin. Simila di e ences we e seen in en iched lysosome- ela ed e ec o esicles
(LREVs). The p o eomics p o iling o en iched EVs om an NK-LGLL line (NKL) and a T-LGLL line
(MOTN-1), con i med indi idual p o iles o e ec o molecules. Conclusion: Ou analyses unde sco e
he indi idual dis ibu ion o e ec o p o eins bu also open new ou es o de ine he ole o in a-
and ex acellula g anules in he disease mani es a ion o pa hology o LGLLs.
Keywo ds: T cells; NK cells; la ge g anula lymphocy e leukemia (LGLL); cy o oxic g anules;
cy o oxic
e ec o p o eins; lysosome- ela ed e ec o esicles (LREV); ex acellula esicles (EV);
exosomes; p o eomics p o iling
1. In oduc ion
La ge g anula lymphocy e leukemia (LGLL) is a a e ch onic lymphop oli e a i e
diso de wi h he e ogeneous clinical p esen a ion. I is cha ac e ized by clonal expansion
o lymphocy es wi h g anula ed cy oplasm [
1
]. The i h edi ion o he WHO classi ica ion
o hema olymphoid umo s places LGLL in he ca ego y o ma u e T-cell and NK cell
leukemias wi h h ee majo sub ypes: T-la ge g anula lymphocy ic leukemia (T-LGLL),
NK-la ge g anula lymphocy ic leukemia (NK-LGLL), and agg essi e NK-cell leukemia
(ANKL). A u he dis inc ion can be made be ween CD4
+
o CD8
+
T-LGLL and
αβ
- o
γδ
-
T-LGLL [
2
]. LGL leukemia accoun s o app oxima ely 2–5% o ch onic lymphop oli e a i e
diseases in he US and Eu ope [
1
]. The incidence is epo ed o be 0.2–0.72 pe one million
pe yea [
3
,
4
]. The median age o onse is 60 yea s and bo h sexes a e simila ly a ec ed [
5
].
Ch onic T-LGLL accoun s o 85% o cases, while NK-LGLL accoun s o less han 10%.
Cells 2024,13, 1310. h ps://doi.o g/10.3390/cells13161310 h ps://www.mdpi.com/jou nal/cells
Cells 2024,13, 1310 2 o 21
Agg essi e NK-LGL leukemia (ANKL) occu s mo e equen ly in Asia and a ec s sligh ly
younge pa ien s, pa icula ly hose wi h a his o y o EBV in ec ion [6].
Diagnosis and cha ac e iza ion o LGLL-sub ypes a e based on cy ology, immunophe-
no ype, and e idence o monoclonali y [
7
]. The pheno ype o T-LGLL is he e ogeneous,
being CD2
+
, CD3
+
, CD8
+
, CD57
+
, CD45RA
+
, CD16
+
, and CD4
−
, wi h o wi hou exp es-
sion o CD56 [
7
,
8
]. As men ioned,
αβ
-TCRs a e o en exp essed, bu
γδ
-TCR
+
cases ha e
also been desc ibed [
6
,
9
,
10
]. O e all, his T-cell pheno ype co esponds o a cons i u i ely
ac i e ma u e memo y T e ec o cell [
8
]. In T-LGLL clones, cy o oxic e ec o p o eins
including g anzymes and pe o in a e equen ly exp essed, al hough in di e en combi-
na ions. A less equen sub ype occu s in 10–15% o cases and is CD4
+
, CD57
+
, CD8
−
o
CD8
+dim
[
9
]. NK-LGLL a e mos ly CD2
+
, CD3
−
, CD8
+
, CD16
+
, CD56
+
and also exp ess
cy o oxic e ec o molecules [8].
Al hough signi ican p og ess has been made in ou unde s anding o gene ic and
molecula al e a ions associa ed wi h LGLL (see ecen o e iews [
1
,
9
,
11
–
14
] o de ails),
he con ibu ion o he LGLL-associa ed cy o oxici y and espec i e e ec o molecules
has only been poo ly add essed. Ini ially, i became e iden ha dys egula ed apop osis
may ep esen a key e en in he de elopmen o LGL malignancy and au oimmuni y.
Pe zo a and Lough an epo ed ha se en ou o he se en LGLL pa ien s es ed displayed
cons i u i e exp ession o FasL gene ansc ip s [
15
]. I was hen obse ed ha leukemic
cells om 9 ou o 11 T-LGLL pa ien s cons i u i ely exp essing bo h Fas and FasL a
high le els we e comple ely esis an o an i-Fas- and an i-CD3-media ed apop osis [
16
].
In e es ingly, he LGLL T cells did no show appa en mu a ions in Fas and in mos cases
unde wen apop osis a e PHA and IL-2 s imula ion [
16
]. O e he yea s, FasL was also
associa ed wi h o he LGLL symp oms, including neu openia. The se um le el o (soluble)
sFasL, which is in ol ed in p e en ing Fas-media ed apop osis, was p oposed as a ma ke
o LGLL ac i i y [17,18].
Fo o he cy o oxic e ec o s, da a poin ing o dys egula ion a e ha dly a ailable.
In ac , g anzyme (G z) B and pe o in (P ) a e o en men ioned as addi ional ma ke s
o asce ain a diagnosis o LGLL, especially o his ological s aining o bone ma ow
biopsies [
5
]. Su p isingly o g anulysin (Gnly), ano he key e ec o o T and NK cells, he
dis ibu ion, elease, o impac in LGLL has appa en ly ne e been add essed. The p esence
o ele a ed le els o sFasL in LGLL pa ien s’ se a migh , howe e , indica e a subs an ial
p oduc ion and elease o cy o oxic g anules om leukemic cells. Al hough sFasL has
been co ela ed wi h he p e en ion o apop osis in leukemic cells, he exposu e o elease
o esicles ca ying he dea h ligand migh ha e b oade implica ions. In pa icula , he
unsa e exposu e o molecules anspo ed in sec e o y esicles o a elease o esicles
ca ying e ec o s as ex acellula esicles migh con ibu e o immune dys unc ion and he
de elopmen o au oimmune diseases.
As a i s a emp o ackle hese issues, we add essed he dis ibu ion and elease
o cy o oxic e ec o molecules in es ablished LGLL cell lines. P e iously, we compa ed
in acellula e ec o esicles om he pa ien -de i ed NK LGLL line NKL [
19
] wi h hose
om he NK cell lymphoblas ic leukemia/lymphoma line YTS [
20
,
21
] and om un ans-
o med cells. We epo ed he e ogeneous p o iles o unc ionally ele an p o eins including
cy o oxic e ec o p o eins in leukemic and ac i a ed human NK cells [22].
We now ollowed his ou e and analyzed pa ien -de i ed LGLL cells, in pa icula
MOTN-1 [
23
], ep esen ing a T-cell LGLL; NKL [
19
] and NK-92 [
24
], ep esen ing wo
NK-LGLLs; and KHYG-1 [
25
], ep esen ing an ANKL. Fo hose cell lines, all indi idual
cellula cha ac e is ics ha e been desc ibed in de ail be o e [
19
,
23
–
25
]. As s a ed abo e,
indi idual LGLL sub ypes di e in he exp ession o lineage-speci ic su ace ma ke s,
display di e en mu a ions in genes o ei he he STAT3/5B o he NOTCH1 pa hway, and
migh p oduce di e en immunomodula o y cy okines.
In e es ingly, in ou hands, all cell lines did exp ess compa able le els o CD26,
bu did no exp ess (memb ane) mFasL. When we analyzed he dis ibu ion o e ec o
p o eins by con en ional and imaging low cy ome y o Wes e n blo ing, we de ec ed
Cells 2024,13, 1310 3 o 21
majo di e ences in he abundance o g anzyme A, g anzyme B, pe o in, and g anulysin.
Simila di e ences we e also seen in cy o oxic g anules en iched as lysosome- ela ed
e ec o esicles (LREVs). In un ans o med T and NK cells, such LREVs, p e iously also
desc ibed as sec e o y lysosomes [
26
], o m he basis o a di e en ial elease o indi idual
cy o oxic e ec o p o eins depending on he s eng h o s imula ion [
27
]. As an example,
we demons a ed ha g anulysin (Gnly) species migh be used as ma ke s o dis inc
en i ies o lysosome- ela ed e ec o esicles (LREVs) since in un ans o med T-cell blas s,
hey seg ega e o di e en sec e o y compa men s wi h g anzyme B o FasL and become
mobilized by ei he classical o non-classical deg anula ion [28].
Due o a po en ial ela ion o LREVs o sec e o y lysosomes o ex acellula esicles
eleased as exosomes o mic opa icles [
27
], we pe o med a compa a i e p o eome p o-
iling o isola ed EV om MOTN-1 and NKL o ob ain a deepe insigh in o indi idual
p o eins’ composi ion and o add ess he po en ial ole o LREVs as in a- and ex acellula
componen s o disease mani es a ion o pa hology.
2. Ma e ials and Me hods
2.1. Cell Lines
No ably, only a ew es ablished cell lines a e a ailable o he s udy o NK-LGLL
biology [
29
,
30
]. Fo ou s udies, we used NK-92 (ACC 488), which was es ablished in 1992
om he pe iphe al blood o a 50-yea -old male wi h LGL-non-Hodgkin lymphoma [
24
]
and NKL (CVCL_0466), which was es ablished in 1996 om he pe iphe al blood o a
63-yea -old
pa ien wi h a mo e agg essi e CD3
−
, CD16
+
, and CD56
+
LGL leukemia [
19
].
Bo h cell lines a e now ca ego ized as NK-LGLL and a e supposed o display s ong
cy o oxici y. This also holds ue o KHYG-1 (ACC 725), an ANKL cell line es ablished in
1997 om he pe iphe al blood o a 45-yea -old woman wi h agg essi e NK leukemia [
25
].
The T-LGLL line MOTN-1 (ACC 559) was es ablished in 2001 om he pe iphe al blood o
a 65-yea -old woman wi h ch onic T-LGLL; in his case, no cy o oxic ac i i y o expanded
cells was desc ibed [
23
]. All cell lines we e kep a 37
◦
C in a humidi ied a mosphe e
wi h 5% CO
2
. The cul u e medium was RPMI 1640 wi h 2 mM glu amine and 25 mM
HEPES (The mo Fishe Scien i ic, Wal ham, MA, USA), supplemen ed wi h 100 U/mL
penicillin/100
µ
g/mL s ep omycin (The mo Fishe Scien i ic) and 10% ( / ) e al cal
se um (FCS, The mo Fishe Scien i ic) o NKL and KHYG-1 o 20% ( / ) FCS o MOTN-1.
NK-92 cells we e cul u ed in Minimal Essen ial Medium
α
(MEM
α
, #32561029, The mo
Fishe Scien i ic) wi h 12.5% ( / ) ho se se um (The mo Fishe Scien i ic) and 12.5% ( / )
FCS. Since g ow h o LGLL cell lines is s ic ly IL-2-dependen , ecombinan IL-2 ( IL-2,
No a is, Basel, Swi ze land) was added a 50 U/mL o NKL, 100 U/mL o NK-92 and
MOTN-1, and 200 U/mL o KHYG-1 expansion.
2.2. Immuno luo escen S aining
FasL and CD26/DPP4 su ace exp ession was checked by con en ional low cy om-
e y [
31
]. A o al o 10
5
cells we e cen i uged in 96-well V-bo om pla es, washed, and
s ained wi h PE-conjuga ed an i-FasL monoclonal an ibody (mab) (clone NOK-1, Fishe
Scien i ic) o an i-CD26/DPP4 mab (clone BA5b, BioLegend, San Diego, CA, USA). A e
incuba ion o 30 min on ice, he cells we e washed wice, ixed in 1% pa a o maldehyde,
and analyzed on a FACS Can o low cy ome e (BD Biosciences, F anklin Lakes, NJ, USA).
Fo s aining o in acellula an igens by low cy ome y, cells we e washed, ixed, and
pe meabilized using he Cy o ix/Cy ope m ki (BD-Biosciences) acco ding o he man-
u ac u e ’s ins uc ions. A e ixa ion, he cells we e analyzed on an FACS Can o low
cy ome e . The ollowing an ibodies we e used: PE-conjuga ed an i-g anzyme A mab
(clone CB9, BioLegend), PE-conjuga ed an i-g anzyme B ecombinan ab (clone QA16A02,
BioLegend), PE-conjuga ed an i-pe o in mab (clone dG9, BioLegend). Fo g anulysin s ain-
ing, we also used an unconjuga ed an i-Gnly mab (clone RF10, MBL In e na ional, Wobu n,
MA, USA) wi h Alexa-Fluo 555-conjuga ed goa an i-mouse polyclonal an ibody (pab)
(The mo Fishe Scien i ic) and unconjuga ed an i-Gnly pab om goa (RD Sys ems, Min-
Cells 2024,13, 1310 4 o 21
neapolis, MN, USA) wi h Alexa-Fluo 555-conjuga ed donkey an i-goa pab, espec i ely.
PE-conjuga ed iso ype-ma ched an ibodies om BioLegend and unconjuga ed an ibodies
om Abcam (Camb idge, UK) se ed as con ols [28].
Fo imaging low cy ome y, 0.5
×
10
6
cells we e washed and suspended in 100
µ
L
LIVE/DEAD Fixable Fa -Red s ain (The mo Fishe Scien i ic) o 15 min, washed again,
and pe meabilized in 50
µ
L o Cy o ix/Cy ope m. A e 30 min, he cells we e washed and
incuba ed wi h a FITC-conjuga ed an i-CD107a mab (clone H4A3, BioLegend) o ano he
30 min. Two addi ional washes we e ollowed by addi ion o PE-conjuga ed an i-g anzyme
A, an i-g anzyme B, an i-pe o in, o an i-CD107a as a con ol. Fo g anulysin s aining, we
also used unconjuga ed an i-Gnly mab RF10 o unconjuga ed an i-Gnly pab wi h Alexa-
Fluo 555-conjuga ed seconda y eagen s and modi ied he s aining p ocedu e acco dingly.
Finally, cells we e ixed in 1% pa a o maldehyde.
2.3. Imaging Flow Cy ome y
Imaging low cy ome y was pe o med exac ly as desc ibed be o e wi h an Im-
ageS eam X Ma k II (Me ck Millipo e, Bu ling on, MA, USA) one-came a sys em wi h
351, 488, 562, 658, and 732 nm lase s [
31
]. Analy e-posi i e cells we e disc imina ed based
on con ols. The B igh De ail Simila i y R3 ea u e was used o quan i y he deg ee o
colocaliza ion in double-posi i e cells only [31].
2.4. Subcellula F ac iona ion
Lysosome- ela ed e ec o esicles (LREVs) we e en iched om expanded cell lines as
desc ibed be o e [
28
,
32
], employing a comme cial lysosome isola ion ki (Me ck/Sigma-
Ald ich) and di e en ial cen i uga ion and ul acen i uga ion.
2.5. Immunop ecipi a ion and Wes e n Blo
To ob ain cell lysa es o Wes e n blo ing, 10
×
10
6
cells we e washed in PBS and lysed
in NP40 lysis bu e (1% ( / ) Nonide
®
P40 (Sigma-Ald ich), 20 mM T is-bu e ,
pH 7.4,
150 mM NaCl, 5 mM EDTA) supplemen ed wi h p o ease and phospha ase inhibi o s o
30 min [28].
Cell deb is was emo ed by cen i uga ion a 14,000
×
g pm and
4◦C
o
10 min
and supe na an s we e boiled in educing sample bu e . To p ecipi a e g anulysin
om supe na an s o uns imula ed o s imula ed LGLL cells, 10
×
10
6
cells we e washed in
PBS and suspended in X- i o medium (The mo Fishe Scien i ic). TPA and/o ionomycin
(bo h om Me ck) we e added a 20 o 500 ng/mL, espec i ely, o s imula e he cells o up
o 2 h in he p esence o absence o 4 mM EGTA and 4 mM MgCl
2
. Following s imula ion,
he cells we e pelle ed, washed wi h PBS, and lysed in NP40 lysis bu e . Supe na an s
we e cen i uged a 3400
×
g o emo e esidual cells and subjec ed o immunop ecipi a ion.
To his end, NP-40 lysa es o supe na an s we e p eclea ed wi h p o ein G-sepha ose beads
(Sigma-Ald ich) o 2 h and incuba ed o e nigh wi h 0.5
µ
g o he polyclonal an i-Gnly
an ibody (R&D Sys ems Inc., Minneapolis, MN, USA) and p o ein G-sepha ose beads a
4
◦
C. A e h ee washes, samples we e boiled in educing sample bu e and subjec ed
o gel elec opho esis on Bis-T is NuPAGE gels (The mo Fishe Scien i ic). P o eins we e
ans e ed o ni ocellulose memb anes (GE Heal hca e, Munich, Ge many) and mem-
b anes we e blocked wi h bo ine se um albumin (Sigma-Ald ich) in TBST (5%, w/ ). The
polyclonal an i-Gnly an ibody was hen used o de ec ion wi h HRP-conjuga ed donkey
an i-goa IgG an ibodies (Abcam) and ECL chemiluminescence eagen s and Hype Film
(GE Heal hca e).
2.6. Gene a ion o Ex acellula Vesicles o P o eome Analyses
To compa e ex acellula esicles om T and NK-LGLL cells, we ocused on NKL and
MOTN-1. Fi s , 4
×
10
8
cells we e washed wice wi h PBS be o e expansion and cul i a ion
o 72 h in 100 mL exosome- educed cul u e medium in he p esence o IL-2. To deple e
exosomes, RPMI 1640 medium supplemen ed wi h 20% FCS and penicillin/s ep omycin
was cen i uged a 4
◦
C and 100,000
×
g(Beckman XL-80 cen i uge, SW 32.1 Ti o o ,
Cells 2024,13, 1310 5 o 21
K e eld, Ge many) o 18 h. Fo NKL cell cul u es, he EV-deple ed supe na an was il e ed
and u he dilu ed wi h se um- ee RPMI 1640 medium con aining penicillin/s ep omycin
o a inal concen a ion o 10% FCS. Pa icle concen a ions in exosome-deple ed cul u e
media we e occasionally es ed ia nanopa icle acking analysis, as de ailed be o e [33].
Ex acellula esicles (EVs) we e isola ed by di e en ial cen i uga ion and ul acen-
i uga ion as desc ibed in [
33
]. B ie ly, cul u e supe na an s o NKL o MOTN-1 cells
kep in EV- educed medium we e cen i uged o 10 min a 300
×
g, 30 min a 2000
×
g
and o 45 min a 10,000
×
g o emo e in ac cells and cell deb is. EVs we e pelle ed
by ul acen i uga ion a 100,000
×
g o 1.5 h, washed once wi h il e ed (0.1 nm) PBS a
100,000
×
g o 1.5 h, and esuspended in 0.3 mL il e ed PBS. EVs’ size and concen a ion
we e de e mined ia nanopa icle acking analysis (NTA) wi h a NanoSigh 300 (NS300)
using NanoSigh so wa e e sion 3.40 (Mal e n Panaly ical, Mal e n, UK).
2.7. LC-MS-Based P o eome Analysis
EV samples we e p o ided in 100
µ
L PBS bu e , and SDS was added o a inal
concen a ion o 1% w/ . Samples we e lysed in a Bio up o Pico (Diagenode, San Diego,
CA, USA) a 4
◦
C (10
×
30 s on, 30 s o ) and 20
µ
L was used o BCA analysis (Pie ce
BCA P o ein Assay Ki , The mo Fishe Scien i ic) acco ding o he manu ac u e ’s p o ocol
(MOTN-1: 0.255
µ
g/
µ
L, NKL: 0.224
µ
g/
µ
L). Pe sample, 10
µ
g p o ein was subjec ed o
educ ion and alkyla ion (12 mM T is(2-ca boxye hyl)phosphine, 40 mM 2-chlo oace amide
o 1 h a
25 ◦C)
and desal ing pe o med acco ding o he SP3 p o ocol [
34
]. SP3 beads
we e added o each sample (1:20 w/wp o ein o beads a io) and e hanol was added o
50% / o induce p o ein binding (15 min a 25
◦
C). Beads we e immobilized using a
magne ic ack, he supe na an s we e disca ded, and he beads washed h ee imes wi h
80% / e hanol. The pelle s we e esuspended wi hin 50 mM TEAB bu e (pH 8.5) wi h
0.2
µ
g ypsin (
1:50 enzyme
o p o ein a io, P omega, Madison, WI, USA), incuba ed o
16 h a 37
◦
C, and he supe na an s we e acidi ied using i luo oace ic acid (TFA) o
LC-MS/MS measu emen .
Samples we e injec ed in iplica e on a Dionex Ul ima e 3000 nano-UHPLC coupled
o a Q Exac i e mass spec ome e (The mo Fishe Scien i ic). Pe injec ion, 1
µ
g p o ein
was loaded on o a ap column (Acclaim Pepmap 100 C-18, 5 mm
×
300
µ
m, 5
µ
m,
100 Å,
Dionex, Sunny ale, CA, USA) and washed o 5 min wi h 3% ACN/0.1% TFA a a low a e
o 30
µ
L/min p io o pep ide sepa a ion using an Acclaim PepMap 100 C-18 analy ical
column (50 cm
×
75
µ
m, 2
µ
m, 100 Å, Dionex). A low a e o 300 nL/min using eluen
A (0.05% o mic acid (FA)) and eluen B (80% ACN/0.04% FA) was used o g adien
sepa a ion (5–50% eluen B). A sp ay ol age o 1.5 kV was applied ia a liquid junc ion
emi e (MS Wil Fused silica emi e , CoAnn Technologies LLC, Richland, WA, USA), wi h
a sou ce empe a u e o 250
◦
C. Full scan MS spec a we e acqui ed be ween 300 and
1800 m/za a esolu ion o 70,000 a m/z200, and op en mos in ense p ecu so ions
we e selec ed o MS/MS analysis (cha ge s a e:
≥
+2, isola ion window:
±
1.5 m/z, HCD
agmen a ion: 27 NCE). MS/MS spec a we e acqui ed a a esolu ion o 17,500 wi h ixed
i s mass a m/z100, lock mass enabled (m/z445.120025), and a dynamic exclusion lis o
40 s.
MS aw iles we e p ocessed by P o eome Disco e e ( e sion 2.2.0.388, The mo
Fishe Scien i ic) using he Seques HT algo i hm agains a human p o ein da abase wi h
addi ional common con amina ing p o eins (only e iewed sequences) om he UniP o
da abase [
35
]. The enzyma ic p ocessing was se o ypsin ( ull-speci ic); maximum
missed clea age e en s: 2; minimal pep ide leng h: 6 AA; p ecu so mass ole ance:
10 ppm;
agmen mass ole ance: 0.02 Da; and alse-disco e y a e se o q = 0.01 by a
Pe cola o node [
36
]. Label- ee quan i ica ion was pe o med by a Mino a ea u e de ec o
wi h a subsequen ea u e mappe (minimum ace leng h: 5; maximum RT shi : 4 min;
mass ole ance: 10 ppm; pa ame e uning: ine). Th ee echnical injec ions we e used
as eplica es o he quan i ica ion s a egy and da a il e ed o high-con idence p o eins
wi h a minimum o wo pep ides/one unique pep ide iden i ica ion. Abundance alues

Cells 2024,13, 1310 6 o 21
we e median no malized and s a is ical es ing was pe o med by a wo-sided Welch - es
wi h Benjamini-Hochbe g FDR calcula ion (4e = 0.01) in Pe seus [
37
]. GO anno a ions
we e added and 1D en ichmen analysis and Fishe ’s exac es we e pe o med wi h a
5% FDR le el. All p o eomics aw da a ha e been uploaded o he P o eomeXchange
Conso ium [38] ia he PRIDE pa ne eposi o y wi h he da ase iden i ie PXD053228.
3. Resul s
3.1. Pheno ypic Peculia i ies o LGLL Cell Lines Analyzed by Flow Cy ome y
I was epo ed ha LGLL cells display a cha ac e is ic su ace deco a ion o lineage-
speci ic ma ke s [
7
] and a cons i u i e exp ession o unc ionally dys egula ed FasLigand
(FasL, CD95L, CD178) and Fas (CD95) [
15
,
16
]. Howe e , acco ding o ou analyses, he ou
well-cha ac e ized cell lines (NK-92, NKL as NK-LGLL, KHYG-1 as ANKL and MOTN-1
as T-LGLL) used in he p esen s udy did no display any (memb ane) mFasL, bu high
amoun s o CD26 (DPP4), which we had p e iously associa ed wi h he deg anula ion o
lysosome- ela ed e ec o esicles (LREVs) [31] (Figu e 1).
To analyze he p esence o cy o oxic e ec o molecules, we pe meabilized he cells o
in acellula s aining o g anzymes (G z) A and B and pe o in (P ). We used PE-conjuga ed
monoclonal an ibodies as speci ied be o e o espec i e iso ype con ol an ibodies (Figu e 1).
The wo NK-LGLLs, NK-92 and NKL, we e posi i e o all e ec o s, wi h a sligh ly highe
luo escence in ensi y o G zA in NKL. In KHYG-1, we de ec ed G zA and P , bu no G zB.
As expec ed om he cha ac e iza ion o MOTN-1, no showing any cy o oxic ac i i y [
23
],
we did no de ec any o he es ed e ec o p o eins by in acellula s aining (Figu e 1).
3.2. Analysis o G anulysin and G anzyme B in En iched LREVs
We ha e shown be o e ha g anulysin (Gnly) species migh be used as ma ke s o
dis inc en i ies o lysosome- ela ed e ec o esicles (LREVs) since, in un ans o med T-cell
blas s, hey seg ega e o di e en sec e o y compa men s and become mobilized by ei he
classical o non-classical deg anula ion [
28
]. In o de o cha ac e ize he dis ibu ion o
LREVs in LGLL cells, we hus analyzed en iched LREVs [
32
] o he p esence o Gnly, G zB,
and LAMP-1 (Figu e 2). To ou su p ise, he Wes e n blo esul s we e qui e he e ogeneous
and indica ed a di e en ial dis ibu ion o LREVs ca ying indi idual e ec o molecules. In
all cells, we de ec ed LAMP-1 as a ma ke o lysosomes in he comple e lysosomal ac ion
and in all indi idual ac ions a e densi y g adien and ul acen i uga ion. No ably,
in NK-92 cells, mos LAMP-1 was de ec ed in ac ions 3 and 4, whe eas in he o he
p epa a ions, mo e LAMP-1 was de ec ed in ac ions 2 and 3. The dis ibu ion o G zB
and Gnly was qui e di e en in he indi idual cell lines. G zB was no de ec ed in MOTN-1
bu was p esen in he c ude lysosomal ac ions o he wo NK-LGLLs and he ANKL
cell lines. Howe e , he subcellula dis ibu ion o G zB di e ed in NK-92 and NKL o
KHYG-1. Whe eas in NK-92, G zB was en iched in hea y ac ions and especially in
ac ion 6 esicles, i was much less in ense in KHYG-1 and ha dly de ec able in NKL
LREVs. Fo Gnly, he pa e ns seen in NK-92 co esponded o wha has been desc ibed o
un ans o med T cells [
28
]. The 9 kDa Gnly, ep esen ing he ma u e o m, was de ec ed
p ima ily in hea ie ac ions associa ed wi h G zB, whe eas he 15 kDa o m was mo e
abundan in he ligh e ac ions 1 o 3. No ably, bo h Gnly species we e s ained in he c ude
lysosomal ac ion o all ou cell lines. In e es ingly, in KHYG-1 and in NKL, al hough in
dis inc ac ions, bo h 9 and 15 kDa Gnly we e associa ed wi h G zB. In NKL, KHYG-1
and MOTN-1, 9 kDa Gnly, howe e , was p ima ily associa ed wi h he ligh e ac ions 1
o 4. Gi en he high simila i y in p o ein dis ibu ion in LREVs om un ans o med cells,
hese esul s migh indica e ha subcellula so ing and dis ibu ion o e ec o p o eins a e
al e ed in LGLL cells.
Cells 2024,13, 1310 7 o 21
Cells 2024, 13, x FOR PEER REVIEW 7 o 24
Figu e 1. Exp ession o FasL, CD26 and cy o oxic e ec o p o eins on/in LGLL cells. Analysis o
su ace FasL and CD26 and in acellula G zA, G zB and P on/in NK-92 (A), NKL (B), KHYG-1
(C) and MOTN-1 (D). Fi s , 105 cells we e s ained di ec ly wi h PE-conjuga ed mAb agains FasL
(NOK1, IgG1) and CD26 (BA5b, IgG2a) o a e pe meabiliza ion wi h Cy o ix/Cy ope m, wi h PE-
conjuga ed mAb agains G zA (CB9, IgG2b), G zB (QA16A02, IgG1), and P (dG9, IgG2b) o wi h
Figu e 1. Exp ession o FasL, CD26 and cy o oxic e ec o p o eins on/in LGLL cells. Analysis o
su ace FasL and CD26 and in acellula G zA, G zB and P on/in NK-92 (A), NKL (B), KHYG-1
(C) and MOTN-1 (D). Fi s , 10
5
cells we e s ained di ec ly wi h PE-conjuga ed mAb agains FasL
(NOK1, IgG1) and CD26 (BA5b, IgG2a) o a e pe meabiliza ion wi h Cy o ix/Cy ope m, wi h
PE-conjuga ed mAb agains G zA (CB9, IgG2b), G zB (QA16A02, IgG1), and P (dG9, IgG2b) o
wi h espec i e iso ype con ol an ibodies. S ained cells we e analyzed a e ixa ion on a BD FACS
Can o low cy ome e . One ep esen a i e expe imen ou o h ee is shown.
Cells 2024,13, 1310 8 o 21
Cells 2024, 13, x FOR PEER REVIEW 9 o 24
Figu e 2. Cy o oxic e ec o p o eins in LGLL cells. Analysis o in e nal G zB and Gnly in NK-92 (A),
NKL (B), KHYG-1 (C) and MOTN-1 (D). Cells we e mildly homogenized wi h a balch homogenize
and subjec ed o di e en ial and densi y cen i uga ion as desc ibed. Whole cell lysa e (WCL), en-
iched o ganelles (EO), c ude lysosomal ac ion (CLF), and he cy osol (CYT) we e sepa a ed o-
ge he wi h ac ions 1–6 o he densi y g adien on Bis-T is NuPAGE gels, ans e ed o ni ocel-
lulose, and es ed o he p esence o LAMP-1 and G zB wi h espec i e mab om BD Bioscience
and BioLegend and HRP-conjuga ed an i-mouse IgG an ibodies om Cy i a. Gnly was s ained wi h
a polyclonal goa an i-Gnly an ibody om R&D ollowed by HRP-conjuga ed an i-goa IgG an i-
bodies om Abcam.
3.3. Imaging o he In acellula Dis ibu ion o Cy o oxic E ec o P o eins
We nex employed imaging low cy ome y o analyze he subcellula localiza ion o
LAMP-1, G zB, and Gnly in NK-92 and NKL cells (Figu es 3 and 4). No ably, almos all
cells s ain posi i e o LAMP-1 (NK-92 97.7%, NKL 99.4%), and 97.1% o NK-92 cells and
97.3% o NKL cells s ain posi i e o G zB. To di e en ia e be ween he 15 and 9 kDa o ms
o Gnly, we employed es ablished Gnly de ec ion eagen s wi h di e en ial binding p op-
e ies. The monoclonal an ibody (mab) RF10 exclusi ely binds o he ull-leng h 15 kDa
a ian , whe eas he polyclonal an ibody (pab) e med pc almos exclusi ely ecognizes
he 9 kDa o m in PFA- ixed samples [28,39]. The 15 kDa a ian o Gnly is exp essed by
only 3.2% o NK-92 cells and 83.1% o NKL cells, whe eas he 9 kDa o m is ound in 28.7%
o NK-92 cells and 54.3% o NKL cells. We also quan i ied he deg ee o colocaliza ion o
cy o oxic e ec o p o eins wi h LAMP-1 in double-posi i e cells in NK-92 (Figu e 3) and
NKL (Figu e 4) cells.
Figu e 2. Cy o oxic e ec o p o eins in LGLL cells. Analysis o in e nal G zB and Gnly in
NK-92
(A),
NKL (B), KHYG-1 (C) and MOTN-1 (D). Cells we e mildly homogenized wi h a balch homogenize
and subjec ed o di e en ial and densi y cen i uga ion as desc ibed. Whole cell lysa e (WCL), en iched
o ganelles (EO), c ude lysosomal ac ion (CLF), and he cy osol (CYT) we e sepa a ed oge he wi h
ac ions 1–6 o he densi y g adien on Bis-T is NuPAGE gels, ans e ed o ni ocellulose, and es ed
o he p esence o LAMP-1 and G zB wi h espec i e mab om BD Bioscience and BioLegend and
HRP-conjuga ed an i-mouse IgG an ibodies om Cy i a. Gnly was s ained wi h a polyclonal goa
an i-Gnly an ibody om R&D ollowed by HRP-conjuga ed an i-goa IgG an ibodies om Abcam.
3.3. Imaging o he In acellula Dis ibu ion o Cy o oxic E ec o P o eins
We nex employed imaging low cy ome y o analyze he subcellula localiza ion
o LAMP-1, G zB, and Gnly in NK-92 and NKL cells (Figu es 3and 4). No ably, almos
all cells s ain posi i e o LAMP-1 (NK-92 97.7%, NKL 99.4%), and 97.1% o NK-92 cells
and 97.3% o NKL cells s ain posi i e o G zB. To di e en ia e be ween he 15 and 9 kDa
o ms o Gnly, we employed es ablished Gnly de ec ion eagen s wi h di e en ial binding
p ope ies. The monoclonal an ibody (mab) RF10 exclusi ely binds o he ull-leng h 15 kDa
a ian , whe eas he polyclonal an ibody (pab) e med pc almos exclusi ely ecognizes
he 9 kDa o m in PFA- ixed samples [
28
,
39
]. The 15 kDa a ian o Gnly is exp essed by
only 3.2% o NK-92 cells and 83.1% o NKL cells, whe eas he 9 kDa o m is ound in 28.7%
o NK-92 cells and 54.3% o NKL cells. We also quan i ied he deg ee o colocaliza ion o
cy o oxic e ec o p o eins wi h LAMP-1 in double-posi i e cells in NK-92 (Figu e 3) and
NKL (Figu e 4) cells.
As expec ed, LAMP-1 is loca ed in g anula s uc u es wi hin bo h NK-92 and NKL
cells and, as a posi i e con ol, nea ly all cells display a b igh de ail simila i y sco e >2
o he colocaliza ion o FITC- and PE-labeled an i-LAMP-1 ab. Su p isingly, he cy o oxic
e ec o p o ein G zB ha dly colocalizes wi h LAMP-1 since less han 10% o double-posi i e
NKL and ha dly any NK-92 cells show a BDS sco e >2 o he G zB/LAMP-1 cos aining. In
NK-92 cells ha exp ess bo h he 15 and he 9 kDa o m o Gnly, only abou 10% o double-
posi i e cells s o e 9 kDa Gnly in LAMP-1-posi i e in acellula g anula, whe eas 44% o
he cells display a BDS >2 o RF10/LAMP-1-cos ainings, indica ing a s o age o
15 kDa
Gnly in LAMP-1
+
LREVs (Figu e 3). No ably, in Gnly
+
NKL cells, bo h he
15 and 9 kDa
o m colocalize wi h LAMP-1, wi h he 15 kDa Gnly/LAMP-1 colocaliza ion being mo e
p onounced (Figu e 4). Thus, compa ed o un ans o med cy o oxic CD8
+αβ
-TCR
+
and
o
γδ
-TCR
+
T cells whe e g anzymes and he 9 kDa Gnly bu usually no he 15 kDa Gnly
associa e wi h in acellula LAMP-1
+
g anula s uc u es, he LGLL cells analyzed he e
appa en ly ha e a mo e he e ogeneous and al e ed e ec o p o ein s o age machine y.
Cells 2024,13, 1310 9 o 21
Cells 2024, 13, x FOR PEER REVIEW 10 o 24
Figu e 3. Colocaliza ion o in acellula LAMP-1 wi h G zB and he 15 kDa (RF10) o 9 kDa (pc)
o m o Gnly in NK-92 cells. Following ixa ion and pe meabiliza ion, cells we e s ained wi h FITC-
conjuga ed an i-LAMP-1 mab. A e washing, cells we e addi ionally s ained wi h PE-conjuga ed
an i-LAMP-1 mab (as a posi i e con ol o colocaliza ion), PE-conjuga ed an i-G zB mab, o wi h
an i-Gnly mab RF10 o a polyclonal an i-Gnly pab (pc) and app op ia e Alexa Fluo 555-conjuga ed
seconda y an ibodies. A o al o 10,000 cells we e acqui ed wi h an ImageS eam Ma k II imaging
low cy ome e . Only ocused, single cells we e conside ed o u he analyses. (A) His og ams dis-
play he geome ic mean alue o he BDS sco e o espec i e s ainings and he pe cen age o cells
displaying a BDS sco e >2. (B) Rep esen a i e images o s ained cells. Scale ba s ep esen 7 μm.
As expec ed, LAMP-1 is loca ed in g anula s uc u es wi hin bo h NK-92 and NKL
cells and, as a posi i e con ol, nea ly all cells display a b igh de ail simila i y sco e >2 o
he colocaliza ion o FITC- and PE-labeled an i-LAMP-1 ab. Su p isingly, he cy o oxic
e ec o p o ein G zB ha dly colocalizes wi h LAMP-1 since less han 10% o double-pos-
i i e NKL and ha dly any NK-92 cells show a BDS sco e >2 o he G zB/LAMP-1 cos ain-
ing. In NK-92 cells ha exp ess bo h he 15 and he 9 kDa o m o Gnly, only abou 10%
o double-posi i e cells s o e 9 kDa Gnly in LAMP-1-posi i e in acellula g anula,
whe eas 44% o he cells display a BDS >2 o RF10/LAMP-1-cos ainings, indica ing a s o -
age o 15 kDa Gnly in LAMP-1+ LREVs (Figu e 3). No ably, in Gnly+ NKL cells, bo h he
15 and 9 kDa o m colocalize wi h LAMP-1, wi h he 15 kDa Gnly/LAMP-1 colocaliza ion
being mo e p onounced (Figu e 4). Thus, compa ed o un ans o med cy o oxic CD8+ αβ-
TCR+ and o γδ-TCR+ T cells whe e g anzymes and he 9 kDa Gnly bu usually no he 15
kDa Gnly associa e wi h in acellula LAMP-1+ g anula s uc u es, he LGLL cells ana-
lyzed he e appa en ly ha e a mo e he e ogeneous and al e ed e ec o p o ein s o age ma-
chine y.
Figu e 3. Colocaliza ion o in acellula LAMP-1 wi h G zB and he 15 kDa (RF10) o 9 kDa (pc)
o m o Gnly in NK-92 cells. Following ixa ion and pe meabiliza ion, cells we e s ained wi h FITC-
conjuga ed an i-LAMP-1 mab. A e washing, cells we e addi ionally s ained wi h PE-conjuga ed
an i-LAMP-1 mab (as a posi i e con ol o colocaliza ion), PE-conjuga ed an i-G zB mab, o wi h
an i-Gnly mab RF10 o a polyclonal an i-Gnly pab (pc) and app op ia e Alexa Fluo 555-conjuga ed
seconda y an ibodies. A o al o 10,000 cells we e acqui ed wi h an ImageS eam Ma k II imaging
low cy ome e . Only ocused, single cells we e conside ed o u he analyses. (A) His og ams
display he geome ic mean alue o he BDS sco e o espec i e s ainings and he pe cen age o cells
displaying a BDS sco e >2. (B) Rep esen a i e images o s ained cells. Scale ba s ep esen 7 µm.
Cells 2024, 13, x FOR PEER REVIEW 11 o 24
Figu e 4. Colocaliza ion o in acellula LAMP-1 wi h G zB and he 15 kDa (RF10) o 9 kDa (pc)
o m o Gnly in NKL cells. Samples we e p ocessed as de ailed in he legend o Figu e 3. (A) His o-
g ams display he geome ic mean alue o he BDS sco e o espec i e s ainings and he pe cen age
o cells displaying a BDS sco e >2. (B) Rep esen a i e images o s ained cells. Scale ba s ep esen 7
μm.
3.4. Di e en ial Ac i a ion-Induced Release o 9 o 15 kDa G anulysin
We p e iously epo ed ha in un ans o med T cells, indi idual LREVs con aining
ei he 15 kDa o 9 kDa Gnly u ilize di e en ou es o mobiliza ion and elease e e ed
o as Ca2+-independen non-classical and Ca2+-dependen classical deg anula ion, espec-
i ely [40]. Gi en he he e ogenous and al e ed e ec o p o ein s o age pa e ns in LGLL
cells, we nex analyzed he signal equi emen s o he elease o he wo o ms o Gnly.
The elease o Gnly in o cell cul u e supe na an s was add essed by immunop ecipi a ion
and Wes e n blo ing a e s imula ion wi h pho boles e (TPA, non-classical deg anula-
ion) o pho boles e and calcium ionopho e (TPA/ionomycin, classical deg anula ion) in
he absence o p esence o he Ca2+ chela o EGTA (Figu e 5).
We used he polyclonal an i-Gnly an ibody (pc) as a p ima y an ibody o Gnly de-
ec ion in Wes e n blo s a e immunop ecipi a ion o Gnly om supe na an s o de e -
mine which o m o Gnly is sec e ed in o he cul u e supe na an upon s imula ion wi h
ei he pho bol es e and/o calcium ionopho e. NKL cells ha dly eleased any Gnly, al -
hough bo h o ms we e appa en ly exp essed, as e idenced by he Wes e n blo o whole
cell lysa es in Figu e 5A. In con as , we eadily p ecipi a ed Gnly wi h he polyclonal an i-
Gnly (pc) an ibody om cul u e supe na an s o uns imula ed o TPA- and TPA/ionomy-
cin-s imula ed NK-92 cells. TPA ac i a ion esul ed in an inc eased and selec i e elease
o he 15 kDa a ian in o he cul u e supe na an , whe eas TPA in combina ion wi h iono-
mycin also induced he elease o he 9 kDa o m. Impo an ly, his Ca2+-dependen elease
igge ed by TPA and ionomycin could be pa ially ab oga ed by Ca2+-chela ion wi h
EGTA. These esul s again highligh he mo e he e ogenous and al e ed s o age and e-
lease pa e ns o cy o oxic e ec o p o eins in LGLL cells.
Figu e 4. Colocaliza ion o in acellula LAMP-1 wi h G zB and he 15 kDa (RF10) o 9 kDa (pc) o m
o Gnly in NKL cells. Samples we e p ocessed as de ailed in he legend o Figu e 3. (A) His og ams
display he geome ic mean alue o he BDS sco e o espec i e s ainings and he pe cen age o cells
displaying a BDS sco e >2. (B) Rep esen a i e images o s ained cells. Scale ba s ep esen 7 µm.
3.4. Di e en ial Ac i a ion-Induced Release o 9 o 15 kDa G anulysin
We p e iously epo ed ha in un ans o med T cells, indi idual LREVs con aining
ei he 15 kDa o 9 kDa Gnly u ilize di e en ou es o mobiliza ion and elease e e ed
o as Ca
2+
-independen non-classical and Ca
2+
-dependen classical deg anula ion, espec-
i ely [
40
]. Gi en he he e ogenous and al e ed e ec o p o ein s o age pa e ns in LGLL
cells, we nex analyzed he signal equi emen s o he elease o he wo o ms o Gnly.
The elease o Gnly in o cell cul u e supe na an s was add essed by immunop ecipi a ion
and Wes e n blo ing a e s imula ion wi h pho boles e (TPA, non-classical deg anula ion)
o pho boles e and calcium ionopho e (TPA/ionomycin, classical deg anula ion) in he
absence o p esence o he Ca2+ chela o EGTA (Figu e 5).
Cells 2024,13, 1310 16 o 21
e ec o molecules and/o cy okines in o he bone ma ow, since he deg ee o ma ow
in il a ion wi h leukemic cells does no co ela e wi h he deg ee o cy openia in pe iphe al
blood [6].
G anzyme B and pe o in a e o en men ioned as addi ional ma ke s o asce ain LGLL
diagnosis, e.g., in his ological s aining o bone ma ow biopsies [
5
]. Fo g anulysin, ano he
key e ec o o T and NK cells, he dis ibu ion, elease, o impac in LGLL has no been
in es iga ed so a . We he e o e add essed he dis ibu ion and elease o LREV-associa ed
cy o oxic e ec o molecules in es ablished LGLL cell lines. P e iously, we compa ed
in acellula e ec o esicles om he pa ien -de i ed NK LGLL line NKL wi h hose om
he NK cell lymphoblas ic leukemia/lymphoma line YT and om un ans o med cells.
We epo ed he e ogeneous p o iles o unc ionally ele an p o eins (including cy o oxic
e ec o p o eins) in indi idual leukemic and ac i a ed human NK cell popula ions [
22
]. In
he p esen s udy, we ollowed his ou e and analyzed pa ien -de i ed LGLL cell lines wi h
a p ima y ocus on cy o oxic e ec o molecules. We chose he well-cha ac e ized T-LGLL
MOTN-1 [
23
], he NK-LGLL lines NKL [
19
] and NK-92 [
24
], and he ANKL KHYG-1 [
25
]
o ou compa a i e s udies. We analyzed he dis ibu ion o o he lysosome-associa ed
e ec o p o eins by con en ional and imaging low cy ome y o Wes e n blo ing. In
con as wi h FasL, high amoun s o dipep idyl pep idase 4 (DPP4, CD26) we e de ec ed on
all es ed LGLL cells. No ably, we ecen ly epo ed ha CD26 is no only a ansmemb ane
signal ansduce bu is also associa ed wi h LREVs and eleased by ac i a ion-induced
deg anula ion [
31
]. No su p isingly, in he p esen s udy, we also ound DPP4 in EVs o
bo h MOTN-1 and NKL cells (P27487, dipep idyl pep idase 4), highligh ing ha DPP4 is
eleased as an ac i e enzyme wi h ex acellula esicles om LGLL cells. Fu he s udies
migh add ess he ole o his dipep idase in disease de elopmen and pa hology [47].
To add ess he p esence o cy o oxic e ec o molecules in LGLL-de i ed cell lines, we
ini ially pe o med in acellula s aining o g anzymes (G zA and G zB) and Pe o in (P ).
The wo NK-LGLL lines NK-92 and NKL we e posi i e o bo h g anzymes and pe o in,
wi h a sligh ly highe luo escence in ensi y o G zA in NKL. In KHYG-1, we de ec ed
G zA and P , bu no G zB. As expec ed, MOTN-1, a T-LGLL p esen ing no cy o oxic
ac i i y [23], did no s ain o any o he es ed e ec o p o eins.
In p e ious analyses, he p ema u e 15 kDa and he ma u e 9 kDa species o Gnly
se ed as ma ke s o dis inc LREV en i ies [
28
]. We i s en iched LREVs by subcellu-
la ac iona ion and s ained he esul ing Wes e n blo s o he p esence o g anulysin,
g anzyme B, and LAMP-1. These analyses al eady poin ed o a di e en ial dis ibu ion
o LREVs ca ying indi idual e ec o molecules. In all cells, we de ec ed LAMP-1 as a
ma ke o lysosomes in he comple e lysosomal ac ion and in mos indi idual subcel-
lula ac ions wi h occasional quali a i e di e ences. The dis ibu ion o G zB and Gnly
p esen ed a he he e ogeneously. G zB was p esen in he c ude lysosomal ac ions o
bo h NK-LGLL and he ANKL cell line. Howe e , whe eas in NK-92, G zB was en iched in
hea y ac ions (i.e., ac ion 6 esicles), i was much less in ense in KHYG-1 and ha dly
de ec able in NKL LREVs. As expec ed, G zB was no de ec ed in LREVs om MOTN-1.
No ably, bo h Gnly species we e s ained in he c ude lysosomal ac ion o all ou cell
lines. The dis ibu ion obse ed in NK-92 co esponded o wha we desc ibed o un ans-
o med T cells [
28
]. The ma u e 9 kDa Gnly was p ima ily de ec ed in hea ie ac ions
associa ed wi h G zB, whe eas 15 kDa Gnly was mo e abundan in he ligh e ac ions
1 o 3.
In e es ingly, in NKL, KHYG-1 and MOTN-1, he ma u e Gnly was associa ed wi h
he ligh e ac ions 1 o 4 and in KHYG-1 and NKL, al hough p esen in dis inc ac ions,
bo h 9 and 15 kDa Gnly we e associa ed wi h G zB. Gi en he epo ed high simila i y in
p o ein dis ibu ion in LREVs om un ans o med cells, hese esul s indica e ha subcel-
lula p o ein so ing and dis ibu ion o e ec o p o eins migh be al e ed in ans o med
LGLL cells. These appa en di e ences a e also e lec ed by he analyses o in acellula
p o ein s o age ia imaging low cy ome y. I is well es ablished ha in un ans o med
cy o oxic lymphocy es e ec o p o eins including g anzymes, pe o in and 9 kDa g an-
ulysin a e sa ely s o ed in in acellula sec e o y g anules whe e hey colocalize wi h he

Cells 2024,13, 1310 17 o 21
lysosomal ma ke p o ein LAMP-1 [
27
,
28
,
40
]. In e es ingly, G zB ha dly colocalizes wi h
LAMP-1 in bo h NKL and NK-92 cells. Mo eo e , in NK-92 cells, 9 kDa is ha dly de ec able
in LAMP-1-posi i e in acellula g anula, whe eas in con as wi h un ans o med cells,
15 kDa
Gnly is s o ed in in acellula lysosomal s uc u es in a subs an ial numbe o cells.
Al hough he o e all Gnly exp ession is much lowe in NKL cells, Gnly
+
cells seem o
s o e bo h 15 and 9 kDa in sec e o y g anules. Thus, compa ed o un ans o med cy o oxic
lymphocy es, bo h NKL and NK-92 cells migh display an al e ed e ec o p o ein so ing
o s o age machine y.
4.2. Di e en ial Ac i a ion-Induced Release o 9 o 15 kDa G anulysin
We epo ed be o e ha in un ans o med T cells, indi idual LREVs con aining ei he
15 kDa o 9 kDa Gnly u ilize ei he Ca
2+
-independen non-classical o Ca
2+
-dependen
classical deg anula ion o hei mobiliza ion and elease [
40
]. In iew o he al e ed LREV
s o age pa e ns in LGLL cells, we in es iga ed signal equi emen s o he elease o he
wo Gnly o ms om NKL and NK-92. We analyzed he elease in o cul u e supe na an s
by immunop ecipi a ion and Wes e n blo ing a e s imula ion wi h TPA o igge non-
classical o TPA/ionomycin o igge classical deg anula ion. Immunop ecipi a es wi h he
polyclonal an i-Gnly an ibody om cell lysa es o NKL and NK-92 e ealed ha he o al
le els o bo h o ms o in acellula Gnly migh be compa able. In e es ingly, we ound
ha NKL cells ha dly elease any Gnly upon s imula ion wi h TPA and/o ionomycin. In
con as , TPA ac i a ion esul ed in a selec i e elease o he 15 kDa a ian om NK-92
cells, whe eas TPA and ionomycin induced he elease o bo h Gnly o ms. Impo an ly,
his Ca
2+
-dependen elease o 9 kDa Gnly igge ed by TPA/ionomycin was educed by
Ca
2+
chela ion wi h EGTA. These esul s again highligh he mo e he e ogenous and al e ed
s o age and elease pa e ns o cy o oxic e ec o p o eins in LGLL cells.
4.3. P o eomic P o iling o LGLL-De i ed Ex acellula Vesicles
The MS-based p o eomic cha ac e iza ion o EV samples is gene ally challenging,
due o he high numbe o expec ed in eg al memb ane p o eins and he limi ed sample
amoun s. While he dep h o analysis, i.e., he numbe o p o ein iden i ica ions is depen-
den on mul iple ac o s, such as he cell ype, he EV elease o he en ichmen me hod, he
p esen ed da ase wi h 1742 iden i ied and 1174 quan i ied p o eins p o ides a compa able
size o o he publica ions in he ield [
48
,
49
]. No ably, o ou compa a i e analysis o T-
and NK-LGLL-de i ed ex acellula esicles, we collec ed cons i u i ely eleased pa icles
om MOTN-1 and NKL cells o e a cul u e pe iod o 72 h in exosome- educed medium.
The isola ed EVs p esen ed as homogeneous popula ions o small- o medium-size esicles
o 170–200 nm in diame e in NTA analyses. In e es ingly, bo h cell ypes eleased compa-
able amoun s o esicles o simila size, esul ing in compa able sample ma e ial o he
subsequen MS analyses.
The s a is ical es ing e ealed ha mo e han hal o he iden i ied p o eins (660)
p o ided no signi ican abundance di e ence in he EV p epa a ions o MOTN-1 and NKL,
while 186 p o eins (119 s ic and 67 mode a e) p esen ed signi ican ly highe abundance
le els in MOTN-1-de i ed EV and 263 p o eins (173 s ic and 90 mode a e) highe le els
in NKL-de i ed EV. Mo eo e , 44 p o eins we e exclusi ely iden i ied in MOTN-1 EV and
21 p o eins exclusi ely in NKL-de i ed esicles (see Supplemen a y Table S2 o indi idual
p o ein anno a ions).
A o al o 766 (43.9%) o he iden i ied p o eins we e associa ed wi h he GO e m
“ex acellula exosomes” (GO:0070062). These included common ma ke p o eins o ming
he co e p o eome o small ex acellula esicles/exosomes such as he p o eins in ol ed
in exosome biogenesis including TSG101 o he p og ammed cell dea h 6-in e ac ing p o-
ein/Alix and syn enin-1, which was ecen ly p oposed as a pu a i e uni e sal bioma ke
o exosomes [
49
]. No ably, in line wi h ou p e ious analyses o T o NK cell-de i ed exo-
somes, we iden i ied he e aspanins CD63 and CD81, bu no CD9 in EVs om MOTN-1
o NKL. Howe e , se e al Rab GTPases, which a e commonly associa ed wi h lysosomal
Cells 2024,13, 1310 18 o 21
a icking and EV o ma ion (i.e., Rab5B, Rab7A, Rab11B and Rab27A) and he lysosome-
associa ed memb ane p o eins LAMP-1 (CD107a) and LAMP-2 (CD107b), we e ound
in bo h EV popula ions. He e, LAMP-1 showed inc eased abundance le els in MOTN-1
samples, whe eas no signi ican di e ence was obse ed o LAMP2. O he cha ac e is ic
and commonly EV-associa ed p o eins such as lo illins, annexins, hea shock p o eins, cell
adhesion molecules o in eg ins (i.e., in eg in
β
1), CD26/DPP4, CD47, and ADAM p o-
eases we e also de ec ed in compa able abundances in EVs om bo h NKL and MOTN-1.
Fo hese p o ein amilies, a equen associa ion o en ichmen in exosomes om di e en
cellula sou ces has been documen ed [49].
Unexpec edly, 2B4 (CD244 [
50
]) and 2DL4 (CD158d [
51
]), wo molecules which ha e
been desc ibed as ac i a ing o inhibi ing NK cell ecep o s, we e iden i ied in he EVs o
bo h NKL and MOTN-1 wi hou signi ican di e ences in abundance, while he inhibi o y
ecep o LILRB1 [
52
] was de ec ed wi h a highe abundance le el in NKL-de i ed esicles.
Impo an ly, p o eins wi h highe abundance le els in NKL-de i ed EVs also included
he men ioned cy o oxic e ec o p o eins G zA, G zB and P . This is in line wi h ou own
analyses and co esponds o he cy o oxic po en ial o his NK-LGLL cell line. In con as ,
MOTN-1-de i ed EVs p esen ed he T-cell su ace glycop o ein CD4 as an indica o o he
o iginal T-cell lineage and he TNF- ecep o 2 in highe abundance. Also, he dea h ecep-
o Fas (CD95) was de ec ed in EVs o bo h cell lines; howe e , i s p esence signi ican ly
inc eased in he MOTN-1-de i ed esicles. In e es ingly, G zK was exclusi ely iden i ied
in EV o MOTN-1. This g anzyme is usually p esen in g anules o NK cells and cy o oxic T
lymphocy es and supposedly ope a es as a cy o oxic p o-apop o ic se ine p o ease owa ds
o eign, in ec ed, o malignan cells. Howe e , nume ous o he in acellula and ex acellu-
la oles o G zK ha e been iden i ied since i s ini ial cloning as g anzyme
3 in 1995 [53,54].
G zK is he only o he yp ase wi hin he g anzyme amily besides G zA and has long
been ega ded as a edundan eplacemen o G zA. No ably, G zK is ac i a ed by emo -
ing a signal dipep ide which di ec s he p e-p o-G zK o he endoplasmic e iculum (ER).
Upon ac i a ion by g anula ca hepsin (o dipep idyl-pep idases?), G zK clea es di e se
subs a es ha we e also iden i ied in EVs om LGLL cells, including nucleosome assembly
p o ein, he e ogeneous nuclea ibonucleop o ein (hnRNP) K,
β
- ubulin, and
α
- ubulin.
Simila o he o he g anzymes, howe e , he “ adi ional” ole o G zK is deba ed and
ex acellula unc ions o G zK in p omo ing in lamma ion and in ec ions a e eme ging
(see [
54
] o a e iew). As men ioned, G zK was exclusi ely iden i ied in MOTN-1 esicles,
which in u n con ained much less G zA, G zB, and P compa ed o NKL-de i ed EV. In
essence, MOTN-1 ep esen s a CD4
+
T-LGLL ha does no display signi ican cy o oxici y
while ca ying an a ypical se o g anula e ec o molecules.
Along his line, when GO e ms we e linked o each indi idual p o ein, MOTN-1 EVs
we e signi ican ly en iched o GO e ms such as p o ein olding and an igen p esen a ion.
In e es ingly, p o eins associa ed wi h memb anes (ex e nal side o plasma memb ane,
in insic o endoplasmic e iculum memb ane, in eg al o o ganelle memb ane) we e
also inc eased in MOTN-1. In con as , in EVs o NKL, he GO e ms p o ein–DNA
complex and nucleosome we e en iched, poin ing o DNA-in e ac ing p o eins such as
his ones. Mo eo e , e ms a ibu ed o he cy oskele on and o cell mo ili y we e en iched
in NKL samples.
In conclusion, as p e iously epo ed o in acellula LREVs om di e en NK cell
lines and ac i a ed NK cells [
22
], ou da a collec ion poin s o a clea ly clono ypic dis ibu-
ion o dis inc e ec o molecules in indi idual LGLL cell lines.
5. Conclusions
La ge g anula lymphocy e leukemias (LGLL) o igina e om ma u e CD3
+
T e ec-
o memo y cells o CD3
−
NK cells and a e cha ac e ized by con aining la ge g anules
ha s o e and anspo cy o oxic e ec o molecules in e alia. LGLL cells equen ly
display uncon olled p oli e a ion and cy o oxici y, bo h esul ing in ch onic malignancy
and au oimmuni y. We demons a ed ha indi idual pa ien -de i ed cell lines o di e -
Cells 2024,13, 1310 19 o 21
en LGLL-sub ypes ca y o elease indi idual and clono ypic combina ions o e ec o
molecules including, e.g., g anzymes, pe o in, and g anulysin. Impo an ly, he indi-
idual dis ibu ion o e ec o s was also de ec ed in p o eome p o iles o cons i u i ely
eleased ex acellula esicles (EVs) o wo ep esen a i e LGLL cell lines. We conclude
ha LGLL-de i ed EVs migh be ega ded as aluable a ge s o he analysis o p o eins
ha con ibu e o LGLL pa hology in indi idual pa ien s and hus migh open new ou es
o indi idualized he apeu ic in e en ions.
Supplemen a y Ma e ials: The ollowing suppo ing in o ma ion can be downloaded a h ps://
www.mdpi.com/a icle/10.3390/cells13161310/s1, Supplemen a y_P o eomics_Tables, con aining
Supplemen a y Table S1—Lis o high-con idence p o ein iden i ica ions; Supplemen a y Table S2—
Resul s o s a is ical Welch - es wi h Benjamini-Hochbe g FDR, q = 0.01; Supplemen a y Table S3—
Resul s o Fishe ’s exac es wi h Benjamini-Hochbe g FDR, q = 0.05; Supplemen a y Table S4—Resul s
o 1D anno a ion en ichmen analysis wi h Benjamini-Hochbe g FDR, q = 0.05;
Supplemen a y Table S5
—
Resul so Fishe ’s exac es wi h Benjamini-Hochbe g FDR, q = 0.05 (selec ed EV p o eins).
Au ho Con ibu ions: Concep ualiza ion, O.J., M.L. and A.T.; me hodology, L.P., M.L. and P.K.;
alida ion, all au ho s; o mal analysis, L.P., M.L. and P.K.; esou ces, O.J. and A.T.; da a cu a ion, L.P.
and P.K.; w i ing—o iginal d a p epa a ion, O.J.; w i ing— e iew and edi ing, L.P., M.L., P.K. and
A.T.; isualiza ion, all au ho s; supe ision, O.J., M.L. and A.T.; unding acquisi ion, O.J. All au ho s
ha e ead and ag eed o he published e sion o he manusc ip .
Funding: This s udy was unded by he Ge man Resea ch Council (DFG) p ojec JA610/7-3 o O.J.
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : All p o eomics aw da a ha e been uploaded o he P o eomeXchange
Conso ium [38] ia he PRIDE pa ne eposi o y wi h he da ase iden i ie PXD053228.
Acknowledgmen s: P.K. and A.T.: DFG-Clus e o Excellence “P ecision Medicine in In lamma ion
(PMI)”, RTF-V.
Con lic s o In e es : The au ho s decla e no con lic s o in e es .
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