Di e se popula ions o T cells wi h NK cell
ecep o s accumula e in he human in es ine in
heal h and in colo ec al cance
Joan O’Kee e1,De ekG.Dohe y
2, Tony Kenna3,Kie anSheahan
4,Dia muidP.
O’Donoghue4,5,JohnM.Hyland
4and Cliona O’Fa elly3,5
1Depa men o Biochemis y, Na ional Uni e si y o I eland, Galway, I eland
2Ins i u e o Immunology, Na ional Uni e si y o I eland, Maynoo h, I eland
3Educa ion and Resea ch Cen e, S . Vincen ’s Uni e si y Hospi al, Dublin, I eland
4Cen e o Colo ec al Disease, S . Vincen ’s Uni e si y Hospi al, Dublin, I eland
5Conway Ins i u e o Biomedical & Biomolecula Resea ch, Uni e si y College Dublin, I eland
T cells exp essing NK cell ecep o s (NKR) display apid MHC-un es ic ed cy o oxici y and
po en cy okine sec e ion and a e hough o play oles in immuni y agains umo s. We ha e
quan i ied and cha ac e ized NKR+T cells eshly isola ed om epi helial and lamina p op ia
laye s o duodenum and colon om 16 indi iduals wi h no e idence o gas oin es inal dis-
ease and om umo and unin ol ed issue om 19 pa ien s wi h colo ec al cance . NKR+T
cell subpopula ions we e di e en ially dis ibu ed in di e en in es inal compa men s, and
CD161+T cells accoun ed o o e one hal o T cells a all loca ions es ed. Mos in es inal
CD161+T cells exp essed
§ g
TCR and ei he CD4 o CD8. Signi ican p opo ions exp essed
HLA-DR, CD69 and Fas ligand. Upon s imula ion in i o, CD161+T cells p oduced IFN-
+
and
TNF-
§
bu no IL-4. NKT cells exp essing he V
§
24V
g
11 TCR, which ecognizes CD1d, we e
i ually absen om he in es ine, bu colonic cells p oduced IFN-
+
in esponse o he NKT
cell agonis ligand
§
-galac osylce amide. NKR+T cells we e no expanded in colonic umo s
compa ed o adjacen unin ol ed issue. The p edominance, he e ogenei y and di e en ial
dis ibu ion o NKR+T cells a di e en in es inal loca ions sugges s ha hey a e cen al o
in es inal immuni y.
Key wo ds: Human / In es ine / T lymphocy es / NKT cells / Tumo immuni y
Recei ed 24/1/04
Re ised 25/3/04
Accep ed 14/5/04
[DOI 10.1002/eji.200424958]
Abb e ia ions:
>
-GalCe :
§
-Galac osylce amide LP: Lam-
ina p op ia NKR: NK cell ecep o
1 In oduc ion
A mino subse o bo h mu ine and human T cells
exp esses NK cell ecep o s (NKR) and displays unc-
ional cha ac e is ics o NK cells and classical T cells
[1–4]. NKR+T cells become ac i a ed apidly a e s imu-
la ion, a e capable o MHC-un es ic ed cy o oxici y in
i o and can sec e e la ge amoun s o cy okines includ-
ing IFN-
+
,TNF-
§
, IL-2 and IL-4 [5–7]. They accoun o
abou 5% o pe iphe al blood lymphocy es bu a e
en iched in li e and bone ma ow, accoun ing o
35–50% o lymphocy es in hese o gans [8, 9]. NKR can
ac as inhibi o y molecules on cloned T cells as shown
by in i o models in CD3-TCR media ed cy olysis [10,
11]. Recen ly, NKR+T cells ha ecoginize HLA-E mole-
cul es in associa ion wi h pep ides de i ed om MHC
class I molecules o i uses we e desc ibed [12]
Al hough he mechanisms ha con ol he exp ession o
NKR on T cells a e no well unde s ood, hese ecep o s
a e belie ed o unc ion as cos imula o y molecules o o
egula e e ec o T cell ac i i y [13–16].
In mice, he majo i y o NKR+T cells a e NKT cells. NKT
cells exp ess NK1.1 and an in a ian TCR
§
-chain,
V
§
14J
§
18 ( o me ly V
§
14J
§
281), which p e e en ially
pai s wi h a limi ed numbe o
g
chains and ecognizes
glycolipid an igens p esen ed by he MHC class I-like
p o ein CD1d [17–19]. The na u al ligand(s) o NKT cells
is unknown, bu hey espond o
§
-galac osylce amide
(
§
-GalCe ), an ex ac o he ma ine sponge Agelas mau-
i anius, in a CD1d- es ic ed manne [20, 21]. NKT cells
play key oles in immuni y agains umo s in some
mu ine models. Injec ion o mice wi h ei he IL-12 o
§
-
GalCe can esul in umo ejec ion by a mechanism ha
is dependen on IFN-
+
p oduc ion and/o an i- umo
cy o oxici y by NKT cells [22, 23]. Fu he mo e, mice
de icien in NKT cells ail o media e IL-12-induced ejec-
ion o umo s [24].
2110 J. O’Kee e e al. Eu . J. Immunol. 2004. 34: 2110–2118
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de
CD1d- es ic ed NKT cells exp essing in a ian
V
§
24J
§
18 TCR
§
-chains ( o me ly V
§
24J
§
Q) pai ed wi h
V
g
11 ha e been iden i ied in humans [25, 26], bu hey
a e ound in much lowe numbe s in blood (
˚
0.02% o
lymphocy es), li e and bone ma ow (
X
1%) compa ed
o mice [9, 27]. Howe e , human T cells exp essing a i-
ous NKR, including CD56, CD57, CD161, CD94 and
kille Ig-like ecep o s, ha e been epo ed o sha e unc-
ional simila i ies wi h in a ian NKT cells, such as apid
MHC-un es ic ed cy o oxici y and po en cy okine
sec e ion [3, 4, 7]. Human NKR+T cells a e also hough
o play oles in an i- umo immuni y [5, 28, 29].
The human gas oin es inal ac con ains se e al pheno-
ypically and unc ionally dis inc popula ions o T cells
[30–33]. Many o hese cells a e hough o play oles in
an i- umo immuni y. T cells isola ed om he epi helial
laye and he lamina p op ia (LP) o he human colon a e
cy o oxic and kill ca cinoma cell lines in i o [34, 35],
and T cell in il a ion in o umo issue is associa ed wi h
imp o ed p ognosis o colonic ca cinoma [36, 37].
Recen s udies ha e indica ed ha NKR+Tcellsa ealso
p esen in mu ine and human in es ine [38–40], bu o
da e no s udies ha e add essed he po en ial oles o
hese cells in in es inal cance .
We ha e pheno ypically and unc ionally cha ac e ized
NKR+T cells in he epi helial and LP laye s o non-
diseased human duodenum and colon and e alua ed
changes in hei numbe s and pheno ypes in pa ien s
wi h colo ec al cance . Ou esul s indica e ha dis inc
subse s o NKR+T cells, bu no in a ian NKT cells,
accumula e a di e en in es inal loca ions. Many o
hese cells exp ess Fas ligand and ma ke s o ac i a ion
and sec e e in lamma o y cy okines upon s imula ion in
i o. Colonic CD56+,CD57
+and CD161+T cells we e no
expanded in umo issue compa ed wi h adjacen unin-
ol ed issue.
2Resul s
2.1 Compa men aliza ion o NKR+T cells in
human in es ine
Flow cy ome ic analysis o single-cell suspensions p e-
pa ed om human colon and duodenum e ealed di e -
en ial dis ibu ion o NKR+T cell popula ions in he epi-
helium and LP (Fig. 1A). App oxima ely one hal o all T
cells in bo h he epi helial (median 44.6%) and LP
(58.4%) laye s o he colon exp essed CD161 (Fig. 1B).
CD56 was exp essed by smalle p opo ions o colonic T
cells, being p esen on a median o 35.1% o epi helial
laye T cells and on signi ican ly lowe numbe s (22.4%;
p
X
0.05) o LP T cells. CD57 was exp essed by simila
p opo ions o colonic epi helial laye (8.2%) and LP
(9.8%) T cells as in pe iphe al blood (9.6%) (Fig. 1B).
When T cell subpopula ions in duodenal issue samples
we e examined, CD161 was also ound o be exp essed
by he majo i y o epi helial laye (58.5%) and LP (57.5%)
T cells (Fig. 1B). Smalle p opo ions o T cells exp essed
CD56, wi h signi ican ly highe le els ound in he LP
(19.6%) compa ed wi h he epi helium (10.9%; p
X
0.05).
T cells exp essing CD57 we e less equen ly de ec ed in
bo h he epi helium (4.3%) and he LP (12.2%). The high
equency o CD161+cells in bo h he epi helial laye and
LP o human colon and he co-localiza ion o CD161 and
CD3 we e con i med by immunohis ochemis y and dou-
ble immuno luo escence (Fig. 1C, D).
2.2 Pheno ypic cha ac e iza ion o in es inal
CD161+T cells
In bo h human colon and duodenum, CD8 was
exp essed by he majo i y o epi helial laye CD161+T
cells (89.5% in colon and 59.9% in duodenum; Fig. 2A,
B), while smalle equencies exp essed CD4. In con-
as , CD4 was exp essed mo e equen ly by CD161+T
cells in he LP (58.5% in colon and 81.2% in duodenum;
Fig. 2A, B). Less han 5% o epi helial laye and LP
CD161+T cells in bo h colon and duodenum exp essed
nei he CD4 no CD8, a double-nega i e pheno ype
commonly associa ed wi h NKT cells [17]. Analysis o
TCR pheno ypes indica ed ha almos all CD161+T cells
in bo h duodenal and colonic epi helium and LP laye s
exp ess
§ g
TCR, while less han 3% exp essed
+ ˇ
TCR
(Fig. 2A, B). CD56 was exp essed by
X
20% o CD161+T
cells a all in es inal loca ions es ed (Fig. 2A, B).
The majo i y o o al colonic CD161+T cells exp essed
he ea ly ac i a ion ma ke CD69 (median 78.2%), and
up o 38.2% (median 18.4%) exp essed he la e ac i a-
ion ma ke HLA-DR (Fig. 2C). CD25 was exp essed by a
smalle p opo ion o colonic CD161+T cells (median
7.2%). Signi ican popula ions o in es inal CD161–T
cells also displayed ac i a ed pheno ypes (Fig. 2C).
Exp ession o Fas ligand (CD95L) was signi ican ly mo e
equen in CD161+T cells han in CD161–T cells (14.5%
s. 1.8%; p
X
0.05; Fig. 2C).
2.3 V
>
24+V
I
11+NKT cells in human colon
I has p e iously been shown ha , in mos issues, he
co-exp ession o he V
§
24 and V
g
11 TCR chains de ines
in a ian CD1d- es ic ed V
§
24J
§
18+NKT cells [25, 26,
41, 42]. We used low cy ome y o de ec V
§
24J
§
18+
NKT cells in human colon. The pe cen age o colonic
CD3+T cells ha exp essed V
§
24 anged om 0.3% o
3.3% (median 1.0% in epi helium and 1.6% in LP;
Fig. 3A). Howe e , hese colonic epi helial and LP V
§
24+
T cells did no co-exp ess he V
g
11 chain. In he li e , up
Eu . J. Immunol. 2004. 34: 2110–2118 NK ecep o -posi i e T cells in human in es ine 2111
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de
P
Fig. 1. NK ecep o -posi i e T cells accumula e in he human
in es ine. (A) Flow cy ome ic analysis o he co-exp ession
o CD3 wi h CD56, CD57 o CD161 on ga ed lymphocy es
om a non-diseased colon specimen. The numbe s in he
uppe igh quad an s show he pe cen ages o CD3+cells
ha exp ess CD56, CD57 o CD161. (B) Pe cen ages o
CD3+cells ha exp ess CD56, CD57 o CD161 in he epi he-
lial (E) and LP laye s o human colon ( op, n=8) and duode-
num (middle, n=10) and in pe iphe al blood (bo om, n=7).
Ho izon al ba s indica e he median alues. (C) Localiza ion
o CD161+cells by immunohis ochemical s aining in no mal
colonic mucosa. CD161+cells a e indica ed by b own
s aining (×200). (D) Immuno luo escence s aining o
CD3+CD161+T cells in no mal colon. Posi i e s aining is
indica ed by he g eenish-yellow s aining (×400).
o 90% (median 64.2%) o V
§
24+T cells exp essed V
g
11
(Fig. 3B) [27]. These da a indica e ha , while up o 1.6%
o hepa ic T cells exp ess V
§
24V
g
11 TCR, hese in a i-
an NKT cells a e p esen in ex emely low numbe s in
he human colon.
V
§
24V
g
11+NKT cells speci ically ecognize
§
-GalCe
p esen edbyCD1d[41,42]whichisexp essedandis
unc ionally ac i e in he mu ine and human in es ine
[43–46]. We examined eac i i y o
§
-GalCe in he
colonic cell p epa a ions by cul u ing 1×106cells wi h
§
-
GalCe and analyzing he cul u e supe na an s o IFN-
+
p oduc ion. Le els o IFN-
+
we e signi ican ly up-
egula ed by
§
-GalCe beyond le els de ec ed in uns im-
ula ed cul u es (Fig. 3C).
2.4 Cy okine p oduc ion by CD161+T cells
The abili y o eshly isola ed colonic epi helial and LP
laye CD161–and CD161+T cells o p oduce cy okines
upon s imula ion in i o was assessed by low cy o-
me ic analysis o pe meabilized cells. Signi ican p o-
po ions o CD161+T cells p oduced IFN-
+
and TNF-
§
,
bu no IL-4, upon ac i a ion wi h PMA and ionomycin
(Fig. 4A). TNF-
§
was p oduced by signi ican ly highe
equencies o CD161+T cells compa ed wi h CD161–T
cells om colonic epi helium (39.8% s. 21.2%; p
X
0.05)
and LP (69.7% s. 57.1%; p
X
0.05). In con as , he e-
quencies o colonic epi helial and LP CD161+and
CD161–T cells ha p oduced IFN-
+
in esponse o s imu-
la ion we e simila . The p opo ions o bo h CD161+and
CD161–T cells ha p oduced IL-4 upon PMA/ionomycin
s imula ion was
X
5% in all dono s examined (Fig. 4B).
2.5 NKR+T cells a e no expanded in colonic
umo issue
Immuno luo escence s udies on pai ed umo and unin-
ol ed colon issue sec ions om six pa ien s wi h
colonic ca cinoma (Fig. 1D) e ealed ha T cell numbe s
we e inc eased in he umo s in ou pa ien s and
dec eased in wo. The median numbe s o CD3+cells/
mm2in umo and unin ol ed issue om i e pa ien s
we e 640 and 487, espec i ely (pno signi ican ;
Fig. 5A). Flow cy ome ic analysis o hese T cells
e ealed ha NKR+T cells we e no expanded in umo s.
The p opo ions o T cells ha exp essed CD161 and
2112 J. O’Kee e e al. Eu . J. Immunol. 2004. 34: 2110–2118
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de
Fig. 2. Pheno ypic cha ac e iza ion o CD161+T cells eshly
isola ed om human in es ine. (A, B) Pe cen ages o
CD3+CD161+lymphocy es ha exp ess CD8, CD4, double-
nega i e CD8–CD4–(DN),
§ g
TCR,
+ ˇ
TCR and CD56 in he
epi helial and LP laye s o human colon (A, n=7) and duode-
num (B, n=5). (C) Pe cen ages o o al CD3+CD161+and
CD3+CD161–lymphocy es om no mal colon (n=7) exp ess-
ing HLA-DR, CD25, CD69 and Fas ligand. Ho izon al ba s
indica e median alues.
Fig. 3. In a ian NKT cells do no accumula e in he human
colon. (A) Pe cen ages o CD3+cells ha exp ess V
§
24 (le )
and V
§
24V
g
11 ( igh ) TCR chains in epi helial (E) and LP lay-
e s o human colon (n=7) and blood (n=4). Ho izon al ba s
indica e median alues. (B) Rep esen a i e low cy ome y
do plo s showing V
§
24 and V
g
11 TCR chain exp ession by
ga ed CD3+cells eshly isola ed om no mal colon, blood
and li e . Numbe s show pe cen ages o CD3+cells ha
exp ess he V
§
24V
g
11 TCR. (C) Mean IFN-
+
le els eleased
by uns imula ed,
§
-GalCe -s imula ed and PHA-s imula ed
o al colonic cells (n=5). *p
X
0.05.
CD56 we e signi ican ly educed in umo s compa ed
wi h adjacen unin ol ed issue (medians 31.9% s.
49.7% o CD161+Tcellsand8.6% s. 19.9% o CD56+
Tcells;p
X
0.05; Fig. 5B). Howe e , he absolu e num-
be s o CD161+and CD56+T cells did no di e signi i-
can ly (medians 187 s. 239 and 27 s. 68 cells/mm2in
umo s and adjacen unin ol ed mucosa, espec i ely;
Fig. 5B). The p opo ions and absolu e numbe s o
CD57+T cells we e also unchanged in colonic umo s
(Fig. 5B).
V
§
24+T cells we e also compa ed in umo issue and
adjacen mucosa in ma ched issue specimens om i e
pa ien s. The pe cen ages o V
§
24+T cells in no mal
mucosa we e low (0–2%), and he le els emained i u-
ally unde ec able in umo issues (da a no shown).
3Discussion
NKR+T cells a e p edominan ly localized in pe iphe al
issues and ha e he capaci y o apidly kill umo cells
and p oduce cy okines wi hou he need o p io p iming
wi h an igen [1–9]. We epo he e ha he heal hy adul
in es ine is a si e o accumula ion o se e al dis inc
NKR+T cell popula ions ha a e di e en ially dis ibu ed
in he duodenum and colon and in he epi helial and LP
laye s.Incon i ma iono p e iouss udies[38,39],we
Eu . J. Immunol. 2004. 34: 2110–2118 NK ecep o -posi i e T cells in human in es ine 2113
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de
Fig. 4. In acellula cy okine p oduc ion by CD161+and
CD161–T cells om no mal colonic epi helium (E) and LP. (A)
Rep esen a i e low cy ome y do plo s showing IFN-
+
,
TNF-
§
and IL-4 exp ession by colonic CD161+T cells s imu-
la ed o 4 h wi h PMA and ionomycin. The pe cen ages o
CD161+cells ha exp ess he cy okines a e indica ed in he
uppe igh quad an s. (B) Median pe cen ages o CD161+
and CD161–cells om epi helial and LP laye s o 9 no mal
colonic specimens ha exp ess IFN-
+
,TNF-
§
and IL-4 upon
s imula ion (*p
X
0.05).
Fig. 5. Colonic NKR+T cells a e no expanded in colonic
cance . (A) Quan i ica ion o CD3+cells pe mm2o pai ed
colonic ca cinoma and adjacen unin ol ed colonic mucosa
issue sec ions in 5 pa ien s using immunohis ochemis y.
(B) Pe cen ages (le ) and absolu e numbe s ( igh ) o CD3+
cells ha exp ess CD161, CD56 o CD57 in pai ed speimens
o umo -in ol ed issue and adjacen unin ol ed mucosa.
Ho izon al ba s indica e he median alues (n=19 o pe -
cen ages, n=5 o absolu e numbe s; *p
X
0.05).
ound ha CD161+T cells a e among he mos abundan
NKR+T cell popula ion in he in es ine. These cells we e
ound o accoun o app oxima ely one hal o all T cells
in bo h he epi helial and LP laye s o he duodenum and
colon. Compa ed o pe iphe al blood, CD56+T cell num-
be s we e ele a ed in he LP o he duodenum and colon
and he epi helial laye o he colon. CD57+T cells we e
ound a simila equencies in blood and he in es inal
compa men s.
Ou esul s indica e ha almos all in es inal CD161+T
cells exp ess
§ g
TCR. They can exp ess CD4 o CD8,
bu he majo i y in he epi helial laye s o bo h he duode-
num and colon a e CD8+, while CD4+cells p edomina e
among CD161+Tcellsin heLP.CD161
+T cells wi h he
double-nega i e (CD4–CD8–)pheno ypecommonly
associa ed wi h NKT cells [17–19] we e a ely ound in
he in es inal compa men s es ed. Colonic CD161–and
CD161+T cells showed simila equencies o exp ession
o he T cell ac i a ion ma ke s CD25, CD69 and HLA-
DR, bu Fas ligand (CD178) was ound o be exp essed
by highe equencies o CD161+ han CD161–Tcellsin
he colon. Colonic CD161+and CD161–Tcellswe e
capable o apid sec e ion o IFN-
+
and TNF-
§
, bu li le
IL-4, upon s imula ion ex i o wi h PMA and ionomycin.
The pe cen age o CD161+T cells capable o p oducing
TNF-
§
wasma kedlyhighe whencompa edwi h
2114 J. O’Kee e e al. Eu . J. Immunol. 2004. 34: 2110–2118
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de

CD161–T cells. These indings indica e ha he majo i y
o CD161+T cells in he in es ine p oduce cy okines o
he Th1/Tc1 p o ile, consis en wi h a cy o oxic pheno-
ype.
In he p esen s udy, we show ha while he human
in es ine con ains la ge numbe s o NKR+T cells, e y
low p opo ions o hese exp ess he in a ian V
§
24V
g
11
TCR chain associa ed wi h CD1d es ic ion. Simila ind-
ings ha e been epo ed o mu ine NKR+T cells de ined
by he co-exp ession o CD3 and NK1.1, which a e a e
in he small in es ine bu accumula e in he colon [40]. In
con as o mu ine li e , in es inal NKR+Tcellsdono
exp ess in a ian V
§
14J
§
18 TCR o equi e CD1d o
hei de elopmen [40]. Howe e , CD1d is cons i u i ely
exp essed by mu ine and human in es inal epi helial cells
[43–46], sugges ing ha o he non-in a ian NKT cells
may be p esen in he in es ine. In suppo o his, we
ound ha colonic mononuclea cells eleased signi ican
amoun s o IFN-
+
a e s imula ion wi h
§
-GalCe in i o.
§
-GalCe eac i i y by human in aepi helial lymphocy es
(IEL) has also been ecen ly demons a ed by Van de Wal
and co-wo ke s [43], who showed ha ligand ecogni ion
is CD1d-dependen . The
§
-GalCe - eac i e T cells in
human colon could ei he be in a ian V
§
24J
§
18+NKT
cells ha a e p esen in e y low numbe s bu can apidly
ac i a e o he cells, such as NK cells and con en ional T
cells, o p oduce IFN-
+
. Al e na i ely, o he non-in a ian
NKT cells ha ecognize
§
-GalCe p esen ed by CD1d
maybep esen in hecolonandmaybe hep ima y
sou ce o IFN-
+
. Non-in a ian CD1d- es ic ed NKT
cells ha exp ess CD56 and/o CD161 ha e been
de ec ed in human bone ma ow [9] and in hepa i is C
i us-in ec ed li e [47]. Howe e , ecen s udies using
CD1d-
§
-GalCe e ame s ha e p o ided e idence ha
§
-GalCe eac i i y is es ic ed o V
§
24V
g
11+NKT cells
[41, 42], which would a gue agains he no ion o non-
in a ian hepa ic NKT cells ecognizing
§
-GalCe . NKT
cells eac i e wi h CD1 iso ypes ha a e no ound in
mice, namely CD1a, CD1b and CD1c [18], may also
eside wi hin he CD56+/CD161+T cell compa men o
he in es ine.
CD56+and CD161+T cells can pa icipa e in an i- umo
immune esponses, being capable o po en an i- umo
cy oly ic ac i i y and he p oduc ion o la ge amoun s o
in lamma o y cy okines in i o [5–7]. Flow cy ome ic
analysis o lymphocy es isola ed om umo s and adja-
cen his ologically no mal colonic issue om19pa ien s
showed ha he p opo ions o T cells ha exp ess CD56
o CD161 a e signi ican ly educed in umo s. Howe e ,
while o e all numbe s we e also educed in umo s om
4 ou o 5 pa ien s, his educ ion was no signi ican . We
also ound ha CD161+T cells mo e equen ly exp ess
Fas ligand and p oduce mo e TNF-
§
han CD161–T
cells, sugges ing ha hey a e specialized o an i- umo
cy o oxici y. Ou obse a ions ha NKR+T cells a e no
expandedin umo sa eno consis en wi ha ole o
hese cells in umo immuni y, bu he educ ion in he
p opo ions o NKR+T cells could esul in insu icien
ac i a ion o NK o cy oly ic T cells equi ed o e icien
clea ing o umo cells [48]. Al e na i ely, i could be he
esul o ac i a ion-induced cell dea h o umo -
in il a ing lymphocy es; apop osis o umo -in il a ing
lymphocy es was p e iously ound o co ela e wi h
lymph node me as ases and poo p ognosis in p ima y
colo ec al cance [49]. Changes in human NKR+T cell
numbe s, pheno ypes and unc ions ha e also been
desc ibed in o he malignancies [27–29, 50], al hough
hei impo ance is less clea han in s udies in mice
[22–24].
In conclusion, his s udy has shown ha he human
in es ine is a si e whe e CD161+T cells p e e en ially
accumula e, sugges ing ha hese cells a e impo an in
local immuni y including esis ance o umo cell in a-
sion. CD161+T cells equen ly exp ess Fas ligand and
p oduce IFN-
+
and TNF-
§
in esponse o s imula ion.
Ve y small numbe s o hese cells exp ess he in a ian
V
§
24V
g
11 TCR, bu eac i i y agains
§
-GalCe is
de ec able among colonic cells, sugges ing he p es-
ence o o he CD1d- es ic ed NKT cells.
4 Ma e ials and me hods
4.1 Subjec s
Six een pa ien s (9 emales and 7 males; mean age 45 yea s;
ange 24–69 yea s) who we e being in es iga ed o uppe
gas oin es inal symp oms we e s udied. Small in es inal
disease was excluded by endoscopy and ou ine his ology.
Nine een pa ien s (11 emales and 8 males; mean age 69
yea s; ange 56–84 yea s) wi h colo ec al cance we e s ud-
ied. All endoscopic samples we e ob ained wi h in o med
consen , and he s udy was app o ed by he Resea ch and
E hics commi ee o S . Vincen ’s Uni e si y Hospi al, Dub-
lin.
4.2 P epa a ion o in es inal cells
Single-cell suspensions o epi helial laye and LP we e p e-
pa ed om colonic and duodenal biopsy specimens as p e-
iously desc ibed [51, 52]. B ie ly, biopsy samples we e
o a ed o 1 h a 37°C in calcium- and magnesium- ee
Hanks Balanced Sal Solu ion (Gibco-BRL, Paisley, GB) sup-
plemen ed wi h 5% FCS, 1 mM DTT and 1 mM EDTA. This
esul s in emo al o he epi helial laye , lea ing he LP in ac
and a ached o he basemen memb ane. The esul ing
single-cell suspension was washed in RPMI 1640 medium
Eu . J. Immunol. 2004. 34: 2110–2118 NK ecep o -posi i e T cells in human in es ine 2115
© 2004 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim www.eji.de
supplemen ed wi h 10% FCS and an ibio ics, and iable
cells we e enume a ed by e hidium b omide and ac idine
o ange s aining. To ob ain LP cells, he emaining mucosal
issue was placed in 5 ml supplemen ed RPMI 1640 medium
con aining 130 U/ml collagenase (Type 1A, Sigma-Ald ich,
I eland) and o a ed o 3 h a 37°C. The esul ing single-cell
suspension was washed in RPMI 1640 medium, and iabili y
coun s we e pe o med as abo e.
Su gically- esec ed in es inal issue, which included unin-
ol edcolonicmucosaaswellas umo issue,wasp e-
pa ed simila ly excep ha he issue specimens we e i s
cu in o ine pieces using a s e ile scalpel and hen ea ed
wi h 200 U/ml collagenase. The samples we e subsequen ly
il e ed h ough a nylon mesh and washed in RPMI medium
con aining 10% FCS. Cell numbe and iabili y we e de e -
mined as abo e. PBMC we e p epa ed by s anda d densi y
g adien cen i uga ion (Lymphop ep, Nycomed Pha ma,
Oslo, No way).
4.3 An ibodies and low cy ome y
Fluo och ome-labeled monoclonal an ibodies (mAb) speci ic
o human CD3, CD4, CD8, CD56, CD57, CD161,
§ g
TCR,
+ ˇ
TCR, HLA-DR, CD25, Fas ligand, IFN-
+
,TNF-
§
, IL-4 and
iso ype ma ched con ols we e ob ained om BD Pha Min-
gen(Ox o d,GB).An i-V
§
24 and an i-V
g
11 mAb we e
ob ained om Coul e Immuno ech (Ma seilles, F ance).
Single-cell suspensions (1×105) de i ed om in es inal is-
sue and pe iphe al blood we e s ained using speci ic mAb
as p e iously desc ibed [8, 51, 52] and analyzed by low
cy ome y using a FACScan and CellQues so wa e (Bec on
Dickinson).
Fo measu emen o in acellula cy okines (IFN-
+
,TNF-
§
,
IL-4), eshly isola ed in es inal cell suspensions we e s imu-
la ed wi h 10 ng/ml PMA and 1
?
g/ml ionomycin (Sigma-
Ald ich) o 4 h a 37°C in he p esence o 10
?
g/ml b e el-
din A (Sigma-Ald ich). Cells we e s ained o su ace exp es-
sion o CD161 and CD3 and subsequen ly ixed and pe -
meabilized be o e s aining wi h mAb speci ic o he cy o-
kinesandanalysisby lowcy ome y,asdesc ibedp e i-
ously [7].
4.4 Immunohis ochemis y and immuno luo escence
Fo immunohis ochemical s aining, esec ed colonic issue
specimens we e ixed in o malin and embedded in pa a in.
Sec ions we e subsequen ly dewaxed, mic owa e-hea ed
(3–5 min a 750 W in bu e con aining 0.1 M sodium ci a e
and0.1Mci icacid,pH6), insedinPBSand henincu-
ba ed wi h monoclonal an i-CD161 (5
?
g/ml) (Se o ec,
Ox o d, GB) o iso ype-ma ched con ol an ibodies. S aining
was e ealed wi h a Vec s ain Eli e ki (Vec o labo a o ies,
Pe e bo ough, GB) and indi ec immunope oxidase s aining.
Fo immuno luo escence s udies, biopsy specimens we e
moun ed in c yop ese a i e embedding (OCT, Tissue Tec,
Fine ec Eu ope, The Ne he lands) and snap- ozen in liquid
ni ogen. Se ial sec ions, cu a 5
?
m, we e labeled wi h
50
?
g/ml monoclonal an i-CD161, 50
?
g/ml polyclonal an i-
CD3 (Dako, Ely, GB) o iso ype-ma ched con ol an ibodies.
Seconda y an ibodies consis ed o Cy3-labelled goa an i-
bio in (Sigma-Ald ich) o CD161 and an i- abbi FITC
(Sigma-Ald ich) o CD3. Fluo escen mic oscopy was
pe o medonaNikonTE300mic oscope.
4.5 Cy okine esponse o
>
-GalCe
In es inal cells (1×106) we e cul u ed in 24-well issue cul u e
pla es in he p esence o 100 ng
§
-GalCe (Ki in Pha maceu-
ical Resea ch Labo a o y, Gunma, Japan) [20–22], PHA o
ehicle as a con ol. A e 48 h incuba ion, cul u e supe na-
an s we e collec ed, and IFN-
+
p oduc ion was assayed by
ELISA acco ding o he manu ac u e ’s ins uc ions (R&D
Sys ems, Oxon, GB).
4.6 S a is ical analyses
Flow cy ome ic esul s we e exp essed as he median al-
ues and ange. The Mann-Whi ney U es o non-pa ame ic
da a was used o analyze esul s. p alues o less han 0.05
we e conside ed signi ican .
Acknowledgemen s: The
§
-GalCe used in his s udy was
a kind gi om Pha maceu ical Resea ch Labo a o y (Ki in
B ewe y Co. L d., Gunma, Japan). We a e indeb ed o he
gas oin es inal and su gical eams a S . Vincen ’s Uni e -
si y Hospi al, Dublin o p o iding issue specimens, o Ma -
ina Goga y o help wi h immunohis ochemis y and o D .
Lau a Mad igal Es ebas o help ul discussions. This wo k
was suppo ed by g an s awa ded by En e p ise I eland and
he I ish Heal h Resea ch Boa d.
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Co espondence: Cliona O’Fa elly, Educa ion and
Resea ch Cen e, S . Vincen ’s Uni e si y Hospi al, Elm
Pa k, Dublin 4, I eland
Fax: +353-1-2838123
e-mail: cliona.o a elly
—
ucd.ie
2118 J. O’Kee e e al. Eu . J. Immunol. 2004. 34: 2110–2118
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