ILA, the Human 4-1BB Homologue, Is Inducible in Lymphoid - Blood

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ILA, the Human 4-1BB Homologue, Is Inducible in Lymphoid and
Other Cell Lineages
By Herbert Schwarz, Jean Valbracht, Julia Tuckwell, Johannes von Kempis, and Martin Lotz
We recently identified a genethat isinduced by lymphocyte
activation (ILA).The sequenceof the full-length 1.4-kb cDNA
characterized ILA as a new member of the nerve growth
factor/tumor necrosisfactor (NGFITNF) receptor family and
the human homologue of the murine T-cell-specific receptor
4-IBB. Thepresent study demonstrates ILA mRNA isoforms
at 4.4,4.0, and 1.8 kb in poly-A+ RNA from activated, but
not from resting human peripheral blood T lymphocytes.
A reverse transcriptase-polymerase chain
reaction (RT-PCRI
assay was used t o study tissue distribution and regulation
of ILA expression. The gene was induced in T lymphocytes
by phytohemagglutinin (PHA), phorbol myristate acetate
(PMA), and antibody t o CD3, in B lymphocytes by PMA and
antibodies t o cell surface Ig, and in blood monocytes by
interleukin-1/3 (IL-lp), lipopolysaccharide (LPS), and PMA. In
T lymphocytes, ILA mRNA was detectable 1.5 hours after
stimulation, reachedmaximal levels at 8 hours, and declined
t o background levels by 48 hours. Induction of ILA mRNA
required protein synthesisand was primarily due to in-
creasedtranscription. Actinomycin D reduced ILA mRNA levels in activated lymphocytes50% within 30 minutes,demonstrating a relatively short half-life of this mRNA. Analysis of
nonlymphoid cells showed that ILA mRNA was not detectable in resting cells. However, in contrast t o the lymphoidspecificexpression of the murine ClBB gene, ILA was
detected in nonlymphoid cells, including epithelial and hepatoma cells after stimulation with IL-lP. ILA was not detectable in several brain derived cell lines. TheILA cDNA encodes
a 30-kD protein as demonstrated by in vitro
translation, and
this protein is immunoprecipitated by antisera that were
raised against ILA peptides or a glutathione-S-transferase
fusion protein. Flow cytometry showed expression of ILA
protein on a subsetof activated T or B lymphocytes.
In conclusion, activation-dependentexpression ofILA is found not
only in T lymphocytes, but also in B lymphocytes, monocytes, and diversenonlymphoid cell types.
0 1995 by The American Societyof Hematology.
T
as previously described.26Chondrocytes*вЂ™ and synoviocytes** were
HE BIOLOGIC ACTIVITY of cytokines as mediators
isolated from tissues obtained from patients undergoing joint reof host defense responses is controlled through reguplacement or at autopsy from donors without known history of joint
lated expression of cytokines, as well as their receptors.
disease. Human thymocytes were isolated from tissue that was reTissue distribution of cytokine receptors and the production
moved during cardiac surgery as previously described.вЂќ Human Tof soluble receptors are additional mechanisms that detercell leukemia virus (HTLV)-1 transformed T-cell lines were obtained
mine cytokine function.
by coculture of peripheral blood T cells with an irradiated HTLVCloning of cytokine receptor genes has resulted in the
1 -infected cell line (kindly provided by Dr W. Wachsman, University
identification of several receptor families that contain strucof California, San Diego, CA). B cells from the peripheral blood of
turally related receptors.вЂ™ The nerve growth factor receptor
normal donors were transformed with Epstein-Barr virus (EBV) by
(NGFR)/tumor necrosis factor receptor (TNFR) family is
infection with EBV-containing supernatants from the B954 cell line.
The following cell lines were obtained from ATCC (Rockville,
characterized by the presence of 3 to 6 cysteine-rich motifs of
approximately 40 amino acids in the extracellular d ~ m a i n . ~ . ~MD): U373, glioblastoma; SK-N-MC, neuroblastoma; Wen, retinoblastoma; HS683, glioma; HEp-2 epithelial; HepG2 hepatoma;
Members of this family include the low-affinity NGFR;
A549, lung carcinoma; U937, promonocyte; Raji, EBV-transformed
TNFR-I,вЂ™.вЂ�j TNFR-II,7 CD40,вЂ™ CD30,9 CD27,вЂ™вЂќ Fas/APOB cells; HUl78, Jurkat, and Molt4, T-cell lines.
1,вЂќвЂќвЂ™ 0X-40,13.14and 4-1BB.IвЂ™ These receptors recognize
RNA preparation and Northern blot analysis. Total RNAwas
soluble or cell-surface bound ligands, which also form a
isolated by the acid phenol method.30 Poly A+ RNA was prepared
family of structurally related proteins and mediate diverse
with PolyATract mRNA isolation system (Promega, Madison, WI).
cellular respon~es.~
TNF and NGF regulate cell proliferation
RNA was fractionated on 1% agarose-formaldehyde gels and transand secretory functions in different cell
Stimulation
ferred to Hybond N membranes (Amersham, Arlington Heights,
of cells through the TNFRI6 and Fas/APO- 117,вЂќ can induce
IL). Gel-purified DNA fragments were labeled with 32Pby random
apoptosis. CD40 is expressed on B lymphocytes and medipriming and used as probes. The 1.4-kb full-length E A cDNA was
ates T-cell-dependent B-cell maturation and Ig class
used as probe.
s ~ i t c h . ~Stimulation
~,вЂ™~
of CD27,21,ZZ
which is expressed on
most peripheral blood T cells and mature thymocytes, and
From the Sam and Rose Stein lnstitute for Research on Aging
of the rat T-cell subset antigen OX-40 by specific antibodies
and Department of Medicine, University of California. San Diego,
increases lymphocyte proliferation. 4-1BB has recently been
La Jolla, CA.
shown to function as an accessory signaling molecule during
Submitted June 23, 1994; accepted October 17, 1994.
T-cell activationz3 and associate with the tyrosine kinase
Supported by National Institutes of Health Grants No. CA81406
p56вЂ�ck.24
and AR39799. J.v.K. was supported byafellowship
of the
We recently cloned the cDNA encoding ILA, a new memвЂњDeutscher Akademischer Austauschdienst ( D U D ) вЂ™ вЂ™ .
ber of the human NGFRNFR family, which is homologous
Address reprint requests to Martin Lotz, MD, University of Calito 4-1BB.вЂќ The present study shows that this receptor gene
fornia at San Diego, 9500 Gilman Dr, La Jolla, CA 92093-0663.
can be induced in lymphoid and different nonlymphoid cell
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
types.
MATERIALS AND METHODS
Cells and cell culture. Human peripheral blood mononuclear
cells (PBMC), monocytes, T, and B lymphocytes were prepared
Blood, Vol 85, No 4 (February 15). 1995: pp 1043-1052
вЂњadvertisementвЂќ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1998 by The American Society of Hematology.
0006-4971/98/8804-0009$3.~~
1043
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1044
SCHWARZ ET AL
RNA loads on thefilters were determinedby control hybridizations
tor for the T7 RNA polymerase 5вЂ� to the ILA coding region, were
with a full-length cyclophilin cDNA probe.вЂќ In some experiments, a
used as templates. A plasmid lacking a T7 promotor was used as a
G3PDHcDNAfragment was used for control hybridizations. The
control. ?-cysteine was included into the reaction for detection of
fragment was amplified by reverse transcriptase-polymerase chain rethe in vitro translation products by autoradiography of sodium dodeaction (RT-PCR) using G3PDH primers described below and subcy1 sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)separated proteins.
cloned into pGEM3z (Promega).
Autoradiographs were scanned on a Microtek MSF-300G scanner
Producrion of un eukuryotic ILA-Ig fusion protein. An expres(Microtek International Inc, Hsinchu, Taiwan) and bands were subse-sion vector containing the codingregion for the extracellular domain
quently analyzed using NIH Image1.44 software (National Institutes
of ILA and human Ig Fc constant domains was prepared and used
of Health, Bethesda, MD). Relative densities
of hybridization signals
to transfectCOScells.FusionproteinwasisolatedonproteinA
were calculated in comparison to controls (unstimulated cells) for
sepharose columns from the conditioned media and shown to have
both ILA and cyclophilin hybridization signals. Calculations from
the expected molecular mass on silver-stained polyacrylamide gels
ILA hybridizations werethen corrected on the basis of those obtained and Western blots. Sera from rabbits, which were immunized with
for cyclophilin to correct for variable RNA loads.
this purified protein,containedantibodies that specificallyrecognized the ILA-Ig as determined by enzyme-linked immunosorbent
RT-PCR. RNA (up to 5 pg) was reverse-transcribed in a 20 p,L
assay (ELISA) and immunocytochemistry. The antisera
were purivolumecontaining 4 p L 5XRT-buffer(BRL),
10 mmol/L DTT,
fied on protein A and the reactivity to human Fc was removed by
500 pmol/L dNTPs, I p L random hexanucleotides (2 mglmL) (Pharabsorption on human lg.
macia, Alameda, CA), 200 U MoMLV-RT (BRL), and 20U RNasin
Preparurion I$ f L A antibodies. Antibodies
were
prepared
(Promega) for 60 to 120 min at 37В°C.
against ILA peptides and the GST-ILA fusion protein. Several pep0.1 to 3
Polymerasechainreaction(PCR)wasperformedwith
pL (depending on the primers) of the RT reaction product in 25 pL
tidesweresynthesized,coupledtokeyholelimpethemocyanin
volume with 1 U AmpliTaq (Perkin Elmer Cetus, Emeryville, CA),
(KLH) and used to immunize New Zealand white rabbits. The rabbits
produced antibodies to the peptides, and the antibodies were aftinity
140 pmoUL dNTPs, 1.5 pmoVL MgClz, 10 mmol/LTris pH 8.3.
purified. AntibodytopeptideILA4
(pos 49to65:Pro-Pro-Asn50 mmol/L KCI, and 10 pmollL of each primer. After a 5-minute
Ser-Phe-Ser-Ser-Ala-Gly-Gly-Gln-Arg-Thr-Ser-Asp-lle-Cys)
and to
denaturation step at 94T, the reaction proceeded in 35 cycles of30
peptide ILAS (pos 124 to 139): Thr-Phe-Asn-Asp-Gln-Lys-Arg-Glyseconds at 94вЂќC, 30 seconds at 55вЂќC, and 50 seconds at 72В°C. folIle-Ser-Arg-Pro-Trp-Thr-Asn-Cys)
were used fortheexperiments
lowed by 5 minutes at 72В°C.
described here. Rabbits were immunized with GST-ILA fusion proPrimers used for ILA PCR were ILA-START: GAG AAT TCC
tein. After three immunizations in biweekly intervals, the sera conATG GGA AAC AGC TGT TAC, SKR6-SEN: AGG AGC AAG
tained antibodies that recognizedtheGST-ILA
fusion proteinon
GACCTGAGACAT,SKR6-AS:AGCAGCAGGTCACAG
Western blot. Theseantibodyreactivitieswere
not present in the
AG, ILA-BAC5вЂ™ CAT TCC CGG GTC CTT GTA GTA AC, ILApreimmune sera from the same animals.
BAC3вЂ™: CGG TGA TCA TCC TGG CTC TCT CGC AGG GGC,
Affinity; purification
ILA-DEL1 : TGC CTG CAT ATG TCA CAG, and ILA-DEL2: CAT Affinity purijicution of peprideuntiseru.
was performed by membrane affinity chromatography (MAC; AmiATGCAGGCAGACCCTGGACAAA.
con, Beverly, MA) according to manufacturersвЂ™ instructions.
Briefly,
Primersusedforglyceraldehyde3-phosphatedehydrogenase
peptides were dissolved in 0.5 mol/L NaHCO,, pH 9.0, at a concen(G3PDH) PCR were sense: TGG TAT CGT GGA AGG ACT CAT
tration of 1 mgfmL. Membranes were soakedi n this peptide solution.
GAC and antisense: ATG CCA GTG AGC TTC CCG TTC AGC.
air dried for 2 hours at RT. treated with 1 mg/mL Na-borohydride
They amplify a 190-bp fragment.
in phosphate-buffered saline (PBS), pH 7.4, for 1 hour at 4В°C. and
Quunriturive PCR. A ILA deletionclone(ILA-DEL)wasconrinsed with PBS. Serum was diluted 1.3 in PBS and filtered through
structed by PCR-based mutagenesis, which resulted in a deletion of
0.45-pm Acrodisc filters (Gelman Sciences, Ann Arbor, MI). Mem200 bp between nucleotide positions 340 to 539.
branes with boundpeptidewereinserted
in aMACholderand
The ILA cDNA from start to the Hue111 site at position 921 was
washed with PBS. The diluted serum was passed over
the filters.
subclonedintothe
EcoRl and SmaI site of pGEM7z(Promega)
washed with PBS,andeluted with 100 mmol/Lglycineand
100
between the SP6 and T7 polymerase transcription start site. A fragment containing the 5вЂ™part of ILA was amplified using a SP6 primer mmol/L NaCl in PBS. Eluted material was concentrated and reconstituted in PBS by centrifugation through Centricon 30 (Amicon).
and ILA-DELI. In a parallel reaction, the 3вЂ™ ILA part was amplified
Immunojluorescence staining undjow cvtomety. Cells were anwith a T7 primer and ILA-DEL2. ILA-DEL2 contained at its 5вЂ™ end
alyzed using a FACScdn (Becton Dickinson, Mountain View. CA)
22 nucleotides identical to the 3вЂ� end of the SвЂ™ TLA fragment. In a
and LYSIS11 software (Becton Dickinson). Laser excitation was at
third PCR with the SP6 andT7 primers, the 5вЂ� and3вЂ™ ILA fragments
488 nm (argon laser) for fluorescein isothiocyanate (FITC). A total
served as templates for the generation of a ILA deletion fragment,
of 4 X 10вЂ™ cells were used per condition. Cells were washed three
which lacksthe200nucleotideslocatedbetweenILA-DELIand
times in PBS, resuspended in 100 pL staining buffer (RPM1 1640.
ILA-DEL2. This fragment was subcloned into the EcoRI Hind111
and
3% FBS, 0.02% NaN,), and stained
with affinity purifiedpeptide
sites of pGEM4z.
Serial dilutions of this deletion clone were coamplified with single ILA5 serum for 30 minutes on ice. After two washes with staining
buffer. the cells were incubated with FTTC-labeled goat antirabbit
stranded cDNA as described earlier. PCR products were separated
IgG (1:40)(BoehringerMannheim,Indianapolis,IN)
in staining
on a I .3% agarose gel and stained with ethidium bromide. Pictures
the cells were
buffer for I hour on ice. After three more washes,
were taken with a Land MP4 Polaroid camera on positivehegative
analyzed by flow cytometry.
Polaroid film #665 (Polaroid Corp, Cambridge, MA), scanned, and
Ruugents. The recombinant human cytokines interleukin-10 (1Levaluated as described for Northern blot analysis.
IO), interferon y (IFNy), and TNFcv were purchased from R & D
f n virrotrunslution offLA prorein. In vitro translated ILA proSystems (Minneapolis, MN). Endotoxin content was less than 0 . 1
tein was prepared with the TNT coupled reticulocyte lysate system
ng per mg of cytokine protein. Lipopolysaccharide (LPS, from Sal(Promega). ILA cDNA from position - 139 to 1021 containing the
monella Minnesota), phorbol 12-myristate 13-acetate (PMA), phytocomplete ILA coding region but lacking the remaining 3вЂ� end was
I site of pRc/CMV (Invitrogen, San Diego. hemagglutinin (PHA), the calcium ionophore A23187, and actinocloned into the EcoRIINot
mycin D (actD) were obtained from Sigma (St Louis, MO). OKT3
CA). Two microgramsof two different plasmids, eachwith a promo-
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ILA, THE HUMAN 4-186 HOMOLOGUE
1045
ILIA
1 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPP 50
4-1BB
1 MGNNCYNVW1VLLLVGCEKVGAVQNSCDNCQPGTFCRKY.NPVCKSCPP 49
~ ~ ~ . ~ ~ ~I:. : .~
: I : . I ..I I:. : I~I I I~ .: :I . :. I . . I I I
ILA
51 NSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECIXTPGFHCLGAGCS 100
4-1BB
50 STFSSIGGQPNCNICRVCAGYFRFKKFCSSTHNAECECIEGFHCLGPQCT 99
..Ill l l l . - l : l l l
l I I I : I Ill1 I I I I : I . . I I I I I l : ~ I ~
4-1BB
101 MCEQDCRQGQELTKKGCKiXCFGTFNDQK.RGICRPViTNCSLlXK~W 149
.ll.lll.lIlIII.III.1::IIIIII. 1:lllllllllll:lll .
100 RCEKDCRPGQELTKQGCKTCSLGTFNDQNGTGVCRPWTNCSLERSVLKT 149
ILA
150 GTKERDWCGPSPADLSPGAS.SVTPPAPAREPGHSPQIISFFLALTSTA
ILA
198
l l . l ~ l l l l l i ~l l~l l .~: . :~ ~. I l l l l ~
: : '~: l l~l l l l l
Fig 1. Amino acid sequence homologyof IIA and
ClBB. The deduced amino acid sequencesof IIA and
the murine 4-IBB were aligned,using the BestFit
program of GeneticsComputerGroupInc(Madison,
WII sequencoanalysis software. Identicalamino
acids are indlcated by verticallines.Aminoacids
with high, low and no similarity are indicated by coblanks,
respectively.
points,
and
lons,
4-1BB
150 GTIEKDWCGPPWSFSPSTTISVTPEGGP..GGHSLQVLTLFLALTS.A 196
ILIA
199 LLFLLFFLTLRFSWKRGRKKLLYIFKQPFMRPVQ'ITQEEDGCSCRFPEE 248
4-1BB
197 LLLALIFITLLFSVLKWIRKKFPHIFKQPFKKTTGAAQEEDACSCRCPQE246
ILA
249 EEGG . . . CEL 255
4-1BB
241 EEGGGGGYEL 256
1 1 : l:l:ll l l l : l :
was recovered as a hybridoma supernatant and titrated
for optimal Tlymphocyte proliferation. Antibody to cell surface immunoglobulin
(anti-p) was purchased from Cappel (Durham, NC).
RESULTS
Characterization of the ILA cDNA. The 1,439-bp ILA
nucleotide sequencez5 was obtained from several separate
library screenings and independent sequencing reactions.
The open reading frame encodes a protein of 255 amino
acid(s) (aa)with a molecular mass of approximately 28 k D .
Hydropathicity analysis32predicted a putative signal peptide
(aa 1 to 17) and a 27 aa hydrophobic region, which is a
potential transmembrane domain (aa 187 to 213), and flanked
by charged residues. Following the 17 aa leader peptide, it
contains an extracellular domain of 169 aa, a transmembrane
region of 27 aa, and a short intracellular domain of 42 aa.
Based on these features, ILA can be classified as a type I
transmembrane protein.
Two potential N-glycosylation sites at positions 138 and
149 are based on the presence of the NXS/T motif (with X
being any amino acid except D or P). The serine at position
242 is a potential site for phosphotylation by protein kinase
C. The two threonines at positions 234 and 235 are potential
sites for phosphorylation by casein kinase 11. In addition,
ILA contains a potential binding site for the tyrosine kinase
~56''~'.
The extracellular part of L A contains three cysteine-rich
repeats, characteristic of members of the NGF/TNFR superfamily. The transmembrane domain is preceded by a serinethreonine-proline rich region (aa 160 to 185), and these three
aa account for 50% (14 of 27 aa) of this domain. Similar
domains are found in other members of the NGF/TNFR
family.'3
Comparison of ILA with sequences in the NBRF database
showed that it is probably the human homologue of the
murine cDNA sequence 4-1BB (Fig l), which is expressed
by specific T-lymphocyte subset^.'^ The deduced amino acid
Ill: .lIIlII.:.....lIII:IlII
I:)
IIIll1
sequences of ILA and 4-1BB display 73.6% similarity and
59.6% identity.
Regulation of ILA mRNA expression in lymphocytes.
ILA was not detectable in total or poly A' RNA from unstimulatednormal human peripheral blood lymphocytes from
more than 20 normal donors. Mitogen stimulation of PBMC
with PHA or PMA induced expression of ILA mRNA. By
Northern blot analysis of poly A+ RNA, three forms of ILA
mRNA could be detected at 4.4, 4.0, and 1.8 kb (Fig 2),
while analysis of total RNA from stimulated PBMC showed
only the 4.4-kb form. Studies on kinetics showed that ILA
mRNA was rapidly induced by stimulation with PHA and
PMA within 1.5 hours, increased to maximal levels by 8
hours, and declined to near background levels by 48 hours
(not shown).
Stimulation of lymphocytes with an antibody to CD3 also
induced LLA mRNA. As compared with stimulation with
PHA and PMA, the effects of anti-CD3 had a slower onset
but a longer duration. L A mRNA was first detected at 4
hours, reached the maximum at approximately 19 hours, and
was still strongly expressed after 48 hours (Fig 3).
Induction of ILA mRNA was dependent on protein synthesis. ILA mRNA was induced in primary T lymphocytes
by treatment withPHA, PMA, and OKT3. Coincubation
with the protein synthesis inhibitor cyclohexamide blocked
ILA mRNA synthesis in both cases (Fig 4).
Treatment of activated PBMC with actD, a transcriptional
inhibitor, was used to determine L A mRNA stability. Cells
were stimulated with PHA (1 pg/mL) and PMA (1 ng/mL,)
for 4 hours before actD (5 pg/mL) was added. Cells were
collected at different time points for analysis by RT-PCR.
For quantitative analysis of ILA mRNA levels, a competitive
PCR was performed by coamplifying cDNA with an ILADEL, lacking a 200-bp region. Figure SA shows a titration
of ILA-DEL against constant amounts of cDNA from the
control and the 15- and 30-minute actD samples. Ethidium
bromide stained gels showed the points of equal intensity
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SCHWARZ
1046
A
1
2
3
4
k b
1 --
4.4
4.0
-
1.8
B
1
2
3
4
ILA mRNA was not expressed in response to IL-lp (10 ng/
mL), leukemia inhibitory factor (LIF; 10 ng/mL), IFNy (500
U/mL), LPS ( 1 pg/mL) or TNFa( 1O
, OO U/mL) after 7 hours
(not shown).
Analysis of T-cell lines showed ILAexpression in HTLVI transformed T cells and in Jurkat cells after stimulation
with PHA and PMA. ILA
could not be detected in the Tcell lines Hut78 andMOLT4,even after stimulation with
PHA and PMA (not shown).
I L A can be induced in normal B lymphocytes and is constitutivels expressed in rran.sformed B cells. In B lymphocytes purified from human peripheral blood,ILAwas detected by RT-PCR in cells that hadbeen activated with
anti-p (12.5 pg/mL) or PMA ( I O ng/mL) for 6 hours. Analysis of B-cell lines showed the presence of ILA mRNA in
unstimulated Raji cells, an EBV-transformed B cell line, as
well as in B cells from normal donors that had been transformed byEBV in vitro. Stimulation withPMA ( I O ng/
mL) had no detectable effect on the constitutive ILA mRNA
expression in EBV-transformed B cells (Fig 6 ) .
I U expression in mononuclear phagocytes. In human
CO
Fig 2. ILA mRNA expression in lymphocytes. RNA was isolated
from unstimulated cells (lanes 1 and 2) or from cultures that had
been activated with PHA (l pg/mL) and PMA (1ng/mL) for11 hours
(lanes 3 and 4). (A) TotalRNA (15 pg/lanes 1 and 3) or polyA' RNA
(1 pg/lanes 2 and 41 was analyzed for thepresence of ILA transcripts
by Northern blotting. (B) To document the amount of RNA loaded,
the filters were subsequently analyzed for cyclophilin mRNA. The
differences in intensity of the cyclophilinsignals in (B) are related t o
the use of total RNA (lanes 1 and 3) versus poly A+ RNA (lanes 2
and 4).
between target and competitor products shifting from 4 to 8
X IO4 in the sample not treated with actD to 4 X IO4 and 2
X IO4 competitor molecules in the samples treated with actD
for 15 and 30 minutes, respectively. To obtain a quantitative
analysis of these results, negatives of the photographs were
scanned and the ratios of target to competitor products were
plotted against the number of competitor molecules present
in the reaction (Fig SB). The equivalence point (1:l ratio)
of target to competitor products was reached at 3.2 X IO4
competitor molecules for the control, at 2.4 X IO4 competitor
molecules for the 1 S-minute actD sample and at I .6 X IO4
Competitor molecules for the 30-minute actD sample. These
values represent a half-life time ofILAmRNAof
30
minutes.
Normal human thymocytes were studied as a source of
immature T lineage cells. They exhibited a similar time
course of ILA mRNA induction as peripheral blood lymphocytes when stimulated with PHA and PMA.Stimulation with
PHA or PMA alone was sufficient for ILA mRNA induction.
ET AL
0
4
5
7
19 24 44
hours
ILA
G3PDH
Fig 3. Time course of ILA expression in response t o T-cell activation byanti-CD3. PBMC were stimulated with anti-CD3 (OKT3). RNA
was isolated at the indicated time points and analyzed by RT-PCR
using the primersILA-BAC5' and 3'. which result in amplification of
a fragment with an expected size of 604 bp. To document an equal
amount of template, the samples were also analyzed for G3PDH
mRNA.
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1047
ILA, THE HUMAN 4-1BB HOMOLOGUE
X
I
V
lated cells but increased strongly in response to IL-Io (10
ng/mL). Among the nonlymphoid cell types tested, the lung
carcinoma A549 expressed ILAmRNA constitutively (not
shown). These findings demonstrate that ILA is inducible
by the proinflammatory cytokine IL-Io in diverse cell types,
A
control
15 min ActD
30 min ActD
Fig 4. Cyclohexamide inhibits ILA mRNA induction in T lymphocytes. Primary T lymphocytes were activated with PHA (2 gg/mLI,
PMA (5 ng/mL), or OKT3 (1:5000 dilution of hybridomasupernatant)
and cultured for 16 hours in the absence or presence of cyclohexamide (50 gg/mL). RNA was isolated and analyzed by RT-PCR using
the primers ILA-BAC5' and 3'. The same templates were analyzed
for G3PDH. Marker: PhiX 174 Haelll; control: unstimulated cells.
blood monocytes ILA was induced by stimulation with ILIp or PMA (0.1 ng/mL) for 4 hours. The effects ofPMA
were dose-dependent with induction occurring at 0.1 ng/mL.
The same three mRNA isoforms of 4.4, 4.0, and 1.8 kb as
in lymphocytes were also detected in monocytes (Fig 7).
In addition toPMAand IL-Io, ILA was also induced in
monocytes by stimulation with LPS (not shown). ILA was
also found in the human premonocytic cell line U937 and
in vitro-derived macrophages after induction with IL-Ip ( I O
ng/mL) and PMA (10 ng/mL) (not shown).
Tissue clistrihrction and repdotion of I L A expression in
nonlymphoid cells. To determine whether ILA expression
is restricted to the immune system, primary cells and cell
lines from other tissues were examined. ILAmRNA was
not detectable by Northern blotting or PCR in unstimulated
human rheumatoid synoviocytes and chondrocytes and in
several human tumor cell lines, including the hepatoma
HepG2. However, after activation with IL-ID, ILA mRNA
was detected in several nonlympoid cell types. In epithelial
and hepatoma cells, ILA mRNA was not present in unstimu-
B
I
loglo competitor (molecules)
Fig 5. ILA mRNA stabilii. PHA/PMA-stimulated and actD-treated
PBMC were analyzed for expression of ILA mRNA, using ILA-START
and ILA-BAC3' as primers. Known amounts of
an ILA-DELwere coamplified in the same reactions. (AI Products were run on a 1.30/0agarose
gel and stained by ethidium bromide. IBI
Plot of data obtained from
scanning of photographs of (Al. Control, U; 15-minute actD, V; 30min actD, 0.
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SCHWARZ ET AL
1048
primary
EBV transformed
0)
U
-aQ
c,
e
I
6 hour stimulation:
-
ILA
-
GSPDH
PMA 10 ng/ml
antiy 12.5 pg/ml
Fig 6. ILA expression in primary and in EBV-transformed B lymphocytes. Primary and EBV-transformed B lymphocytes were stimulated for 6 hours and analyzed for ILA mRNA by RT-PCR using SKRGSEN and -AS as primers. To document an equal amount of template,
the samples were also analyzed for GBPDH mRNA.
and thus has a much wider tissue distribution than described
for 4-1
To determine expression of ILA in the nervous system,
several brain derived cell lines were tested for presence and
inducibility of ILA. Using RT-PCR, ILA was not found in
RNA from glioma (HS683) cells, resting or activated for 8
hours with IL-I(I0 U/mL) or IFNy (500 U/mL) or PMA (5
ng/mL) plus the calcium ionophore A23 187 (1 nmol/L) (not
shown). Identical results were obtained for a glioblastoma
and a retinoblastoma cell line (U373 and WERI, respectively) and for two neuroblastoma cell lines (SK-N-MC and
IMR-32).
Production of recombinant IL.4 protein and immunoprecipitation with spec@ antibodies. To characterize ILA
protein, rabbit antibodies were raised against ILA peptides
and GST-ILA and ILA-Ig fusion proteins. For the production
of recombinant ILA protein, the full-length ILA cDNA was
cloned into the expression vector pRc/CMV. RNA derived
from this plasmid was translated in vitro with reticulocyte
lysate in the presence of вЂќS-cysteine. As a specificity control,
we also synthesized luciferase (luc) protein by in vitro trans-
lation. Analysis of the in vitro translation products by SDSPAGE and autoradiography (no precipitation) shows the luciferase band at 60 kD and the ILA band at 30 kD as the
most prominent bands (Fig 8).
Aliquots of the identical in vitro translation products were
precipitated with pre- and postimmune sera, generated by
immunization of rabbits with the GST-ILA fusion protein
or a synthetic ILA peptide. Figure 8 shows that luciferase
was precipitated equally by the preimmune and immune antisera to ILA. This was due to binding of the proteins to the
sepharose beads, which were used in the immunoprecipitation experiments (not shown). The antisera to GST-ILA and
ILA peptide specifically precipitated the recombinant ILA
protein. The low levels of ILA precipitated by the preimmune sera were also due to ILA binding to the sepharose
beads.
These results suggest that the ILA cDNA encodes a protein, whichis recognized by antisera, that were raised against
ILA peptides or GST-ILA fusion protein. The predicted molecular mass of ILA is 28.4 k D . In vitro-translated ILA protein has an apparent mass of approximately 30 kD.
Flow cytometry analysis of I L A protein expression on primary lymphocytes. Specificity and titer of the antisera for
staining of native ILA proteinwas determined by immunocytochemistry. COS cells transfected with the eukaryotic expression vector pRc/CMV-ILA, but not untransfected cells,
showed strong and specific staining (not shown). The same
antiserum was used for flow cytometric analysis ofILA
protein expression on primary lymphocytes. Lymphocytes
were activated with PMA (5 ng/mL), IL-2 ( I O 0 p/mL), and
OKT3 (1:5,OOO)for 48 hours. Staining was performed with
affinity purified antiserum to peptide ILA5 (32 pg/mL) and
FITC-labeled antirabbit IgG (Boehringer Mannheim). Approximately 25% of the activated cells showed specific staining (Fig 9).
DISCUSSION
Members of the NGFRNF receptor family are characterized by the presence of three to six cysteine-rich extracellular
domains. ILA contains three cysteine-rich domains, and the
second is composed of only 32 instead of the usual 41 to 44
amino acids. This domain structure is very similar in ILA
and 4-IBB. Through the conservation of the cysteine-rich
extracellular domains, the members of this receptor family
share a common structure for ligand binding.Similar to other
members of the NGFRNFR family, ILA contains a serinethreonine-proline-rich hinge region between the transmembrane domain and the cysteine-rich domains. For the 75-kD
TNFR, it has been shown that this domain has an extended
structure, which is likely to facilitate ligand binding to the
cysteine-rich domains..вЂќ The interspecies identity for members of this receptor family is significantly higher than the
identity of different members from the same species. For
example, the human and murine type 1 and type 2 TNFR
share 64% and 62% identity, respectively.3hThe identity
between the human type 1 and 2 TNFRislimitedtothe
ligand binding domain and is only 27%.вЂ™ Similarly, the identity of murine to human CD27 is 65%. Its identity to 4-1 BB
is 39%.вЂќ The identity between 4-IBB andILA is59.6%
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
I U . THE HUMAN 4-1BB HOMOLOGUE
1049
A
1
2
3
4
5
6
kb
.-.
- 4.4
-
,R
e
ai
.-e
ai
- 4.0
$
- 1.8
B
1
2
3
4
5
6
Fig 7. ILA induction in monocytes by PMA and IL-lp. Peripheral blood monocytes
were stimulated with PMA and IL-lp at the indicated concentrations. (A) RNA was
isolated after 4 hours and analyzed for I L A mRNA by Northern blotting. (B1 Because
the load of RNA differed considerably in thisexperiment, a rehybridization of the blot
with GBPDH cDNA is shown. 1, control; 2, PMA (0.1 ng/mL); 3, PMA (lnglmL1; 4,
PMA (10 nglmL1; 5, PMA (50 nglmL1; 6, IL-lp 10 ng/mL. (Cl shows the results after
densitometry and normalization of the ILA signals on the basis of GBPDH signals.
and corresponds to the interspecies identity of other members
of this receptor family. Based on this identity, ILA is the
likely human counterpart of 4-1BB.
Signal transduction through these receptors has only partially been characterized and diverse mechanisms appear to
be used. The NGFR itself displays only low-affinity of NGF.
High-affinity binding of NGF is obtained upon association
with the trk oncogene product, which has tyrosine kinase
activity.'* TNFR appear to use oxygen radicals, the phosphatidic acid, and the sphingomyelin pathways for intracellular
signaling. The intracellular region of 4-IBB contains the
consensus site for association with p56Ickoriginally identified
in CD4 and CD8.39 This site appears to be functional as 4IBB and ~ 5 6 "were
~ coimmunoprecipitated with anti-41BB or anti-p56"' antibodies24suggesting the possibility that
4-1BB on ligand binding might activate the tyrosine kinase
~ 5 6 " In
~ . 4-1BB. the sequence of the potential p56ICkbinding
site is CSCRCP (aa 239 to 244). The corresponding sequence
in ILA, CSCRFP (aa 241 to 244), contains only one amino
acid substitution. Site-directed mutagenesis showed that the
two cysteine residues are the essential elements for association with p561ck.39
This sequence in ILA was confirmed with
two independently isolated cDNAs and suggests that this
potential signaling mechanism is conserved between 4-1BB
and ILA. A threonine residue is present in the intracellular
domains of CD40, Fas/APO-l, NGFR and TNFR. This residue has been implicated in CD40 signaling and may represent an additional conserved mechanism of signal transduction. In the intracellular part of ILA, as well as 4-1BB, there
are two consecutive threonine residues which constitute a
potential phosphorylation site for casein kinase IL4'
Three different isofonns of ILA mRNA were detected in
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
SCHWARZ ET AL
1050
anti ILA
peptideserum
anti GST-ILA
serum
C
0
Q)
C
3
E
.-E
2
kD
n
1
-
"1
69 46
-
- 30 -
-
21.5
ILA
L
activated PBMC. The most abundant form was at 4.4 kb,
and this was the only species ofILA readily detectable in
total cellular RNA. Isolation of Poly A' RNA was necessary
for clear detection of the 4.0 and l .8 kb isoforms. The smallest form at 1.8 kb is of sufficient size to contain the fulllength coding region, while the larger size of the two additional species is probably due to extended 3' untranslated
regions. The difference between the 1.4-kb cDNA and the
smallest transcript of 1.8 kb is probably due to priming of
oligo dT on an internal A-rich region, because six independent cDNA clones of 1.4 kb were isolated.
ILA mRNA was notdetected in nonactivated PBMC from
1
control
1
anti-ILA
Fluorescence intensity
Fig 9. Flow cytometry analysis of ILA protein expression on primary lymphocytes. Lymphocytes (0)resting and (B) activated for 2
days with PMA (5 nglmL),IL-2 (100 plmL1, and OKT3 (1:5000) were
stained and analyzed by FACScan. (AI Antirabbit IgG (H + LI-FITC
alone (B) Affinity purified peptide 5 serum (32pglmL1 + antirabbit
IgG (H + L)-FITC.
Fig 8. Immunoprecipitation of ILA protein. ILA
and luc proteins were synthesizedby invitro translation as described in Materials and Methods. Aliquots
of each reaction were analyzed by SDS-PAGE (no
precipitation). Identical aliquots were precipitated
with pre- and postimmune sera, generated by immunization of rabbits with the GST-ILA fusion protein,
or a synthetic ILA peptide. ILA and luc proteins run at
a molecular weight of 33 kD and 60 kD, respectively.
more than 20 healthy donors using a sensitive PCR assay.
However, ILA wasrapidly inducible byPHA, PMA,and
anti-CD3. Transcripts were detected early (90 minutes) after
stimulation with PHAPMA. Immediate early genes as c-jtcn
and clfos are inducible within 15 minutes." ILA thus appears
to be an early activation gene.
A competitive PCR was performed to determine ILA
mRNA stability. Fifteen and 30 minutes after addition of
actD to activated PBMC, ILA mRNA decreased to 76% and
52% of control levels, respectively. Based on these values,
the half-life ofILAmRNAwas
calculated to be approximately 30 minutes. This characterizes ILA as a highly unstable mRNA. In activated monocytes, the half-lives of the cj u n and clfos transcripts, which are among the most unstable
mRNAs, are 27 and 25 minutes, re~pectively.~'
Following demonstration of ILA expression by activated
PBMC, we analyzed whether ILA is similarly restricted to
the expression in T lymphocytes as 4-IBB. The results
showed that ILA can be inducedin all major cellular subsets
of the immune system and at early, as well as late, stages
of their differentiation. Its expression in all normal lymphoid
cell types tested was activation-dependent.
ILA was induced in blood monocytes in response to their
major activators including LPS, IL-10, and PMA. In addition, it appeared that monocyte adherence is also a stimulus
for the induction of this mRNA (unpublished observation).
U937 cells representing immature monocytes and in vitro
differentiated macrophages expressed ILA in response to
PMA or IL-10.
B lymphocytes wereisolatedfromperipheralblood
of
normal donors. In purified populations of these cells, ILA
expression was induced by antibodies to cell surface Ig or
PMA. EBV-transformed B-cell lines expressed ILA mRNA
constitutively. Collectively, these results showthatwithin
the immune system ILA is more broadly expressed than 4-
From www.bloodjournal.org by guest on December 22, 2014. For personal use only.
ILA, THE HUMAN 4-IBB HOMOLOGUE
1BB. This indicates that ILA may not only function as the
recipient on T lymphocytes for signals derived from other
cells, but potentially serves as a molecule in mediating communication among different cell types of the immune system.
Of further significance to the role of L A in host defense
responses is its expression in several nonlymphoid cell types.
The analysis of tissue distribution performed in the present
study clearly shows that ILA is inducible in human articular
chondrocytes as one example of a normal mesenchymal cell
type, as well as in hepatoma and epithelial tumor cell lines.
It is of interest that ILA was not inducible in any of the
different cell lines established from brain tumors, possibly
indicating that ILA is not a product of neuronal cell types.
The other members of the NGF/TNFR family differ in
their tissue distribution. The rat OX-40 ismost restricted
and only expressed by certain T-cell subsets. CD40 is mainly
expressed by B lineage cells, while CD30 can be found on
activated normal T and B cells, as well as on HodgkinвЂ™s
lymphoma cells. CD27 has been detected on T and B lymphocytes, mature thymocytes, and some chronic B lymphocytic leukemias. In contrast to these receptors that appear to
be restricted to T and B lymphocytes is the broad tissue
distribution of the NGFR and the two TNFR. ILA is a member of this receptor family that has a wide tissue distribution
and is, in this respect, similar to the NGF and TNF receptors.
Biochemical characterization of ILA protein was performed by in vitro translation of RNA, which was derived
from the full-length ILA cDNA. The in vitro translation
product had a molecular mass of 30 kD and wasimmunoprecipitated by antisera against GST/ILA fusion protein or ILA
peptides. The predicted molecular weight of ILA is 28.4k D ,
which is in agreement with the size of the in vitro translated
protein.
The expression and inducibility of ILA protein on primary
cells was demonstrated in lymphocytes where activation with
known inducers of ILA mRNA also induced expression of
ILA protein.
ACKNOWLEDGMENT
We thank J. Shin for excellent technical assistance.
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1995 85: 1043-1052
ILA, the human 4-1BB homologue, is inducible in lymphoid and other
cell lineages
H Schwarz, J Valbracht, J Tuckwell, J von Kempis and M Lotz
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