Decreased Interleukin-15 From Activated Cord Versus Adult Peripheral
Blood Mononuclear Cells and the Effect of Interleukin-15 in Upregulating
Antitumor Immune Activity and Cytokine Production in Cord Blood
By John X. Qian, Sun min Lee, Yu Suen, Eva Knoppel, Carmella van de Ven, and Mitchell S. Cairo
Interleukin-15 (IL-15) is an important lymphokine regulating
natural killer (NK) activity, T-cell proliferation, and T-cell cytotoxic activities. We hypothesized that the reduced expression and production of IL-15 from cord blood (CB) may contribute to the immaturity of CB immunity and potentially
delay immune reconstitution after CB transplantation. We
compared the expression and production of IL-15 from activated cord versus adult mononuclear cells (MNCs) and the
regulatory mechanisms associated with IL-15 expression in
CB MNCs. We have also studied the effect of exogenous IL15 stimulation on CB and adult peripheral blood (APB) MNCs
in terms of NK and lymphokine-activated killer (LAK) activities and cytokine induction. Lipopolysaccharide (LPS)-stimulated CB and APB MNCs were used to determine IL-15 expression and protein production by Northern analysis and
Western immunoblot analysis. IL-15 mRNA expression and
protein accumulation in CB MNC were 25% Ô 2.0% (12 hours,
n ! 4, P Ú .05) and 30% Ô 2.5% (12 hours, n ! 3, P Ú .05),
respectively, when compared with APB MNCs. Nuclear runon assays showed no differences between CB and APB
MNCs during basal levels of transcription and after transcriptional activation. However, the half-life of IL-15 mRNA was
approximately twofold lower in activated CB MNCs than in
activated APB MNCs (CB: 101 Ô 5.8 minutes v APB: 210 Ô
8.2 minutes, n ! 3, P Ú .05). Exogenous IL-15 significantly
enhanced CB NK and LAK activities up to comparable levels
of APB (P Ú .05). IL-15 also significantly induced interferong (IFN-g) and tumor necrosis factor-a (TNF-a) protein production (days 1, 3, and 6, P Ú .05, n ! 3) in CB MNCs. IL15–stimulated LAK cells induced a significant lytic response
against two acute lymphoblastic cell lines and two pediatric
neuroblastoma cell lines. Both NK and LAK activities were
augmented by the combination of IL-12 and IL-15, and the
low-dose combination of IL-12 and IL-15 achieved similar
levels of in vitro NK and LAK cytotoxicity compared with
higher doses of either lymphokine. The present study suggests that IL-15 mRNA and protein expression is decreased
in activated CB, secondary, in part, to altered posttranscriptional regulation. The reduced production of IL-15 from CB
MNCs in response to stimulation may contribute to the decrease in IFN-g and TNF-a production and CB cellular immunity. However, exogenous IL-15 enhanced IFN-g and TNF-a
production and NK and LAK cytotoxicities in CB MNCs. The
reduced production of IL-15 from activated CB may contribute to the immaturity of CB cellular immunity and delayed
immune reconstitution after unrelated CB transplantation.
Exogenous IL-15 administration may compensate for the immaturity of CB immunity. The synergistic in vitro effects of
low-dose IL-12 and IL-15 also implies the possible use of
low doses each of IL-12 and IL-15 for enhancing immune
reconstitution and/or possibly as a form of antitumor immunotherapy after CB transplantation.
q 1997 by The American Society of Hematology.
W
killer (NK) cell function7,8 and decreased type-specific antibody production.9,10 Harris et al11,12 and others have also
reported that CB is composed of phenotypically and functionally immature lymphocytes that are associated with decreased allo-antigen–specific cytotoxic T lymphocytes
(CTLs).13,14 We have previously shown reduced mRNA expression and protein production of specific cytokines including granulocyte-macrophage colony-stimulating factor
(GM-CSF), granulocyte colony-stimulating factor (G-CSF),
interleukin-3 (IL-3), macrophage colony-stimulating factor
(M-CSF), and IL-12 from stimulated human CB versus APB
mononuclear cells (MNCs).15-18 Decreased production
of tumor necrosis factor-a (TNF-a) and interferon-g
(IFN-g) from human CB versus APB MNCs has also been
reported.19-21 These defects in CB T-cell immune function
and specific cytokine production may contribute to the delayed immune reconstitution in patients after unrelated CB
transplantation.
IL-15 was recently identified as a novel cytokine that was
originally cloned from a simian kidney epithelial cell line
(CV-1/EBNA).22 The IL-15 gene has 8 exons and is located
in the human genome on chromosome 4q31.23 Human IL15 cDNA encodes a 162 amino acid peptide with a long
leader sequence of 48 amino acids yielding a 114 amino
acid mature protein with a size of 13 to 14 kD.22 IL-15
mRNA is expressed by a wide variety of tissues, including
placenta, skeletal muscle, kidney, lung, heart, fibroblasts,
epithelial cells, and most abundantly by activated monocytes.
IL-15 shares many biologic properties with IL-2, including
induction of T-cell, B-cell, and NK cell proliferation.22,24-28
E HAVE RECENTLY shown that umbilical cord
blood (CB) could be used successfully as an alternative source of hematopoietic stem cells after myeloablative
therapy for both malignant and nonmalignant disorders.1 Recent results after unrelated CB transplantation have suggested a significant delay in immune reconstitution.2,3 CB
hematopoiesis and cellular immunity are developmentally
immature when compared with adult peripheral blood
(APB). This immaturity may be due, in part, to defects in
CB cellular lymphokine and cytokine production.4 Neonatal
lymphocytes from CB have reduced T-cell5,6 and natural
From the Division of Hematology/Oncology and Blood and Marrow Transplantation Children’s Hospital of Orange County, Orange
CA.
Submitted January 13, 1997; accepted June 19, 1997.
J.X.Q., S.m.L., and Y.S. contributed equally to the manuscript.
Supported by grants from the Pediatric Cancer Research Foundation and the Walden W. and Jean Young Shaw Foundation.
Presented in part at the American Society of Hematology, December 1996, Orlando, FL.
Address reprint requests to Mitchell S. Cairo, MD, Director, Hematology/Oncology Research and Blood and Marrow Transplantation, Children’s Hospital of Orange County, 455 S Main St, Orange,
CA 92868.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
‘‘advertisement’’ in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
q 1997 by The American Society of Hematology.
0006-4971/97/9008-0029$3.00/0
Blood, Vol 90, No 8 (October 15), 1997: pp 3106-3117
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CORD BLOOD IL-15 EXPRESSION AND CYTOTOXICITY
Comparatively, IL-2 is predominantly expressed and produced by activated T cells.22,29 IL-15 binds to only the b and
g subunits of the IL-2 receptor complex without requiring
the use of the a subunit to exert its biologic activities.22,30-32
IL-15 has been reported to enhance nonspecific NK and
lymphokine-activating killer (LAK) cytotoxic activities.22,26-28
IL-15 also induces NK cell production of IFN-g, TNF-a,
and GM-CSF.28 In vitro and in vivo studies have shown IL15 to induce a variety of antitumor effects, including induction of CTL and LAK activities.33,34
We hypothesize that the reduced production of IL-15 from
activated CB MNCs compared with APB MNCs may contribute, in part, to immaturity of CB cellular immunity and
potentially the delay in immune reconstitution after unrelated
CB transplantation. Furthermore, exogenous IL-15 administration may enhance CB cellular immunity and has the potential for enhancing immune reconstitution and for immunotherapy after unrelated CB transplantation. In this study,
therefore, we investigated the expression and production of
IL-15 and regulatory mechanisms associated with IL-15 expression in CB compared with APB MNCs. We sought to
determine if IL-15 could enhance CB IFN-g and TNF-a
production and CB NK and LAK cytotoxicities compared
with APB and also to determine if IL-15 would be additive
or synergistic with IL-12 with regard to in vitro antitumor
immunity.
MATERIALS AND METHODS
Isolation of MNCs from CB and APB. Peripheral blood was
obtained by venipuncture from healthy adult volunteers in accordance with the principles of the Declaration of Helsinki. Blood samples were also obtained from the umbilical cords of the placentas
of normal, full-term, nonstressed infants immediately after scheduled
cesarean section. The samples were collected in heparinized syringes. CB and APB MNCs were isolated from whole blood by density
gradient separation on Ficoll-Hypaque gradients (density Å 1.007
g/mL; Sigma Chemical Co, St Louis, MO) for 30 minutes. The
MNCs at the interface were collected, washed twice, and resuspended in RPMI-1640 (GIBCO, Grand Island, NY) culture medium
supplemented with 10% heat-inactivated human AB serum (Sigma).
MNCs isolated by this density gradient separation were purified to
greater than 98% homogeneity, and cell viability as measured by
trypan blue exclusion was more than 99%. There was no difference
in the MNC differential between CB and APB (CB: 82% { 8.0%
lymphocytes and 8.8% { 4.0% monocytes; APB: 86% { 4.0%
lymphocytes and 7.2% { 3.0% monocytes). The cells were cultured
at a density of 1 1 106 cells/mL in culture medium for the following
assays.
RNA isolation and Northern blotting. To determine IL-15
mRNA expression, CB and APB MNCs (60 1 106 cells) were stimulated with lipopolysaccharide (LPS; from Escherichia coli 0127:B8
at 10 mg/mL; Sigma) for up to 48 hours. Total cellular RNA was
extracted from stimulated and unstimulated cells by the method of
Chomczynski and Sacchi.35 Polyadenylated (A/) RNA from cytoplasmic total RNA was then purified with oligo (dT) cellulose column (mRNA purification kit from Pharmacia Biotech Inc, Piscataway, NJ) and electrophoresed on 1% agarose and 5% formaldehyde
gels. The samples were heated in 40% formamide and 14% formaldehyde at 657C for 15 minutes and then cooled before the addition
of 1 mg/mL ethidium bromide. RNA was transferred to nitrocellulose
and baked for 2 hours. Hybridization with an antisense probe made
by transcription of a human cDNA (kindly provided by Dirk Ander-
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son, Immunex, Seattle, WA) was performed at 637C overnight in
50% formamide, 51 sodium chloride sodium citrate (SSC), 11 Denhardt’s, 50 mmol/L sodium phosphate (pH 6.5), 0.1% sodium dodecyl sulfate (SDS), 250 mg/mL salmon sperm DNA, and 10% dextran
sulfate. Blots were washed with 21 SSC at room temperature and
with 0.11 SSC at 687C for 1 hour. Blots were exposed to Kodak
XAR-5 film (Eastman Kodak, Rochester, NY). The hybridization
signals were quantified by densitometry of autoradiographs. Blots
were then rehybridized with glyceride-3 phosphate dehydrogenase
(GAPDH) probe, 775-bp Pst I/Xba I fragment from phcGAP (ATCC,
Rockville, MD), as an internal standard. The levels of IL-15 mRNA
were calculated by normalizing signal optical densities to those of
GAPDH mRNA. Cord and adult Northern blot analyses were performed simultaneously under identical hybridization conditions and
with the same amount of exposure time of the blot.
Immunoblot analysis. Plastic adherent monocytes (10 to 20 1
106 cells, ú95% CD14/ monocytes) were obtained from MNC culture (5 1 106 cell/mL) after being incubated at 377C for 1 hour
according to the method of D’Andrea et al.36 Stimulated (LPS at 10
mg/mL for 12 hours) and adhered (without LPS) monocyte cultures
were gently washed twice in phosphate-buffered saline (PBS).
Whole cells were then lysed in PBS containing 1% Nonidet P-40,
0.5% sodium deoxycholate, 0.1% SDS, 1 mmol/L EDTA, 1 mmol/
L phenylmethyl sulfonyl fluoride,37 and 20 mg/mL pepstatin A for
30 minutes on ice. Lysates were spun in a microfuge at 12,000 rpm
for 15 minutes. Supernatant was collected and stored at 0707C for
Western blot analysis. Lysates were thawed, and quantitation of total
protein from each sample was performed in duplicate, twice before
loading, using the BCA protein assay (Pierce, Rockford, IL). Equal
amounts (20 mg) of protein were loaded per lane, and proteins were
separated by 8% SDS-polyacrylamide gel electrophoresis (SDSPAGE). Proteins were electrophoretically transferred to nitrocellulose membranes and blocked with 5% nonfat dry milk solubilized
in PBS for 1 hour at room temperature. Blots were then incubated
in 10 mg equivalent of goat polyclonal antibody (AB-247-NA; R &
D Systems, Minneapolis, MN) directed against human IL-15 protein
in 2% milk (vol/vol, solubilized in PBS) overnight at 47C. Incubation
with 0.2% (vol/vol) horseradish peroxidase-conjugated swine antigoat IgG (BioSource International, Camarillo, CA) for 1 hour at
room temperature followed. IL-15 protein bands were visualized
by development with luminol/peroxide chemiluminescent substrate
(Pierce) for 1 minute and exposed to x-ray film for 30 seconds. Twodimensional densitometry of film was performed using the BioImage Model 50S (Millipore, Bedford, MA) automated scanning
system. Immunoblot analyses were performed with three different
samples.
Nuclear run-on transcription assay. Cultures of three different
samples were stimulated with LPS (10 mg/mL; Sigma) for 12 hours
to obtain maximal induction of IL-15. Nuclei isolation and nuclear
run-on assays were performed as previously described38 and involved
modification procedures described by Weber et al39 and Groudine
et al.40 Nuclear run-on assays were performed with cDNA (IL-15,
0.48 kb cDNA; GAPDH, 775-bp Pst I/Xba I fragment from phcGAP)
as targets. The amount of target DNA per slot was 1 mg. The hybridization mixture contained 2.5 to 5 1 107 cpm/5 mL. Run-on signal
strengths were determined by densitometry of autoradiographs. The
density of the bands was calculated by normalizing values with
respect to the signals of internal standards (GAPDH).
mRNA half-life. Cord and adult MNCs (60 1 106 cells) were
stimulated with LPS (10 mg/mL) for 12 hours before exposure to
the transcriptional inhibitor, actinomycin D (10 mg/mL), as described
previously.17 Cells were harvested at intervals of 0, 60, 120, 240,
and 360 minutes. Cytoplasmic RNA was extracted, poly (A)/ RNA
was purified, and Northern blot analysis was performed as described
above for the IL-15 mRNA expression. The amount of IL-15 mRNA
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was normalized to the amount of GAPDH mRNA in each sample
and then expressed as a percentage, setting the amount of mRNA
at time 0 equal to 100%. The data were plotted against the time
after addition of actinomycin D, and the half-life of each transcript
was calculated based on the resultant graphs.
Induction of cytokine production and NK and LAK cytotoxicity.
Recombinant simian IL-15 (specific activity on CTLL proliferation,
3.33 1 105 U/mg) was kindly provided by Dr T. Troutt (Immunex),
and the recombinant human IL-12 (specific activity on phytohemagglutinin blast proliferation, 5.26 1 106 U/mg) was kindly provided
by Dr S. Wolf (Genetics Institute, Cambridge, MA). MNCs at 5 1
106 cells/mL were placed into a plastic petri dish for 1 hour of
incubation at 377C to remove monocytes. Monocyte-depleted MNCs
(MD MNCs) were adjusted to 1 1 106 cells/mL, seeded in 24-well
flat-bottom plates containing varying concentrations of cytokines,
and incubated at 377C in a 5% CO2 humidified incubator. NK activity
was measured against K562 target cells (NK-sensitive, a human
erythroleukemic cell line; ATCC) after 18 hours of stimulation, and
LAK activity was measured against Daudi target cells (LAK-sensitive, a human Burkitt’s lymphoma; ATCC) after 72 hours of stimulation. At the end of the incubation, the effector cells were harvested,
washed, and resuspended in appropriate concentrations based on the
ratios of effector to target cells (E:T ratio) for the cytotoxicity assays.
Cytotoxicity assay. A standard 3-hour 51Cr-release assay41 was
performed to measure the cytotoxicity. Briefly, the target cells were
labeled with 100 mCi of Na2CrO4 , washed twice, and resuspended
in a concentration of 5 1 104 cells/mL. One hundred microliters of
each target and effector cell suspension with E:T ratios (20:1, 10:1,
and 5:1) was added to a V-bottom 96-well culture plate. The mixture
was centrifuged briefly and incubated at 337C for 3 hours. At the
end of the incubation, 150 mL of cell-free supernatant was collected
from each well. The radioactivity was measured in a Beckman LS
1800 Liquid Scintillation Counter (Beckman, Fullerton, CA). All of
the samples were run in triplicate. The percentage of lysis was
calculated at each E:T ratio using the formula ([Experimental 0
Spontaneous Release]/[Maximum 0 Spontaneous Release]) 1 100%
and then converted to lytic units (LU; 30% target cell killing in 107
effector cells) using a computer-assisted program.42 To determine
the tumoricidal spectrum of CB nonspecific cytotoxicity, two acute
lymphoblastic leukemia cell lines, CCRF-CEM (T cells; ATCC) and
CCRF-SB (B cells; ATCC), and two neuroblastoma cell lines, NB100 (ATCC) and SK-N-MC (ATCC), were used as target cells.
The study of effects of IL-15 and IL-12 in combination on CB NK
and LAK activities. A method previously described by DeBlakerHohe et al43 was used to determine if IL-15 and IL-12 in combination
induces a synergistic or additive NK and LAK activity from CB
MNCs. To avoid a deviation of using controls twice while comparing
the cytolytic response from the combination of two cytokines with
the sum of that from each single cytokine, the method was modified
by subtracting the control from each single data before being applied
to the calculation. Each cytotoxicity result of 17 combinations from
5 doses of IL-15 (0.1, 0.5, 1.0, 5.0, and 10 ng/mL) and 4 doses of
IL-12 (0.1, 0.5, 1.0, and 10 U/mL) was compared with the sum of
that from the same dose of each single cytokine using the following
formula: [(Cytotoxicity in Combination 0 Sum of Each Cytokine
Alone)/(Sum of Each Cytokine Alone)] 1 100%. Based on the calculated results, the synergistic effect was arbitrarily defined as the
cytolytic response from the combination of two cytokines exceeding
the sum of that of each single cytokine by more than 10%, additive
as 0% to 10% and nonadditive as less than 0%.
TNF-a and IFN-g enzyme-linked immunosorbent assay (ELISA).
CB and APB MNCs at a concentration of 1 1 106 cells/mL were
stimulated by IL-15 (50 ng/mL) for 24, 72, and 144 hours. The
supernatant was collected and the protein level was measured by
ELISA (Biosource) following the manufacturer’s protocol. All of
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the samples were run in duplicate and data were presented as the
mean { SEM. The sensitivity of the assay was 7.8 pg/mL.
Statistical analysis. Results from cytotoxicity studies and
ELISA were presented as the mean { SEM of three or more samples.
Student’s t-test was used for determining significant differences between two groups, and Kruskal-Wallis nonparametric ANOVA test
was used for comparing multiple groups with Bartlett’s test as the
posttest for determining specific significant subgroups (Instat Graph
Pad Software, San Diego, CA). A P value õ.05 was considered
significant.
RESULTS
Reduced IL-15 mRNA expression in stimulated cord versus adult MNCs. Northern blot analyses of CB and APB
MNCs were performed simultaneously under identical conditions to compare the IL-15 mRNA expression before and
after LPS stimulation. Unstimulated MNCs from both CB
and APB had an undetectable expression of IL-15 mRNA.
In a time course study of IL-15 mRNA expression after
LPS (10 mg/mL) stimulation, IL-15 mRNA expression was
induced within 6 hours upon LPS stimulation and reached
a peak level at 12 hours in both CB and APB MNCs, returning to basal level after 24 hours. However, IL-15 mRNA
expression in CB MNCs was only 25% { 2.0% (mean {
SEM, n Å 4, P õ .05) compared with the level in APB
MNCs after 12 hours of stimulation with LPS (Fig 1).
Decreased IL-15 protein accumulation in cord versus
adult MNCs. IL-15 protein production from whole cell lysates was determined by Western immunoblot analysis with
a goat polyclonal anti–IL-15 antibody (Fig 2). Equal
amounts of protein (20 mg) were loaded per lane after being
quantitated in duplicate twice. Bands with a molecular size
of 13 kD were detected from stimulated cord and adult cells.
IL-15 was not detected in adhered cells without LPS. IL-15
protein production in LPS-stimulated CB monocytes (lanes
5 and 6) was only 30% { 2.5% (mean { SEM) of the level
in APB monocytes (lanes 3 and 4; n Å 3, P õ .05) after 12
hours of LPS stimulation.
Comparable transcriptional rate of the IL-15 gene in cord
versus adult MNCs. Nuclear run-on transcriptional analysis
was performed to determine if the low amount of IL-15
mRNA in stimulated CB MNCs was due to a decreased
transcription rate of the IL-15 gene. Nuclear run-on transcripts from nuclei isolated from CB MNCs and stimulated
with LPS for 12 hours were compared with run-on transcripts
isolated from similarly treated APB MNCs. As shown in Fig
3, unstimulated CB and APB MNCs showed low basal level
signals of IL-15 transcript (optical density [OD], õ 0.1),
which was approximately the same in both cord and adult.
After stimulation with LPS (12 hours), the transcriptional
rate of the IL-15 gene was significantly increased in both
CB and APB MNCs (cord, 20- { 2.5-fold; adult, 22- { 1.7fold; n Å 3, P õ .05). However, there was no appreciable
difference between activated CB and APB MNC in the degree of transcriptional activation (P Å not significant [NS]).
Decreased IL-15 mRNA half-life in cord versus adult. Because the transcriptional rate of the IL-15 gene was virtually
the same for both CB and APB MNCs, the stability of IL15 mRNA was compared in stimulated CB and APB MNCs
by blocking mRNA synthesis with actinomycin D to deter-
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mine whether the differential regulation was occurring at the
posttranscriptional level. CB and APB MNCs were stimulated with LPS (10 mg/mL) for 12 hours before actinomycin
D (10 mg/mL) was added for various time periods (0 to 360
minutes.). Northern blots of poly (A)/ RNA were hybridized
with an antisense riboprobe made by transcription of human
cDNA. The levels of IL-15 mRNA progressively decreased
during actinomycin D exposure in both CB and APB MNCs.
Transcripts were quantitated by densitometric scanning of
the autoradiographs. As shown in Fig 4, the measured
mRNA half-life of IL-15 from stimulated CB MNCs was
approximately twofold lower than that from stimulated APB
MNCs (t1/2 : 101 { 5.8 minutes v 210 { 8.2 minutes, CB v
APB, mean { SEM, n Å 3, P õ .05).
Enhanced CB and APB NK and LAK cytotoxic activities
after IL-15 stimulation. The baseline NK and LAK cytotoxicities were determined after 18 and 72 hours of incubation in the absence of cytokines. The NK activity of CB
against K562 was significantly lower than that of APB MD
MNCs (CB v APB: 60 { 9 v 115 { 18 LU, P õ .05, n Å
10; Fig 5). However, the LAK activity of CB was similar
to APB (CB v APB: LAK, 43 { 10 v 44 { 19 LU, P Å
Fig 2. Detection of IL-15 in stimulated cord (CB) and adult (APB)
monocytes. Immunoblot analysis of LPS (12 hours) stimulated CB
and APB monocytes using anti–huIL-15 goat antibody. Equal
amounts of protein (20 mg) were loaded per lane. Before loading,
quantitation of total protein from each sample was performed in
duplicate, twice using BCA protein assay. Lane 1, rhIL-15; lane 2,
adhered monocytes; lanes 3 and 4, LPS-stimulated adult monocytes;
lanes 5 and 6, LPS-stimulated cord monocytes. The lower panel bar
graph represents comparative IL-15 protein production from three
different LPS-stimulated (12 hours) CB and APB monocytes. The
amount of IL-15 protein was expressed as a percentage, setting the
amount of APB level equal to 100% (APB v CB, 100% v 30% Ô 2.5%,
P Ú .05, n ! 3).
Fig 1. Time course of induction of IL-15 mRNA expression in cord
(CB) and adult (APB) MNCs. CB and APB MNCs were isolated, cultured in RPMI, and stimulated with 10 mg/mL LPS for 0, 3, 6, 12, 24,
and 48 hours. Cells were harvested and poly (A)" RNA samples were
analyzed by Northern blot analysis of IL-15 mRNA (1.5 kb). Results
shown are representative of three different CB and APB RNA blot
hybridizations, normalized to GAPDH signal. The lower panel bar
graph represents comparative IL-15 mRNA expression from LPSstimulated (12 hours) CB and APB MNCs. Four different CB and APB
samples were analyzed after 12 hours of LPS stimulation (10 mg/mL)
for IL-15 mRNA expression by Northern blot analysis. The amount of
IL-15 mRNA was expressed as a percentage, setting the amount of
APB mRNA equal to 100% (APB v CB, 100% v 25% Ô 2.0%, P Ú .05,
n ! 4).
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NS, n Å 10; Fig 6). After incubation of MD MNCs with IL15 (10 ng/mL), both CB and APB NK activities (18 hours)
were significantly increased over control (control v IL-15 at
10 ng/mL: CB, 45 { 9 v 533 { 72 LU, P õ .01, n Å 10;
APB, 115 { 18 v 537 { 68 LU, P õ .05, n Å 6). CB NK
activity reached a comparable level of APB (IL-15 at 10 ng/
mL: CB v APB, 533 { 72 v 539 { 68 LU, P Å NS). IL-15
(10 ng/mL) also induced a significant increase of both CB
and APB LAK activities against Daudi target cells (control
v IL-15 at 10 ng/mL: CB, 43 { 10 v 843 { 64 LU, P õ
.01, n Å 12; APB, 44 { 19 v 420 { 107 LU, P õ .01, n Å
6; Fig 6). However, CB MD MNCs was more responsive to
IL-15 stimulation than APB for induction of LAK activity
(CB v APB: IL-15 at 1.0 ng/mL, 262 { 62 v 144 { 91, P
õ .05; IL-15 at 10 ng/mL 843 { 64 v 420 { 107 LU, P õ
.01; Fig 6).
IL-15 induced nonspecific tumoricidal NK and LAK activities. Four cell lines, CCRF-CEM, CCRF-SB, NB-100, and
SK-N-MC, were used to examine in vitro antitumor activity
of CB NK and LAK cells after IL-15 stimulation. IL-15 (10
ng/mL) stimulation resulted in enhanced CB cytotoxicity
against all of these tumor cell lines. As shown in Fig 7A,
three cell lines, CCRF-CEM, SK-N-MC, and NB-100, be-
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Fig 3. Nuclear run-on analyses of IL-15 transcription in cord (CB) versus adult (APB) MNCs stimulated
with LPS (10 mg/mL for 12 hours). Equivalent
amounts of radioactive labeled RNA were hybridized
to filters containing the indicated target DNA. Results shown are representative of three different experiments. US, unstimulated; S, stimulated; pUC 18,
control vector; GAPDH, 775-bp Pst I/Xba I fragment
from phc GAP; IL-15, 480-bp cDNA (CB 20- Ô 2.5-fold
v APB 22- Ô 1.7-fold, P ! NS, n ! 3).
came more sensitive to CB NK cytotoxicity induced by IL15 (control v IL-15 stimulated: CCRF-CEM, 50 { 34 v 229
{ 34 LU, P õ .05; SK-N-MC, 18 { 17 v 269 { 91 LU, P
õ .01; NB-100, 22 { 4 v 208 { 25 LU, P õ .01, n Å 4).
IL-15–induced CB LAK cytotoxicity produced an enhanced
lytic response against all four tumor cell lines over controls
(control v IL-15 stimulated: CCRF-CEM, 55 { 39 v 318 {
3 LU, P õ .01; CCRF-SB, 18 { 12 v 436 { 114 LU, P õ
.05; SK-N-MC, 50 { 25 v 358 { 13 LU, P õ .01; NB-100,
33 { 31 v 330 { 40 LU, P õ .01, n Å 3; Fig 7B).
Additive and synergistic effects of low doses of IL-15 and
IL-12 on CB NK and LAK cytotoxicity. The effects of the
combination of IL-15 and IL-12 on NK and LAK cytotoxicity
was examined by comparing the cytolytic response from two
cytokines in combination with the sum of that from each
single cytokine. The results in Table 1 show that using lower
doses of IL-15 (0.1, 0.5, and 1.0 ng/mL) and IL-12 (0.1, 0.5,
and 1.0 U/mL) concomitantly generated either synergistic or
additive effects on CB NK cytotoxicity against K562 targets.
The synergy from these lower dose combinations induced a
comparable NK cytotoxicity compared with single higher
doses of each cytokine individually. Specifically, IL-15 (1.0
ng/mL) and IL-12 (0.5 U/mL) induced a lytic response comparable to that induced by 10 ng/mL of IL-15 alone or 10 U/
mL of IL-12 alone (532 { 64 v 473 { 70 LU, P Å NS, and
v 410 { 48 LU, P Å NS). Conversely, higher dose combinations of IL-12 and IL-15 induced only nonadditive effects on
CB NK cytotoxicity. Table 2 summarizes the combined ef-
Fig 4. The half-life of IL-15 mRNA in cord (CB) (A)
versus adult (APB) (B) MNCs. Actinomycin D (10 mg/
mL) was added for the indicated times to cells from
cord (A) and adult (B), stimulated with LPS (10 mg/
mL) for 12 hours. Poly (A)" RNA was analyzed by
Northern blotting for the presence of IL-15 transcript
(1.5 kb). The data were plotted against the time after
the addition of actinomycin D. Results shown are
representative of three different experiments (t1/2 :
101 Ô 5.8 minutes v 210 Ô 8.2 minutes, CB v APB, P
Ú .05, n ! 3).
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Fig 5. IL-15 induction of CB and APB MNC NK
cytotoxicity against K562 line. *CB v APB, 45 Ô 9 v
115 Ô 18 LU, P Ú .05, n ! 10; ( ) CB; ( ) APB.
fects of IL-15 and IL-12 on CB LAK cytotoxicity against
Daudi targets. The synergistic effect of IL-12 and IL-15 is
seen at two combinations of lower doses of IL-15 and IL-12
(0.1 or 0.5 ng/mL of IL-15 / 0.1 U/mL of IL-12) and the
lytic response from the latter combination is comparable to
that of a single higher dose of IL-12 (10 U/mL), but much
lower than that of IL-15 (10 ng/mL) (IL-15 / IL-12 v IL-12
alone v IL-15 alone: 512 { 48 LU v 501 { 54 LU, P Å NS;
and v 823 { 68 LU, P õ .01). The higher dose combinations
of IL-15 and IL-12 induced a suppressive effect on LAK
cytotoxicity when compared with the single higher dose of
IL-12 and IL-15 (10 ng/mL IL-15 / 10 U/mL IL-12 v 10 ng/
mL IL-15 alone v 10 U/mL IL-12 alone, 259 { 77 v 823 {
68 LU, P õ .01; 259 { 77 v 501 { 54, P õ .05, n Å 10).
IL-15 enhancement of IFN-g and TNF-a production from
CB MNCs. IL-15 (50 ng/mL) stimulation induced a significant increase of IFN-g protein production in CB MNCs
(IL-15 stimulated v control: day 1, 112 { 13 v 3.3 { 1.2
pg/mL, P õ .01; day 3, 480 { 75 v 8 { 4 pg/mL, P õ .01;
day 6, 670 { 20 v 135 { 52 pg/mL, P õ .01, n Å 3)
However, the IFN-g levels in IL-15–stimulated CB were
still significantly lower than stimulated APB (IL-15–stimulated CB v IL-15–stimulated APB: day 1, 112 { 13 v 567
{ 44 pg/mL, P õ .01; day 3, 480 { 75 v 866 { 30 pg/mL,
P õ .05; day 6, 670 { 20 v 830 { 20 pg/mL, P õ .05, n
Å 3; Fig 8A). Similarly, IL-15 (50 ng/mL) stimulation also
significantly increased TNF-a protein production in CB
MNCs (IL-15 stimulated v control: day 1, 308 { 81 v 50 {
26 pg/mL, P õ .01; day 3, 423 { 76 v 70 { 30 pg/mL, P
õ .01; day 6, 358 { 44 v 128 { 51 pg/mL, P õ .05, n Å
3). The TNF-a production in CB MNCs was still significantly lower than APB (IL-15–stimulated CB v stimulated
APB: day 1, 308 { 46 v 676 { 88 pg/mL, P õ .01; day 3,
423 { 76 v 983 { 179 pg/mL, P õ .05; day 6, 358 { 44 v
1,125 { 108, P õ .01, n Å 3; Fig 8B).
DISCUSSION
Numerous studies have demonstrated the success of hematopoietic growth factors to enhance hematologic reconstitution after stem cell transplantation.44-51 Vowels et al52 re-
Fig 6. IL-15 induction of CB and APB MNC LAK
activity against Daudi cell line. CB v APB: *IL-15 at 1
ng/mL, 258 Ô 55 v 132 Ô 51, P Ú .05; **IL-15 at 10
ng/mL, 843 Ô 64 v 420 Ô 107 LU, P Ú .01. ( ) CB;
( ) APB.
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Fig 7. (A and B) IL-15 (10 ng/mL) induction of CB NK and LAK cytotoxicity against several tumor cell lines. Acute lymphoblastic leukemia
cell lines: CCRF-CEM (T-lymphoblastoid cells) and CCRF-SB (B-lymphoblastoid cells); neuroblastoma cell lines: SK-N-MC and NB-100. (A) The
tumoricidal spectrum of CB NK activity: IL-15 (10 ng/mL) v unstimulated: CCRF-CEM*, 229 Ô 75 v 50 Ô 34 LU; SK-N-MC**, 269 Ô 91 v 18 Ô 17
LU; NB-100**, 208 Ô 25 v 22 Ô 4 LU; n ! 4. ( ) Medium; ( ) IL-15 (10 ng/mL). (B) The tumoricidal spectrum of CB LAK activity: IL-15 (10 ng/
mL) v unstimulated: CCRF-CEM**, 318 Ô 3 v 55 Ô 39 LU; CCRF-SB*, 436 Ô 114 v 18 Ô 12 LU; SK-N-MC**, 358 Ô 13 v 50 Ô 25 LU; NB-100**,
330 Ô 40 v 33 Ô 31 LU; n ! 3. ( ) Medium; ( ) IL-15 (10 ng/mL). *P Ú .05; **P Ú .01.
ported that the use of GM-CSF post-CB transplantation
resulted in rapid engraftment and mild graft-versus-host disease. However, the delay in immune reconstitution post-CB
transplantation may be due to the defects in CB cellular
immunity and cytokine production.2-4 We have recently reported that the reduced expression and production of IL-12
from activated CB may contribute to the immaturity in CB
cellular immunity.18 The number and functionality of donorderived lymphocytes in patients after CB transplantation remains to be determined. The use of exogenous cytokines
post-CB transplantation that enhance CB immune function
may compensate for the immaturity of CB cellular immunity
and enhance immune reconstitution and CB tumor immunity
post-CB transplantation.
Although there are many similar biologic activities between IL-2 and IL-15, the regulation of IL-15 expression
differs markedly from that of IL-2. IL-15 mRNA is expressed in a variety of tissues, including placenta, skeletal
muscle, kidney, and activated monocytes/macrophages but
not normal T cells.22 Enhancement of IL-15 protein production from monocytes occurs in response to a wide variety
of agonists including LPS, IFN-g, Bacillus Calmette-Guerlin
(BCG), Mycobacterium tuberculosis, Toxoplasma gondii, or
Salmonella cholersesuis.53-55 During states of increased de-
Table 1. The Synergistic and Additive Effects of IL-15 and IL-12 on Cord Blood NK Cytotoxicity Against K562
IL-15 (ng/mL)
IL-12
(U/mL)
0
0.1
0.5
1.0
10
0
156
283
352
410
{
{
{
{
0.1
23
32
54
48
54
227
368
449
509
{
{
{
Ô
{
10*
28 (/8%)†
63 (/9%)
64 ("10%)
67 (/9%)
0.5
133
383
486
554
{
Ô
Ô
Ô
19
63 ("33%)
65 ("17%)
65 ("14%)
ND
1.0
179
405
532
489
559
{
Ô
Ô
{
{
29
59
64
71
65
("21%)
("15%)
(08%)
(05%)
5.0
10
383 { 61
534 { 92 (01%)
ND
514 { 107 (030%)
457 / 92 (042%)
473 { 70
307 { 71 (051%)
ND
495 { 97 (040%)
631 { 91 (029%)
Abbreviation: ND, not done.
* NK cytotoxicity (lytic units) in Table 1 is presented as the mean { SEM (n Å 8).
† (%) indicates the effects (synergistic Û 10%, additive 0% to 10%, not additive õ 0%) of IL-15 and IL-12 in combination on NK activity
compared with the sum of each cytokine alone which is obtained by the formula as below. All the data are normalized by subtracting the control
prior to being applied to calculation:
Cytotoxicity in Combination 0 (Sum of Each Cytokine Alone)
1 100%.
Sum of Each Cytokine Alone
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Table 2. The Synergistic and Additive Effects of IL-15 and IL-12 on CB LAK Cytotoxicity Against Daudi
IL-15 (ng/mL)
IL-12
(U/mL)
0
0.1
0.5
1.0
10
0
198
378
435
501
{
{
{
{
0.1
34
49
54
54
86
313
460
487
375
{
Ô
{
{
{
24*
54 ("10%)†
51 (03%)
56 (07%)
97 (036%)
0.5
234
512
519
516
{
Ô
{
{
1.0
62
48 ("19%)
53 (017%)
63 (023%)
ND
348
542
574
573
354
{
{
{
{
{
58
44
68
75
92
(01%)
(022%)
(027%)
(057%)
10
100
823 { 68
576 { 51 (044%)
ND
403 { 60 (068%)
259 { 77 (080%)
930 { 57
601 { 76 (047%)
ND
372 { 75 (076%)
381 { 60 (078%)
Abbreviation: ND, not done.
* LAK cytotoxicity (lytic units) in Table 2 is presented as mean { SEM (n Å 10).
† (%) indicates the effects (synergistic Û 10%, not additive õ 0%) of IL-15 and IL-12 in combination on LAK activity compared with the sum
of each cytokine alone. All the data are normalized and calculated in the same way as in Table 1.
mand (stimulation), mononuclear phagocytes contribute significantly to the production of IL-15 in both CB and APB.
However, our present results suggest that CB MNCs express
and produce less IL-15 in response to bacterial stimulation
(Figs 1 and 2). The decreased IL-15 mRNA expression in
CB versus APB MNCs is not secondary to alteration in IL15 gene transcription (Fig 3). In comparison, alterations in
posttranscriptional stability appear to account, in part, for
the decrease in IL-15 mRNA expression in CB versus APB
MNCs (Fig 4).
Control of mRNA stability is not well understood, but the
process is thought to involve various factors interacting with
specific mRNA sequences.56,57 The adenosine / uridine
(AU)-rich or AUUUA-repeat elements (ARE) in the 3* untranslated regions (UTR) of many cytokine and protooncogene transcripts are known to be the targets of a pathway
for selective processing and mRNA degradation.58-62 Several
AUUUA repeats of the 3* UTR of IL-15 sequence may
contribute to the instability of IL-15 mRNA.
We previously reported the complex posttranscriptional
regulatory mechanisms associated with several cytokines.
The reduced accumulation of M-CSF, GM-CSF, and IL-12
mRNA in CB MNCs is associated with a reduction in mRNA
half-life compared with APB MNCs, whereas the rate of
gene transcription remains comparable in CB and APB
MNCs.15,17,18 The 3* UTR of these cytokines have multiple
copies of AUUUA-elements.63,64 A reduced mRNA half-life
and comparable transcription rates for GM-CSF, M-CSF,
and IL-12 in CB versus APB MNCs indicate that these AREcontaining transcripts may also be less stable in CB MNCs.
Translational inhibition by cycloheximide after stimulation
of cells causes a superinduction of GM-CSF, as well as MCSF mRNA, which is approximately 2.5-fold greater in CB
versus APB MNCs.17,65 Increased transcript stabilization in
stimulated CB MNCs after CHX treatment suggests that,
before translational inhibition, higher levels of a translational-dependent nuclease may be present in CB MNCs. An
ARE-directed endonuclease66 and several ARE-binding factors61,62,67-71 have been identified. One of these is a 37-kD
protein designated AUF1. We have recently reported that
the decreased GM-CSF mRNA stability in CB versus APB
MNCs was inversely correlated with AUUUA-element bind-
Fig 8. (A and B) IL-15 (50 ng/mL) induction of IFN-g and TNF-a production in CB MNCs. (A) Time course of IFN-g production in CB and APB
MNCs; ( ) CB-control, ( ) CB–IL-15, ( ) APB-control, ( ) APB–IL-15. (B) Time course of TNF-a production in CB and APB MNCs; ( ) CBcontrol, ( ) CB–IL-15, ( ) APB-control, ( ) APB–IL-15.
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ing activity and with the levels of AUF1 binding factor.65 It
seems likely that various protein factors interacting with
specific mRNA sequences exist in vivo and are involved in
the regulation of AU-rich mRNA decay. Any alteration in
the expression and/or biologic activities of these various
protein factors in stimulated CB MNCs could contribute to
the reduction of IL-15 mRNA expression. Further studies
are required to test these possibilities.
The immaturity of CB immunity, which is associated with
decreased production of IL-2, IL-12, IL-15, IFN-g, and
TNF-a, may contribute to diminished CB NK, LAK, and
CTL cytotoxicities. Seki et al7 and others have reported that
NK cytotoxicity is decreased in CB compared with APB.
IL-2 can enhance the NK cytotoxicity of CB MNCs to the
level of APB MNC activity.11 We have recently18 reported
that IL-12 can enhance CB NK cytotoxicity up to levels of
APB MNC activity. IL-15 not only enhances T-cell function,
but also enhances cytolytic function of both CD56dim NK
and CD8/ T cells.22,28,32 Carson et al28 reported that activation
of CD56dim NK cells by IL-15 was similar to that of IL2. However, the IL-15–enhanced NK cytotoxic activity is
completely IL-2 independent. Our present studies suggested
that IL-15 also enhanced CB NK and LAK activities up to
the adult level. CB LAK activity appeared to be more sensitive to exogenous IL-15 compared with APB (Fig 5). Tumoricidal studies showed that IL-15 induced significant CB NK
and LAK activities against a broad range of neuroblastoma,
leukemia, and lymphoma cell lines (Fig 7).
Although IL-2 has been shown to have therapeutic benefits
for some cancer patients,72 the substantial toxicities associated with high doses of IL-2 have limited its use clinically.73
IL-12 has also experienced dose-limiting toxicities.74 Recently, IL-15 has been shown to mimic the antitumor activities of IL-2 with potentially less toxicity in an in vivo animal
model.33 Further studies are required to evaluate this aspect.
Combinations of lower doses of IL-15 and IL-12 might have
the potential of augmenting in vivo antitumor immune function and minimizing toxicity. DeBlaker-Hoke et al43 showed
a synergistic effect on inducing APB NK and LAK activities
by the combination of lower doses of IL-2 and IL-12. Carson
et al28 suggested that the combination of IL-12 and IL-15
had a synergistic effect on augmenting APB NK cytolytic
activity and IFN-g production. In the present study, we demonstrated that low-dose combinations of IL-12 (0.1 U/mL)
and IL-15 (0.1 to 1.0 ng/mL) induced a synergistic or additive effect on CB NK cytotoxicity, except for the combination of IL-12 (1.0 U/mL) and IL-15 (1.0 ng/mL). The synergistic NK activity reached the same levels as a single high
dose (IL-12, 10 U/mL; IL-15, 10 ng/mL) of either individual
cytokine. Although no synergistic or additive effects from
high-dose combinations of IL-12 (5 to 10 U/mL) and IL-15
(5 to 10 ng/mL) on CB NK activity are seen, the cytotoxicity
levels were still higher than that induced by the single dose
of individual cytokine (Table 1). Similarly, low-dose combinations of IL-12 (0.1 U/mL) and IL-15 (0.1 to 0.5 ng/mL)
had a synergistic effect on CB LAK activity that is comparable to the level as a single high dose of IL-12 (10 U/mL),
but lower than a single high dose of IL-15 (10 to 100 ng/
mL). However, the high-dose combination of IL-12 (1.0 to
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10 U/mL) and IL-15 (10 to 100 ng/mL) had a suppressive
effect on CB LAK activity (Table 2). This suppression may
be due to NK cell apoptosis in which decreased numbers of
killer cells resulted in a low level of LAK activity. This
observation is consistent with the report by Ross and Caligiuri75 in which costimulation of IL-12 and IL-15 or IL-12
and IL-2 induced NK cell proliferation and IFN-g production
initially, followed by NK cell apoptosis and a decline in
IFN-g production.
The IFN-g and TNF-a production in IL-15–stimulated
CB MNCs was significantly induced and increased up to
the unstimulated APB level, but far lower than the IL-15
stimulated APB level (Fig 8). This result is consistent with
our earlier observation on IFN-g production in IL-12–stimulated CB and APB MNCs.18 This partial compensation after
IL-15 stimulation suggests that the decreased production of
IFN-g and TNF-a by CB MNCs may be due to at least
two factors: defective CB IFN-g and TNF-a production and
defective CB IFN-g – and TNF-a –inducing cytokines, such
as IL-15, IL-12, and IL-2. Interestingly, the effect of IL-15
on CB NK activity and IFN-g production showed that IL15 alone is capable of inducing CB NK activity up to the
APB level; however, IL-15 alone cannot compensate for
IFN-g production up to the stimulated APB level (Figs 5 and
8). This disparity indicates the important role of intrinsically
deficient cytokine production such as IL-12, IL-15, and IFNg in the pathogenes of the immaturity of CB cellular immunity. Further studies are required to verify these observations.
In summary, the present study showed that IL-15 mRNA
and protein production is decreased in activated CB compared with APB MNCs. This discrepancy in IL-15 production is secondary, at least in part, to altered posttranscriptional regulation. The reduced production of IL-15 from
activated CB MNCs might contribute to the immaturity in
CB cellular immunity. However, exogenous IL-15 stimulation may compensate for the immaturity in CB immunity by
enhancing NK and LAK activities and by inducing IFN-g
and TNF-a production. The additional synergistic effects of
lower doses of IL-15 in combination with IL-12 suggests
the potential benefit of the combination of each cytokine to
increase CB antitumor immunity and potentially decrease
toxicity compared with higher doses of either of the cytokines alone. Further studies are needed to define the clinical
implications of these findings and the potential use of IL-15
to enhance CB cellular immunity and/or accelerate immune
reconstitution after CB transplantation.
ACKNOWLEDGMENT
The authors thank Sally Anderson, Renee Dulak, and Linda Rahl
for their editorial assistance in the preparation of this manuscript.
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