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Blood, Vol. 92 No. 3 (August 1), 1998:
pp. 901-907
Negative Regulation by Interleukin-3 (IL-3) of Mouse Early B-Cell
Progenitors and Stem Cells in Culture: Transduction of the Negative
Signals by  c and IL-3 Proteins of IL-3 Receptor and Absence of
Negative Regulation by Granulocyte-Macrophage Colony-Stimulating
Factor
By
Takuya Matsunaga,
Fumiya Hirayama,
Yuji Yonemura,
Richard Murray, and
Makio Ogawa
From the Department of Veterans Affairs Medical Center and the
Department of Medicine, Medical University of South Carolina,
Charleston, SC; and DNAX Research Institute of Molecular and Cellular
Biology, Palo Alto, CA.
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ABSTRACT |
The receptors for interleukin-3 (IL-3), granulocyte-macrophage
colony-stimulating factor (GM-CSF), and IL-5 share a common signaling
subunit  c. However, in the mouse, there is an additional IL-3
signaling protein, IL-3, which is specific for IL-3. We have
previously reported that IL-3 abrogates the lymphoid potentials of
murine lymphohematopoietic progenitors and the reconstituting ability
of hematopoietic stem cells. We used bone marrow cells from c- and
IL-3-knock-out mice to examine the relative contributions of the
receptor proteins to the negative regulation by IL-3. First, we tested
the effects of IL-3 on lymphohematopoietic progenitors by using
lineage-negative (Lin ) marrow cells of 5-fluorouracil
(5-FU)-treated mice in the two-step methylcellulose culture we reported
previously. Addition of IL-3 to the combination of steel factor (SF,
c-kit ligand) and IL-11 abrogated the B-lymphoid potential of the
marrow cells of both types of knock-out mice as well as wild-type mice.
Next, we investigated the effects of IL-3 on in vitro expansion of the
hematopoietic stem cells. We cultured
LinSca-1-positive, c-kit-positive marrow cells from
5-FU-treated mice in suspension in the presence of SF and IL-11 with
or without IL-3 for 7 days and tested the reconstituting ability of the
cultured cells by transplanting the cells into lethally irradiated Ly-5 congenic mice together with "compromised" marrow cells. Presence of IL-3 in culture abrogated the reconstituting ability of the cells
from both types of knock-out mice and the wild-type mice. In contrast,
addition of GM-CSF to the suspension culture abrogated neither B-cell
potential nor reconstituting abilities of the cultured cells of
wild-type mice. These observations may have implications in the choice
of cytokines for use in in vitro expansion of human hematopoietic stem
cells and progenitors.
© 1998 by The American Society of Hematology.
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INTRODUCTION |
INTERLEUKIN-3 (IL-3) supports the
development of multiple hematopoietic lineages by interacting with
multipotential and lineage-committed progenitors in
culture.1-3 Studies in our laboratory indicated that IL-3,
as a single factor supports proliferation of the progenitors after they
exit from the cell-cycle dormant state (G0).3,4 IL-3 also
synergizes with IL-6,5 IL-11,6,7 granulocyte colony-stimulating factor (G-CSF),8 leukemia inhibitory
factor,9 thrombopoietin (TPO),10,11 and steel
factor (SF, c-kit ligand)12,13 in triggering cell divisions
of the multipotential progenitors in G0.
In contrast to the positive regulation of myeloid lineages, IL-3 seems
to exert negative effects on the early stages of lymphopoiesis. In our
laboratory, we have established a two-step methylcellulose culture
assay for murine lymphohematopoietic progenitors and characterized their cytokine requirement.14 SF-based cytokine
combinations supported the proliferation and differentiation of
lymphohematopoietic progenitors whereas addition of IL-3 to the
permissive cytokine combinations abrogated the B-lymphoid potential of
the progenitors.15 We subsequently observed that
T-cell16 and natural killer-cell17 potential
of the progenitors is also inhibited by IL-3. These observations raised
the possibility that IL-3 may be a stage-specific negative regulator
and that it may suppress the earliest process of hematopoiesis, ie,
self-renewal of the stem cells. This hypothesis was confirmed later by
our observation that IL-3 abrogates reconstituting ability of
hematopoietic stem cells with long-term engraftment capability.18
Both mouse and human IL-3 receptors are heterodimers consisting of  and subunits.19-22 The high-affinity receptors for
human IL-3, granulocyte-macrophage CSF (GM-CSF), and IL-5 share common subunit (c).19-22 The subunits are specific for
each cytokine and bind their ligand with low affinity. Whereas there is
only one type of human subunit, c, mouse has two closely related subunits, c and IL-3.23-25 They have 56%
homology with human c. Like human c, mouse c is the common subunit of the receptors for mouse IL-3, GM-CSF, and IL-5. Although
IL-3 has extensive sequence homology with mouse c (91% at the
amino acid level), IL-3 does not form a high-affinity receptor with
mouse IL-5 or mouse GM-CSF receptors. When transfected into mouse
T-cell line, CTLL-2, both c and IL-3 interacted equally well with
the subunit of mouse IL-3 receptor and transmitted proliferation
signals in the presence of IL-3.25 Independently,
investigators in two laboratories26-28 reported generation
and analysis of the mice lacking either of the two receptor genes.
Mice lacking the c showed pulmonary alveolar proteinosis-like
disease, a phenotype similar to that of GM-CSF-deficient
mice.29,30 The number of peripheral blood eosinophils of
c-deficient mice was markedly reduced, and the mice failed to
produce eosinophils in response to parasitic infections because of
absence of transduction of IL-5 signals.26 The
colony-forming ability of the bone marrow cells from c- or
IL-3-deficient mice was also examined.26-28 Cells from
c-deficient mice did not respond to GM-CSF or IL-5 but responded
normally to IL-3. In contrast, cells from IL-3-deficient mice
responded normally to IL-3, GM-CSF, or IL-5. These results clearly show
the unique redundancy of mouse IL-3 receptor system, which is not
present for the human IL-3 receptor. In this study, we used cells from
c and IL-3-gene knock-out mice to examine the relative
contributions of c and IL-3 to the negative regulation of the
early B lymphopoiesis and the long-term reconstituting ability of stem
cells.
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MATERIALS AND METHODS |
Cytokines.
Purified recombinant murine IL-3 and GM-CSF were purchased from R&D
Systems (Minneapolis, MN). Purified recombinant murine SF was obtained
from Immunex (Seattle, WA). Purified recombinant human IL-6 was a gift
from M. Naruto of Toray Industries (Kamakura, Japan). Purified
recombinant human IL-7 was a gift from Sterling Winthrop Inc
(Collegeville, PA). Purified recombinant human IL-11 was a gift from P. Schendel, Genetics Institute (Cambridge, MA). Purified recombinant
human TPO was prepared by the Cytokine Production Group of Kirin
Brewery (Takasaki, Japan). Recombinant human FLT3/FLK-2 ligand (FL) was
provided by S.D. Lyman of Immunex. Purified recombinant human
erythropoietin (EPO) was provided by the Genetics Institute Clinical
Manufacturing Group (Cambridge, MA). Recombinant human G-CSF was a gift
from A. Shimosaka of Kirin Brewery, Co, Ltd. Unless otherwise
specified, the concentrations of cytokines used were as follows: IL-3,
10 ng/mL; SF, 100 ng/mL; IL-6, 100 ng/mL; IL-7, 200 U/mL; IL-11, 100 ng/mL; TPO, 100 ng/mL; FL, 100 ng/mL; EPO, 2 U/mL; G-CSF, 100 ng/mL;
GM-CSF, 160 ng/mL.
Monoclonal antibodies (MoAbs).
Hybridoma D7 (anti-Ly-6A/E [anti-Sca-1]; rat immunoglobulin G
[IgG]2a) was a gift from P. Kincade of Oklahoma Medical Research Foundation (Oklahoma City, OK). MoAb ACK4 (anti-c-kit; rat IgG2a) was
provided by S.I. Nishikawa of Kyoto University (Kyoto, Japan). Hybridoma RB6-8C5 (anti-mouse granulocytes; rat IgG2b) was provided by
R.L. Coffman of DNAX (Palo Alto, CA). MoAb TER119 (anti-erythrocytes; rat IgG2b) was a gift from T. Kina of Kyoto University. Hybridomas 14.8 (anti-B220; rat IgG2b), M1/70.15.11.5 (anti-macrophages; rat IgG2b),
GK1.5 (anti-CD4; rat IgG2b), and 53-6.72 (anti-CD8; rat IgG2a) were
purchased from American Type Culture Collection (Rockville, MD). 53-2.1 (Biotin-conjugated-anti-Thy-1.2; rat IgG2a), RA3-6B2
(Biotin-conjugated-anti-CD45R/B220; rat IgG2a), RB6-8C5 (Biotin-conjugated-anti-Gr-1; rat IgG2b), and M1/70
(Biotin-conjugated-anti-Mac-1; rat IgG2b) were purchased from
Pharmingen (San Diego, CA). AL1-4A2 (fluorescein isothiocyanate
[FITC]-conjugated anti-Ly-5.2; mouse IgG1) and A20-1.7
(FITC-conjugated anti-Ly-5.1; mouse IgG1) were provided by H. Fleming
of Emory University.
Cell preparations.
Cells from 10- to 15-week-old male and female
c-deficient,26 IL-3-deficient,26 and
wild-type littermates (C57B1/6)26 were used in clonal
cultures and transplantation experiments. 5-fluorouracil (5-FU; Adria
Laboratories, Columbus, OH) was administered intravenously through the
tail vein at 150 mg/kg body weight, and bone marrow cells were obtained
2 days later. Cells prepared from pooled femurs and tibiae were washed
twice and then subjected to density gradient separation by using
Nycodenz (Accurate Chemical and Scientific Corp, Westbury, NY)
solution. Cells with densities ranging from 1.063 g/mL to 1.077 g/mL
were collected.31 Cells reacting to a cocktail of
lineage-specific rat MoAbs (RB6-8C5, 14.8, M1/70.15.11.5, GK1.5,
TER119, and 53-6.72) were removed twice by using immunomagnetic beads
(Dynabeads M-450 coupled to sheep anti-rat IgG; DYNAL, Great Neck, NY).
The resulting lineage-negative cells (Lin) were treated
with normal rat IgG (Jackson ImmunoResearch Laboratories, West Grove,
PA) at 20 µg/106 cells to prevent nonspecific binding of
MoAbs to Fc receptors, and were then stained with FITC-conjugated rat
MoAb D7 (anti-Sca-1)32 and biotin-conjugated rat MoAb ACK4
(anti-c-kit).33 Cells were washed twice before staining
with streptavidin-conjugated R-phycoerythrin (PE) (Jackson
ImmunoResearch Laboratories). Both FITC-conjugated rat IgG2a and
biotin-labeled rat IgG2a (Caltag Laboratories, San Francisco, CA) were
used as isotype controls. Sca-1+ c-kit+ cells
were collected by sorting on FACS Vantage (Becton Dickinson Immunocytometry Systems).
Two-step methylcellulose culture for lymphohematopoietic
progenitors.
Four thousand Lin cells or 100 Lin
Sca-1+ c-kit+ cells of 5-FU-treated mice were
plated in 35-mm suspension culture dishes (Falcon, Lincoln Park, NJ)
containing -medium (ICN, Irvine, CA), 1.2% 1,500-cp methylcellulose
(Shinetsu Chemical, Tokyo, Japan), 30% (vol/vol) fetal calf serum
(FCS) (Intergen, Purchase, NY), 1% deionized Fraction V bovine serum
albumin (BSA) (Sigma Chemical, St Louis, MO), 1 × 104
mol/L 2-mercaptoethanol (2-ME) (Sigma), and designated cytokines. Dishes were incubated at 37°C in a humidified atmosphere
flushed with 5% CO2. On the designated day of
incubation, 20 colonies were picked, pooled, and washed and 1/20 of the
pooled cells were plated in secondary methylcellulose culture
containing SF and IL-7. The number of pre-B-cell colonies was counted
on day 10 of the secondary culture by using criteria previously
described.14
Suspension and clonal cell cultures.
Two hundred Lin Sca-1+ c-kit+
cells were incubated in each well of a 6-well plate (Falcon) in 5 mL
suspension culture. The culture medium contained -medium, 20%
(vol/vol) FCS, 1% deionized BSA, 1 × 104 mol/L 2-ME,
and designated cytokines. On day 7 of incubation, aliquots were
analyzed for colony formation and in vivo reconstituting capabilities.
Clonal culture was performed in 35-mm suspension culture dishes
containing -medium; 1.2% 1,500-cp methylcellulose; 30% FCS; 1%
BSA; and 1 × 104 mol/L 2-ME, SF, IL-3, IL-6,
FL, TPO, and EPO. Colonies were scored on day 8 of incubation by in
situ observation of the plates on an inverted microscope according to
the criteria described previously.34 Megakaryocyte colonies
were scored when the colony contained four or more megakaryocytes.
Abbreviations for colony types are as follows: GM,
granulocyte/macrophage colonies; GEM,
granulocyte/erythrocyte/macrophage colonies; GMM,
granulocyte/macrophage/megakaryocyte colonies; GEMM,
granulocyte/erythrocyte/macrophage/megakaryocyte
colonies34; Meg, megakaryocyte colonies.
In vivo reconstitution experiments.
In studies of knock-out mice, 10- to 12-week-old male C57Bl/6-Ly-5.1
mice were administered with single 850-cGy total-body irradiation via a
4 × 106 V linear accelerator. After irradiation of the
recipient mice, freshly sorted Lin Sca-1+
c-kit+ marrow cells (Ly-5.2 cells) from female wild-type,
c  /, and IL-3 / mice were injected into the tail
vein of the recipients together with 4 × 105
"compromised" marrow cells of male C57B1/6-Ly-5.1 mice.
"Compromised" cells had been subjected to two previous rounds of
transplantation and regeneration in male mice.35 Cells
cultured in suspension were also tested for reconstituting capabilities
after 7 days' incubation with designated cytokines; all of the cells
in each well were injected into male C57B1/6-Ly-5.1 mice together with "compromised" cells. Peripheral blood was obtained from the
retro-orbital venous plexus using heparin-coated micropipettes
(Drummond Scientific Co, Broomall, PA) 2, 4, and 6 months after
transplantation. Red blood cells were lysed by 0.15 mol/L
NH4Cl. The samples were then used for flow cytometric
analysis of donor-derived cells by staining with FITC-conjugated
anti-Ly-5.2 (AL1-4A2). In studies of GM-CSF effects, we used
C57B1/6-Ly-5.1 male mice as donors and C57B1/6-Ly-5.2 female mice as
recipients. The lineage phenotype of the donor cells at 6 months
posttransplantation was determined by staining with biotin-conjugated
anti-Thy-1.2, biotin-conjugated anti-CD45R/B220, biotin-conjugated
anti-Gr-1, and biotin-conjugated anti-Mac-1. For indirect staining of
cells with biotin-conjugated antibodies, cells were first incubated
with biotin-conjugated antibodies, then followed by staining with
streptavidin-conjugated PE.
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RESULTS |
Effects of IL-3 on lymphohematopoietic progenitors.
The results of the studies of lymphohematopoietic progenitors are
presented in Table 1. Regardless of the
origin of the cells, colonies supported by the combination of SF and
IL-11 possessed B-cell potential. Addition of IL-3 to the combination
of SF and IL-11 strongly inhibited the B-cell potential of the primary
colonies of both types of knock-out mice as well as wild-type mice. As we reported previously,14,15 the number of primary colonies was unaffected by IL-3. These results indicated that c and IL-3 are redundant regarding signal transduction in negative regulation of
early B lymphopoiesis by IL-3.
Effects of IL-3 on expansion of total cells and progenitors.
Next we studied the effects of IL-3 on the expansion of cells and
colony-forming cells by plating 200 enriched cells in 7-day suspension
culture in the presence of 100 ng/mL SF and 100 ng/mL IL-11 with or
without 100 ng/mL IL-3. As shown in Table
2, the combination of SF and IL-11
increased the total cell counts by 335- to 600-fold, total
colony-forming units (total CFU) by 172- to 402-fold, CFU-GEMM by 26- to 43-fold, and CFU-Meg by 6- to 19-fold, as compared with freshly
enriched cells. Addition of IL-3 resulted in about 30-fold enhancement
of total cell counts and several-fold increase in the total CFU,
CFU-GEMM, and CFU-Meg. These results confirmed that the
myelostimulatory effects of IL-3 are transduced by both c and
IL-3.
Effects of IL-3 on long-term repopulating cells (LTRC).
We then tested the in vivo reconstituting ability of the cultured
cells. The suspension cultures were initiated with 200 Lin Sca-1+ c-kit+ cells in the
presence of 100 ng/mL SF and 100 ng/mL IL-11 with or without 100 ng/mL
IL-3. After 7 days of incubation, cells in a well were obtained and
injected into a lethally irradiated Ly-5.1 recipient. As a control
group, we also transplanted 200 freshly prepared Lin
Sca-1+ c-kit+ cells from wild-type, IL-3
/, and c / mice. The results of the analyses of
peripheral blood nucleated cells are presented in Fig
1. The enriched cells from all mice
incubated with SF and IL-11 had similar reconstituting ability as
freshly prepared cells. In contrast, incubation in the presence of IL-3
significantly reduced the reconstituting abilities of the cells of all
types of mice. A few repopulating cells survived in SF, IL-11, and IL-3 at 2 months posttransplantation, as evinced by the appearance of small
percentages of donor cells in five, three, and three mice out of a
total of seven in Fig 1A, B, and C, respectively. However,
reconstitution was absent at 4 and 6 months posttransplantation. These
observations confirmed that the negative effects of IL-3 are directed
only to LTRC and that the effects are transduced by both
c and IL-3.
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| Fig 1.
Repopulating abilities of freshly sorted bone marrow
cells and cultured cells. (A) Mice transplanted with wild-type cells. (B) Mice transplanted with IL-3 / cells. (C) Mice transplanted with c / cells. ( ), 2 months posttransplantation;
( ), 4 months posttransplantation; ( ), 6 months
posttransplantation.
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At 6 months posttransplantation, the proportions of donor blood
nucleated cells in each of T-cell, B-cell, and myeloid (granulocyte and
monocyte/macrophage) compartments were determined (Table
3). Multi-lineage cells were detected in
the peripheral blood of engrafted recipients. An example of analysis of
a mouse transplanted with c / cells cultured with SF and IL-11
is shown in Fig 2.
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| Fig 2.
An example of hematopoietic reconstitution by c
/ cells cultured with SF and IL-11. Nucleated blood cells of a
recipient mouse were analyzed using flow cytometry 6 months after
transplantation. Thy-1.2+ cells, B220+
cells, and Gr-1+ Mac-1+ cells of donor
(Ly-5.2) origin are seen.
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Effects of GM-CSF.
The observation that the c chain transduces the negative effects of
IL-3 on the stem cells and lymphohematopoietic progenitors raised the
possibility that c may also transduce negative signals of GM-CSF.
Earlier we reported that, in addition to IL-11, IL-6 and G-CSF can
interact with SF in stimulating proliferation of cell cycle dormant
progenitors3,12,13 and the lymphohematopoietic progenitors.14,15 In the next two experiments, we tested
the effects of GM-CSF and IL-3 in variable concentrations on the
progenitors and stem cells using cells from wild-type mice.
Specifically, we tested the effects of addition of these cytokines to
the cultures supported by SF and one of IL-11, IL-6, and G-CSF. The
results are presented in Table 4. As
reported previously,14,15 the primary colonies supported by
SF plus IL-11, SF plus IL-6, and SF plus G-CSF possessed B-lymphoid
potential. Addition of IL-3 at as low as 1 ng/mL concentration
completely abrogated the B-cell potential of the primary colonies. In
contrast, addition of GM-CSF in concentrates ranging from 10 to 1,000 ng/mL failed to suppress the lymphohematopoietic progenitors.
We then tested the effects of GM-CSF on repopulating abilities of the
bone marrow cells. The enriched marrow cells of Ly-5.1 wild-type mice
were cultured in suspension for 1 week under permissive cytokine
conditions with or without additional GM-CSF and transplanted to
lethally irradiated Ly-5.2 recipients. As presented in Table 5, GM-CSF did not negatively affect the
repopulating abilities of cultured cells.
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DISCUSSION |
There is significant current interest in hematology/oncology fields
regarding in vitro expansion of hematopoietic stem cells and
progenitors.36-51 A number of investigators have already
shown that it is possible to increase the number of hematopoietic
progenitors in culture by using combinations of early-acting
cytokines.36-51 Because of the well-known myelopoietic
effects of IL-3, the majority of preclinical protocols for
murine37-43 and human36,44-51 cells included
IL-3. We previously noted negative effects of IL-3 on the ability of
cultured cells to engraft the marrow of recipient mice.18
Our observation was in agreement with the report from Peters et
al52 that suspension culture of murine marrow cells in the
presence of IL-3, IL-6, IL-11, and SF results in impairment of the
engrafting capability of the cultured cells.
The negative effects of IL-3 observed in murine models may be relevant
to in vitro manipulation of human stem cells. Ten patients with
advanced cancers were transplanted with peripheral blood progenitors
that had been expanded in cultures containing IL-3.53 Recently, Williams et al54 reported transplantation of
peripheral blood CD34+ cells expanded in liquid culture
with PIXY321 (the fusion product of IL-3 and GM-CSF) to 8 patients with
advanced breast cancer. No information was provided in these reports
regarding long-term effects on the recipient's hematopoiesis. Donahue
et al55 studied transduction of glucocerebrosidase genes to
primate CD34+ Thy-1+ cells using 7-day culture
in the presence of IL-3, IL-6, and SF.55 After autologous
transplantation, provirus was detected at all time points in both
B-cell and T-cell lineages, but long-term gene transfer was not
observed in the granulocyte population.55 It is possible
that IL-3 abrogated the long-term reconstitution capability of the
cultured primate stem cells.
Both c and IL-3 transduced the negative signals of IL-3. c and
IL-3 have been shown to have redundant functions. Coexpression of
one of the two proteins and IL-3 receptor protein in an IL-2-dependent mouse T-cell line, CTLL-2, resulted in identical, high-affinity binding for mouse IL-3 and IL-3-dependent proliferation of the cell line.25 Neither c-null nor IL-3-null
mice showed apparent hematopoietic defects.26 However,
careful analysis of the IL-3-null mice revealed hyporesponsiveness
of the hematopoietic progenitors to IL-3, indicating probable
quantitative differences between c and IL-3.28
Although receptors for IL-3 and GM-CSF share common signal-transducing
protein c, neither lymphohematopoietic progenitors nor long-term
reconstituting cells were negatively affected by GM-CSF. These
observations are in agreement with the previous reports on the
differences between IL-3 and GM-CSF. Earlier, we documented that GM-CSF
is significantly weaker than IL-3 in support of murine56
and human57 blast cell colony formation. Recently, McKinstry et al58 reported that GM-CSF receptor is not
expressed by mouse Rhodamine123lo
LinLy6A/E (Sca-1)+ c-kit+ cells
that are highly enriched for long-term repopulating cells. These
observations together with our observations presented in this paper
indicate that the protein of GM-CSF receptor is expressed by
neither the stem cells nor the most primitive progenitors. Further,
these observations may have a significant implication in "in vitro
expansion" of human stem cells and progenitors. It is possible that
the use of GM-CSF may be preferable to IL-3 in cytokine mixtures
because GM-CSF may not abrogate long-term reconstituting stem cells
while it supports expansion of some multipotential progenitors.
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FOOTNOTES |
Submitted November 24, 1997;
accepted April 1, 1998.
Supported by the Office of Research and Development, Medical Research
Service, Department of Veterans Affairs; NIH grants DK32294 and
DK/HL48714; and a contribution from Amgen.
Address reprint requests to Makio Ogawa, MD, PhD, Ralph H. Johnson
Medical Center, 109 Bee St, Charleston, SC 29401-5799.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" is accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
We thank Dr Haiqun Zeng for assistance in cell sorting; Dr Pamela N. Pharr and Anne G. Leary for assistance in preparation of this
manuscript; and the staff of Radiation Oncology Department of the
Medical University of South Carolina for assistance in irradiation of
mice.
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Abstract
Introduction
Abstract