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Prepublished online as a Blood First Edition Paper on July 5, 2002; DOI 10.1182/blood-2002-03-0733.
HEMATOPOIESIS
From the Department of Pathology and Laboratory
Medicine and the Jonsson Comprehensive Cancer Center, University of
California at Los Angeles School of Medicine; and the Department of
Developmental and Clinical Immunology, University of Alabama,
Birmingham.
B-cell precursors are present in the thymus, and the thymic
microenvironment is the source of lymphopoietic factors that include interleukin-7 (IL-7). Despite the fact that intrathymic B-cell progenitors are bone marrow-derived cells, the data in this report demonstrate that these progenitors accumulate at an early pro-B-cell stage of development, cycle less than their bone marrow counterparts, and fail to differentiate efficiently. Additional studies presented herein indicate that these effects are mediated, at least in part, by
soluble factors produced by the thymic microenvironment and suggest
that they affect the ability of pro-B cells to respond optimally to
IL-7. Taken together, these observations demonstrate a specific
inhibition of intrathymic B lymphopoiesis, which in turn may explain
why lymphoid cell production in the thymus is largely restricted to
production of T-lineage cells despite the fact that B-cell precursors
and B-lymphopoietic stimuli are present in that organ.
(Blood. 2002;100:3504-3511) Sustained T-cell development in the thymus is
thought to be dependent on continuous migration of bone marrow-derived
precursors to that organ.1-4 While T lineage-committed
progenitors could be included among these cells,5
experimental evidence suggests that at least some intrathymic lymphoid
precursors are multipotent and retain the capacity to generate B
cells.6,7 The recently characterized bone marrow
(BM) common lymphoid precursor (CLP),8 which has B- and
T-cell developmental potential, could be the BM population that
migrates to the thymus and sustains thymopoiesis.
Despite the fact that B-cell precursors are present in the
thymus, B lymphopoiesis in that organ is minimal. One reason for this
is that entry of lymphoid precursors into the B lineage may be limited.
In this regard, it has been proposed that binding of the Notch1
receptor on multipotential lymphoid precursors, such as the CLP, to
ligands expressed by thymic stromal cells9 results in
commitment to the T lineage and a block in B-cell
development.10-12 Activation of Notch1 signaling pathways
may be a critical means by which entry of multipotential precursors
into the B-cell lineage is blocked within the thymus. However, some
thymic lymphoid precursors may fail to do so, thereby allowing pro-B
cells to develop. Indeed, it has been reported that 10% to 13% of
cells within the
CD3 Nevertheless, B-lineage cells in the thymus account for less than
1% of total lymphocytes.14-20 Such a low frequency is
surprising, because B-cell precursors present among TN thymocytes are
exposed to many of the same microenvironmental stimuli as their
counterparts in the BM. For example, the interaction between lymphoid
cells and stromal cells in both organs is mediated through similar
ligand-receptor complexes, such as very late antigen-4/vascular
cell adhesion molecule-1 (VLA-4/VCAM-1) and stromal cell-derived
cytokines that include interleukin-7 (IL-7).21 IL-7
stimulates pro-B-cell proliferation and potentiates immunoglobulin
heavy chain gene rearrangements in those precursors.22-24
Because it has been established that they are bone marrow-derived
cells,25 intrathymic B-cell progenitors would be expected
to respond to IL-7 and other lymphopoietic factors.
These observations suggest that mechanism(s) that limit the maturation
of intrathymic B-cell progenitors exist. Such regulatory pathway(s)
would ensure that expansion of B-lineage cells able to compete for
microenvironmental niches required for T lymphopoiesis does not occur.
There may be additional reasons for limiting intrathymic B-cell
production. A case has been made that thymic B cells are involved in
selection of the T-cell repertoire.26 Thus, an inordinate increase in their numbers might be detrimental to the thymic education process. Therefore, it is likely that signals from the thymic microenvironment minimize the development of B-lineage cells that develop in that organ.
The data in this report support this hypothesis and demonstrate that
B-cell progenitors in the thymus accumulate at an early pro-B-cell
stage of development and fail to differentiate efficiently. These
effects are consistent with findings demonstrating that intrathymic
pro-B cells respond inefficiently to IL-7. Taken together, these
results document an unappreciated inhibitory effect on B lymphopoiesis
by the thymic microenvironment that helps to explain why lymphoid cell
production in the thymus is largely restricted to production of
T-lineage cells.
Mice
Preparation of cell suspensions
Fetal thymic organ cultures Fetal thymic organ cultures (FTOCs) were established according to the protocol described by Jenkinson et al.27 Briefly, fetal thymic lobes from 15-day-old Thy-1.1 Swiss/Webster embryos were harvested aseptically, dissected free from extraneous tissue, and cultured for 5 days in the presence of 1.35 mM deoxyguanosine to deplete endogenous thymocytes. The lobes were then rinsed and incubated with donor cells in hanging drop cultures in Terasaki plates (Fisher, Tustin, CA) for 48 hours. Subsequently, lobes were transferred to FTOC on filter/gelfoam rafts in RPMI 1640 supplemented with 10% FCS, 5 × 10 5 M 2- mercaptoethanol, 100 U/mL
streptomycin, and 100 U/mL penicillin and placed in a humidified, 5%
CO2/air incubator at 37°C. Cell proliferation and
differentiation in FTOC was assessed at regular intervals following
culture initiation, as described below.
Long-term BM cultures Long-term myeloid BM cultures (LTBMCs) were initiated as described by Dexter et al.28 Briefly, the contents of a femur were flushed into a 25 cm2 tissue culture flask in -MEM supplemented with 20% horse serum and 106 M
sodium hydrocortisone succinate. Cultures were incubated at 33°C in a
5% CO2/air incubator. After 2 weeks, cultures were
recharged with 106 BM cells per flask. B-cell development
was induced by switching cultures to the B-lymphoid permissive
conditions (RPMI 1460 supplemented with 5% FCS and
5 × 105 M -mercaptoethanol) described by
K.D.,29 and by Whitlock and Witte.30
Preparation of BM and thymus stromal cells Confluent, heterogeneous BM stromal cell cultures were established by treating LTBMCs with 5 µg/mL mycophenolic acid for 2 to 4 weeks to eliminate hematopoietic cells as described.31 The adherent stromal cells were then maintained in-MEM supplemented with 10% FCS in a 37°C, 5%
CO2/air incubator and were passaged minimally to retain the
characteristics of primary stroma. Heterogeneous thymic stromal cell
cultures were established as described.32 Thymuses were
digested with trypsin/EDTA (ethylenediaminetetraacetic acid) and
collagenase dispase in Ca++-, Mg++-free medium
for 35 minutes at 37°C. The confluent thymic stromal cells were then
fed weekly with MEM D-valine supplemented with 10% FCS and maintained
in a 37°C, 5% CO2/air incubator. These cells were
passaged minimally to retain the characteristics of primary stroma. The
generation of the S17 stromal cell line has been
described.33 The S17 BM stromal cell line was fed weekly with -MEM supplemented with 10% FCS.
Diffusion chamber cultures In some experiments, heterogeneous BM or thymic stromal cells were grown to confluency in transwells fitted with membranes containing 0.4-µm pores (Becton Dickinson, San Jose, CA). Inserts were inserted into wells of 6-well plates in which myeloid LTBMCs had been established. Cultures were then switched to B-lymphoid permissive conditions, fed twice weekly, and cells assayed for B-cell production by immunophenotyping.Immunofluorescence and cell sorting BM cell suspensions were treated with Tris-ammonium chloride (pH 7.2) to lyse red blood cells. Cells were then incubated with an anti-CD16/CD32 antibody (Fc RII/III, clone 2.4G2; Pharmingen, San
Diego, CA) to reduce nonspecific labeling prior to staining. The
following monoclonal antimouse antibodies used for staining were all
obtained from Pharmingen: CD45R (B220, clone RA3-6B2), CD127 (IL-7R,
clone SB/14), CD43 (clone S7), CD24 (HSA, clone 30-F1), and Ly-51
(BP-1, clone 6C3). These antibodies were conjugated to
fluorescein isothiocyanate (FITC), phycoerythrin (PE), TriColor (TC),
or biotin. Biotinylated antibodies were revealed with TC-conjugated streptavidin (Southern Biotechnology, Birmingham, AL). Optimal working dilutions were determined for each antibody prior to use. All
incubations were performed in Ca++-, Mg++-free
phosphate-buffered saline (PBS) at 4°C for 30 minutes.
Following the last wash, 104 to 105 live cells
per sample were analyzed by flow cytometry on a FACScan (Becton
Dickinson) with CellQuest software (Becton Dickinson).
BM cells were incubated with FITC-conjugated anti-CD45R, PE-conjugated
anti-CD43, or PE-conjugated anti-IgM, as described above, prior to
sorting on a FACStarplus flow cytometer (Becton Dickinson) located in
the Jonsson Comprehensive Cancer Center Flow Cytometry Core at UCLA.
Reanalysis showed that the purity of the sorted CD45R+
IgM Cell cycle analysis Cells were fixed in 0.5% paraformaldehyde for 15 minutes at room temperature and then permeabilized with 70% ethanol and resuspended in Ca++-, Mg++-free PBS supplemented with 1 µg/mL 7-amino actinomycin D (7-AAD; Calbiochem, San Diego, CA). At least 105 cells were acquired with a FACScan, and their cycling status was estimated using Modfit LT software (Becton Dickinson).
Thymic B-cell differentiation is blocked at an early stage in vivo Multiple stages of B-cell differentiation in the BM can be defined based on the differential expression of various cell surface determinants. Because thymic B-lineage cells are bone marrow-derived,25 their expression of the CD45R, CD43, Ly51 (BP-1), and CD24 cell surface determinants was assessed by flow cytometry. This combination of reagents allows the pro-B-cell pool to be subdivided into fractions A, B, C, and C', as defined by Hardy et al.34 Fraction A includes the most immature pro-B cells, while those in fraction C' are completing immunoglobulin (Ig) heavy chain gene rearrangements before their transition into pre-B cells.As expected, cells in the bone marrow pro-B-cell compartment were
present at the frequency described previously35 and
included populations that had matured to the C' stage of development
(Figure 1A). In contrast, very few thymic
pro-B cells had matured to the fraction C' stage (Figure 1A). Because
the most actively proliferating B-lineage cells are those in the
pro-B-cell compartment, the cell cycle status of intrathymic
CD45R+CD43+ cells also was determined. As shown
in Figure 1B, the frequency of cycling thymic pro-B cells was
approximately 3-fold lower than that observed with comparable
populations from the BM.
B-cell development is blocked in FTOC The above results indicate that B-cell production in the thymus is blocked at a relatively early stage of development. In order to assess the fate of isolated B-cell progenitors in the thymic environment in more detail, a modification of the FTOC system was used. Fetal thymic lobes from day-15 embryos were treated with deoxyguanosine to remove endogenous thymocytes prior to seeding with FACS-purified CD45R+sIgM bone marrow cells. Thymic lobes
prepared in this manner are fully functional and support T-cell
development.36 Other aliquots of
CD45R+sIgM cells were seeded on the BM
stromal cell line S17, which has been shown to support B-cell
differentiation in vitro.33 Cells were recovered and
analyzed phenotypically 7 days following initiation of these cultures,
as described above.
Figure 2 demonstrates that while
B-lineage cells were recovered from thymic lobes, they did not
efficiently mature past the fraction C stage. These data are consistent
with observations made on freshly harvested thymic pro-B cells (Figure
1A) and indicate the validity of the FTOC system for analyzing thymic B
lymphopoiesis. The fate of CD45R+ surface
(s)IgM
Exposure to the thymic environment renders B-cell progenitors unresponsive to lymphopoietic signals Because cytokines such as IL-7 are produced in the thymus,37,38 it seemed unlikely that failure of pro-B cells to mature was due to the absence of B-lymphopoietic factors. Instead, the possibility existed that the thymic microenvironment rendered pro-B cells unresponsive to B-lymphopoietic signals. To test this hypothesis, cells harvested from FTOC at various times after seeding lobes with bone marrow-derived CD45R+sIgM cells were reseeded onto the S17
BM stromal cell line under B-cell permissive conditions.
As shown in Table 1 and Figure
3, cells harvested 2 days
after culture in FTOC established vigorous long-term B-cell cultures when reseeded on S17 stroma. However, by 7 days of culture in FTOC,
they were no longer able to do so. These results are consistent with
data from 2 experiments demonstrating that
CD45R+sIgM
Pro-B cells in the thymus are hyporesponsive to IL-7 IL-7 is required for murine pro-B cells to proliferate and complete Ig heavy chain gene rearrangements and mature into pre-B cells.39 Because thymic pro-B cells accumulate in fraction C and cycle at a lower rate than their BM counterparts, their capacity to respond to IL-7 was examined. Following 7 days of culture in FTOC, cells were harvested from the lobes, and their proliferative response to IL-7 was compared to that of CD45R+sIgM cells from fresh BM. The frequency
of CD45R+CD43+ cells, which includes the most
IL-7-responsive cells, in each population was determined by FACS.
Based on this information, cultures were initiated using the same total
number of CD45R+CD43+ cells from each source.
As shown in Figure 4A, cells harvested from thymic lobes responded to IL-7, but the magnitude of their proliferative response was lower than that of the freshly harvested BM population.
This observation prompted an analysis of IL-7 receptor Thymic stromal cell-derived factors inhibit B lymphopoiesis The above observations indicate that the thymic microenvironment is a source of mediators that render pro-B cells hyporesponsive to IL-7. To determine whether such inhibitors were soluble molecules, initial experiments used a modification of the myeloid to lymphoid long-term BM switch culture system29 to test whether thymic stromal cells could inhibit B-cell development in the absence of direct cell contact.Within a week following transfer of long-term BM cultures established
under the myeloid conditions described by Dexter and colleagues28 to B-lymphoid-permissive
conditions,30 pro-B cells emerge, and by 3 weeks
B-lineage cells predominate in the cultures. As shown in Figure
5A, heterogeneous populations of bone
marrow or thymic stroma growing in transwells were introduced into
these cultures. By 3 weeks following transfer of long-term myeloid BM
cultures to B-lymphoid permissive conditions, vigorous cultures
containing CD45R+ cells were established (Figure
5B). This same pattern of growth occurred when empty transwells
(data not shown) or transwells containing heterogeneous BM stroma
(Figure 5B) were placed in the cultures at the time of their transfer
to the lymphoid conditions. However, when transwells containing
heterogeneous preparations of thymic stromal cells were introduced into
the cultures, CD45R+ cell production was dramatically
reduced (Figure 5B), indicating that soluble mediators produced by the
thymic microenvironment can inhibit intrathymic B lymphopoiesis.
Down-regulation of IL-7R
cells from type 1 IFN receptor-deficient
(Ifnar1/) mice were used to seed fetal
thymic lobes. CD45R+sIgM cells from 129 mice,
the background strain of the Ifnar1/mice,
were assayed in parallel. CD127 expression on
CD45R+CD43+ cells harvested from these FTOCs
was compared to that on bone marrow pro-B cells 7 days later. As
shown in Figure 4B, exposure to the thymic microenvironment
resulted in a greater than 90% reduction of CD127 high-expressing
cells in Ifnar1/ mice. This level of
inhibition was comparable to that in BALB/c and greater than that in
129 strain mice. A prediction based on this observation is that the
frequency of B-lineage cells in the thymus of
Ifnar1/ mice should not be elevated. This
is in fact the case, as the frequency of B-lineage cells in the thymus
of Ifnar1/ mice was comparable to that in
control animals (Figure 4C).
Resumption of B-cell maturation in the presence of exogenous IL-7 Taken together, the above data suggest that the growth and development of pro-B cells in the thymus is limited through IL-7R down-regulation. This event may in turn limit pro-B cell capacity to
compete with T-cell progenitors for the IL-7 produced by the thymic
microenvironment. A prediction based on this hypothesis is that
increasing intrathymic IL-7 levels may in turn allow B cell
growth. To test this premise, FTOCs were initiated with
CD45R+sIgM BM cells in the presence of 50 U/mL of exogenous IL-7. As shown in Figure
6A, approximately half of the
CD45R+ cells no longer expressed CD43 after 7 days of
culture, suggesting that they had matured to the pre-B-cell stage.
This result contrasts with the finding that the majority of B-lineage
cells in non-IL-7 supplemented FTOC were
CD45R+CD43+. In addition, although B-lineage
cells harvested from day-7 FTOC were not able to establish long-term BM
cultures on S17 BM stromal cells (Figure 3), they were able to do so if
the medium was supplemented with IL-7 (Figure 6B). These cultures could
be maintained for at least 3 weeks, and phenotypic analysis confirmed
that the cultures contained B-lineage cells (data not shown).
Since IL-7 is required for T-cell development,44 it was
important to determine how levels of IL7R
Both BM and thymic stromal cells express ligands required for direct interactions with developing B-lineage cells and secrete cytokines required for B-cell development. Despite these similarities, B-cell development in the thymus is limited. Studies aimed at investigating this phenomenon revealed that B-cell development is blocked at a relatively early stage of development in the thymus because signals produced by the thymic microenvironment inhibit that process. Initial analyses focused on characterizing B-lineage cells harvested from the thymus. These studies demonstrated that the frequency of pro-B cells in cycle was lower in the thymus than in the BM and that B-lineage cells did not efficiently mature past the fraction C stage of development. When similar analyses were performed on bone marrow B-cell progenitors 7 days after being seeded in fetal thymic lobes in vitro, the same results were obtained. In addition to corroborating the results obtained with primary cells, this result established the fetal thymic organ culture system as a model for analyzing thymic B-cell production. It is paradoxical that culture in the thymic microenvironment in the FTOC system inhibited B-cell development while other studies have reported the isolation of thymic stromal cell lines that can support B-cell differentiation. Indeed, our own laboratory characterized a thymic stromal cell line that efficiently supported the pre-B to B-cell transition.45 However, it is important to emphasize that the thymic stromal cell line in question only potentiated this latter phase of development but not the short- or long-term growth of pro-B cells. In fact, our characterization of numerous thymic stromal cell lines has so far failed to identify any capable of supporting long-term B-cell development. Furthermore, even if thymic stromal cells with such potential were isolated, they do not represent the thymic microenvironment as a whole. That is why, to assess the effects of the thymic microenvironment on B-cell development, primary cultures of heterogeneous thymic stromal cells rather than thymic stromal cell lines were used in the diffusion chamber studies. That a mechanism to inhibit intrathymic B-cell development exists at all might seem puzzling, since the overwhelming majority of lymphoid cells in the thymus are T-lineage cells. Instead, the inefficient expansion and maturation of thymic pro-B cells could result from their failure to compete effectively for microenvironmental niches. However, it is important to focus this discussion on the most immature thymocytes contained within the TN compartment. Although most cells within this population are likely committed to the T lineage, it has been reported that 10% to 13% are CD45R+.13 If even a fraction of these cells did come into contact with thymic stromal cells that supported their growth and development, this in turn could compromise overall levels of T-cell production, particularly if enough mature B cells that affected the process of thymic education subsequently developed. Thus, it is logical to propose the existence of a mechanism(s) to specifically inhibit(s) thymic pro-B-cell development. That such signals exist is supported by recent studies in which selective inactivation of Notch1 by gene targeting was performed. Radtke et al11 and Wilson et al12 demonstrated that T-cell development was effectively blocked in mice in which Notch1 was conditionally inactivated and that there was an expansion in the number of B-lineage cells in the thymus. However, while the incidence of B-lineage cells was higher than in control mice, a critical point is that the total number of B-lineage cells in the thymus of these mice increased to only about 4 million cells. This number is considerably lower than the 100 million thymocytes that are present in the thymus of young animals. More recently, Han et al46 demonstrated that mice in which the RBP-J transcription factor, which associates with the intracellular domain of Notch to allow DNA binding, has been inactivated also exhibit impaired T-cell development and increased intrathymic B lymphopoiesis. However, even though Notch signaling in these mice was effectively blocked, the total number of thymic B-lineage cells was again only about 4 × 106 cells. Taken together, these results indicate that even when not competing for environmental niches with developing T cells, B-cell precursors do not undergo extensive expansion in the thymic microenvironment. Importantly, no differences between bone marrow B-lineage cells in mice in which Notch1 expression was conditionally inactivated and their control littermates were reported. A recent study of mice that expressed a lunatic fringe transgene, which
results in the inhibition of Notch1 activation, showed that their
thymus contained up to 50 million B-lineage cells.13 That
report would also seem to contradict the conclusion that the thymus
actively inhibits B lymphopoiesis. However, while the results of that
study could be interpreted to infer that inhibitors of thymic B
lymphopoiesis do not exist, that conclusion may be too simplistic in
view of the minimal level of B lymphopoiesis in the
Notch1 In any case, the studies presented herein clearly demonstrate that
exposure to the thymic microenvironment renders B-cell precursors
unresponsive to B-lymphopoietic stimuli. After 7 days in thymic lobes,
B-lineage cells could no longer proliferate and differentiate on BM
stromal cells. Further analysis showed that this occurs because pro-B
cells that have been exposed to the thymic microenvironment are
hyporesponsive to IL-7. This seems to occur through IL-7R The long-term BM culture experiments described in this study suggest that soluble factors produced by thymic stromal cell(s) are at least partially responsible for the inhibition of thymic B-cell production. In fact, thymic stromal cells present in diffusion chambers were able to inhibit the emergence of B-lineage cells, even when precursors were in contact with a supporting layer of BM stromal cells. While aspecific effects of thymic stromal cells on B lymphopoiesis in this system through excessive consumption of nutrients cannot be excluded, it does not seem likely. First, there is no a priori reason to assume that confluent thymic and BM stroma differ significantly in the nutrients they consume. Second, cultures were fed twice weekly, and neither pH fluctuations nor excessive cell death were observed in cultures in which thymic stroma were present. Therefore, it is logical to propose that soluble factors from the thymic stroma are able to inhibit B-cell development and that their effects are potent enough to counteract positively acting B-lymphopoietic signals. It has been reported that type 1 IFNs can inhibit the response of B-cell progenitors to IL-7.40-43 Because IFNs are produced in the thymus, it was of interest to determine if they were responsible for the observed effects by seeding pro-B cells from type 1 IFN receptor-deficient mice into fetal thymic lobes. This analysis revealed that the frequency of CD45R+CD43+ cells that expressed CD127 at high levels had decreased by more than 90% after 7 days in FTOC. This finding, combined with the fact that the frequency of B-lineage cells in the thymus of these knockout mice was not elevated, strongly suggests that signaling through the type 1 IFN receptor is not responsible for the inhibition of intrathymic B lymphopoiesis. These findings would also seem to exclude the involvement of a new type 1 IFN family member, limitin, which has been proposed as a selective inhibitor of B-cell production.48 Taken together, the data in this and other studies make it possible to formulate a model in which checkpoints operative at multiple levels act in concert to limit B-cell development in the thymus. Initially, intrathymic lymphoid precursors, such as the CLP, might receive signals that preferentially potentiate their development along the T- rather than the B-cell lineage, and Notch1-activated signaling pathways may be critical at this juncture.10,11,49 Nevertheless, some pro-B cells develop, and their growth and differentiation is severely limited by thymic microenvironmental signals that render them hyporesponsive to IL-7. While IL-7 is not critical during human B lymphopoiesis,50 similar or alternative thymic stromal cell factors can be postulated to limit pro-B-cell expansion in the human thymus. The inhibition of intrathymic B lymphopoiesis may not be absolute, however, and some pro-B cells may mature into pre-B cells from which B lymphocytes may be generated. A few of these may be retained in the thymus as antigen-presenting cells,26 while others are exported to the periphery.19 It is becoming increasingly appreciated that negative regulators play an important role in the regulation of hematopoiesis. In this regard, numerous mediators have been described that specifically inhibit B-cell production.51 However, these factors have generally been considered in terms of their effects on bone marrow B lymphopoiesis. The findings herein describe an unexpected role of negative regulatory factors in the homeostasis of thymic lymphocyte production. These data contribute to the understanding of how an organ that has the potential to support B-cell production limits that process and suggest that further comparisons of BM and thymic lymphopoiesis will provide additional insights into the regulation of primary lymphocyte production.
The authors appreciate the helpful discussions with Drs Ellen Rothenberg, Max Cooper, David Rawlings, and Andrew Farr.
Supported by National Institutes of Health grant HL60658.
Submitted March 7, 2002; accepted June 25, 2002. Prepublished online as Blood First Edition Paper, July 5, 2002; DOI 10.1182/blood-2002-03-0733.
Y.H. and E.M.-R. contributed equally to this work.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Kenneth Dorshkind, Department of Pathology and Laboratory Medicine and the Jonsson Comprehensive Cancer Center 173216, 10833 Le Conte Ave, Los Angeles, CA 90095; e-mail: kdorshki{at}mednet.ucla.edu.
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Abstract
Introduction
