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Exhibit Enhanced Tumor Growth and Metastasis: Impaired NK Cell Development and Recruitment1
,§
*
Laboratory of Molecular Immunoregulation and
Laboratory of Experimental Immunology, Division of Basic Sciences, and
Intramural Research Support Program, Science Applications International Corp.-Frederick, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702, and
§
Laboratory of Molecular Immunology, Engelhardt Institute of Molecular Biology and Belozersky Institute of Physico-Chemical Biology, Moscow, Russia
 |
Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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lack peripheral lymph nodes
and Peyer’s patches and have profound defects in development of
follicular dendritic cell networks, germinal center formation, and T/B
cell segregation in the spleen. Although LT is known to be expressed
by NK cells as well as T and B lymphocytes, the requirement of LT
for NK cell functions is largely unknown. To address this issue, we
have assessed NK cell functions in LT-deficient mice by evaluating
tumor models with known requirements for NK cells to control their
growth and metastasis. Syngeneic B16F10 melanoma cells inoculated s.c.
grew more rapidly in LT-/- mice than in the wild-type
littermates, and the formation of experimental pulmonary metastases was
significantly enhanced in LT-/- mice. Although
LT-/- mice exhibited almost a normal total number of
NK cells in spleen, they showed an impaired recruitment of NK cells to
lung and liver. Additionally, lytic NK cells were not efficiently
produced from LT-/- bone marrow cells in vitro in the
presence of IL-2 and IL-15. These data suggest that LT signaling may
be involved in the maturation and recruitment of NK cells and may play
an important role in antitumor surveillance. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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is a
multifunctional cytokine structurally homologous to TNF (1, 2), which is expressed by activated lymphocytes (3)
as a soluble homotrimer (LT3) or
membrane-associated heterotrimer with LTß
(LT1ß2 and
LT2ß1)
(4). LT3 shares TNF receptors p55
and p75 with TNF, whereas
LT1ß2 heterotrimers
signal through LTßR, a distinct receptor of the same family
(5). Previous studies revealed that LT/LTßR signaling
plays an essential role in secondary lymphoid organ development and
function. Specifically, LT- and LTßR-deficient mice lack Peyer’s
patches and all peripheral lymph nodes and display disrupted splenic
T/B compartmentalization and germinal center formation after
immunization with T cell-dependent Ags and defective Ig class switching
(6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Similar phenotypic alterations are observed in
LTß-deficient mice, although most of these knockout mice have
cervical, sacral, and mesenteric lymph nodes, and T/B areas segregate
more clearly (9, 10, 16). Recent reports showed that
LT/LTßR-mediated signals may be involved in the fetal/neonatal
recruitment of lymphoid precursor cells to lymph nodes (17, 18) and the maintenance of follicular dendritic cell functions
(19, 20). NK cells constitute an important population of
lymphocytes involved in nonspecific host defense mechanisms. Although
LT is expressed by NK cells at significant levels, the contribution
of LT to NK function has not been reported. In this study, the
LT-/- mice (6) were examined
for in vivo and in vitro NK function. LT-/-
mice challenged with NK cell-susceptible experimental tumors exhibited
an enhanced tumor growth and formation of pulmonary metastases. NK
cells from the knockout mice were defective in migratory potential to
the organs and development from in vitro bone marrow (BM) culture in
the presence of cytokines. Our studies revealed a significant role for
LT signaling in the regulation and development of NK activity. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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LT-/- mice (6) were
purchased from The Jackson Laboratory (Bar Harbor, ME), additionally
backcrossed to the C57BL/6 background, and bred as heterozygotes in
specific pathogen-free conditions. The knockout mice and their
wild-type littermate controls were genotyped by PCR and used for
experiments between 6 and 12 wk of age. Animal care was provided in
accordance with the procedures outlined in the Guide for the Care
and Use of Laboratory Animals (National Institutes of Health
Publication No. 86-23, 1985).
Cell lines
The B16F10 melanoma and 3LL Lewis lung carcinoma cell lines syngeneic to C57BL/6N mice were maintained at 37°C in a 5% CO2 incubator and grown in DMEM (Life Technologies, Gaithersburg, MD) supplemented with 5% heat-inactivated FBS, 100 U/ml penicillin/streptomycin, 4 mM L-glutamine, and 2 mM sodium pyruvate. The YAC-1 lymphoma cell line of A/Sn origin was maintained in RPMI 1640 (Life Technologies) supplemented with 10% FBS, antibiotics, L-glutamine, and sodium pyruvate.
Reagents and mAbs
Polyinosinic-polycytidylic acid (polyIC) and polyL-lysine stabilized in carboxymethyl cellulose (polyICLC) (21) was provided by Dr. Hilton Levy (National Cancer Institute, Bethesda, MD). Monoclonal Abs specific for mouse CD3e and NK1.1 cell surface Ags were purchased from PharMingen (San Diego, CA). Recombinant murine IL-2 and recombinant human IL-15 were purchased from PeproTech (Rocky Hill, NJ) under contract by the Biological Resources Branch, Division of Cancer Treatment and Diagnosis, National Cancer Institute-Frederick Cancer Research and Development Center.
Primary tumor growth
B16F10 cells (2 x 105) were injected s.c. into the shaved lateral flank of mice. The size of primary tumors was determined on days 14, 17, and 21 using a caliper. Tumor volume was calculated with the formula, V = (A x B2)/2, where V = volume (mm3), A = long diameter (mm), and B = short diameter (mm) (22).
Experimental metastasis
B16F10 cells (2 x 105) or 3LL cells (3 x 105) were injected i.v. into mice through the lateral tail vein. Twenty-one days later, the mice were euthanized by cervical dislocation, and lungs were harvested for metastatic colony count under a dissecting microscope.
In vitro BM culture
BM cells were obtained from the tibias and femurs of
unstimulated LT+/+ and
LT-/- mice and cultured in RPMI 1640
supplemented with 10% FBS, L-glutamine, and sodium
pyruvate at 5 x 106 cells per well in
24-well plates. The cells were either unstimulated or stimulated with
recombinant murine IL-2 (10 ng/ml), recombinant human IL-15 (10 ng/ml),
or both. Cytotoxicity assay against YAC-1 targets was performed after 4
days in culture.
Cytotoxicity assay
Cytotoxic activity of cultured splenocytes and BM cells was assessed by standard 4-h 51Cr release assay as described previously (23). Briefly, effector cells were seeded at various concentrations in quadruplicate in 96-well round-bottom microtiter plates. YAC-1 target cells were labeled for 2 h at 37°C with 100 µCi of Na51CrO4 (Amersham Life Science, Arlington Heights, IL), and 1 x 104 labeled cells were added to the effector cells. Plates were incubated at 37°C for 4 h, and the supernatant was harvested and evaluated for levels of 51Cr with a gamma scintillation counter. The percentage of specific cytotoxicity was calculated with the formula, % specific lysis = (a – b)/(c – b) x 100, where a = experimental release (cpm), b = spontaneous release (cpm), and c = maximum release (cpm).
Isolation of lung and liver nonparenchymal cells
Nonparenchymal cells of lungs and livers were isolated as described previously (24). Briefly, lungs and livers were perfused with prewarmed HBSS. Cell suspensions from excised organs were generated with a stomacher, and cell debris was removed by passing through nylon mesh. Cells were washed and resuspended in HBSS. Nonparenchymal cells were isolated by density-gradient centrifugation with Lympholyte-M (Cedarlane Laboratories, Ontario, Canada).
Statistical analyses
The statistical significance of all assays was assessed by using the two-tailed Student’s t test.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice
In the first set of experiments, we compared primary tumor growth
in LT-/- mice and wild-type littermate
controls. B16F10 melanoma cells injected s.c. into the lateral flank
formed solid tumors with relatively well-defined margins in either type
of mice. The tumor size was monitored on days 14, 17, and 21, and
enhanced tumor growth in the LT-/- mice was
noted (p < 0.002 on days 17 and 21) (Fig. 1
). Tumor invasion into the abdominal
cavity and considerable peritoneal dissemination were observed in three
of seven LT-/- mice examined (not shown). No
visible metastatic colonies were found in any organs from either
knockout or control mice by day 21. These observations suggested that
the local antitumor response is impaired in
LT-/- mice.
|
-/- mice can generate
an effective defense against systemic tumor metastases, we next applied
experimental metastasis models of syngeneic tumor cells and evaluated
the formation of metastases in the knockout mice.
LT-/- mice exhibited significantly higher
(p < 0.005) metastatic incidence in the form
of experimental pulmonary metastasis following i.v. injection of
B16F10; LT+/+ mice exhibited 46 ± 28
(mean ± SD) foci per lung (n = 6), whereas
LT-/- mice exhibited 117 ± 29 foci per
lung (n = 6). The mean number of melanoma colonies
found in LT-/- mice was 2- to 3-fold greater
than that in the wild-type littermate controls. The lungs from
LT-/- mice were grossly enlarged by the
metastatic disease, and the lung colonies formed in
LT-/- mice were markedly larger in size
(Fig. 2). Additionally, most of the
LT-/- mice, but not wild-type mice, appeared
to be severely cachectic on the day of the harvest. Multiple skin
metastases and moderate to severe peritoneal dissemination were also
observed in all knockout mice examined. Multiple liver metastases were
found in five of six knockout mice and in two of six wild-type mice
examined. Similar results were obtained for experimental metastasis of
3LL Lewis lung carcinoma cells (data not shown).
|
-/- mice exhibit lower NK
cytotoxicity in vitro
Previous studies have demonstrated the involvement of NK cells in
the inhibition of experimental and spontaneous metastases in several
models (25, 26, 27, 28). Therefore, these data suggested a
deficiency in NK activity in LT-/- mice. We
next performed experiments to evaluate the effect of the LT deletion
on in vitro NK cytotoxicity. Splenocytes were isolated from wild-type
and LT-/- mice, and cytotoxicity against
NK-susceptible YAC-1 cells was evaluated. As shown in Fig. 3, unstimulated splenocytes from
LT-/- mice exhibited significantly less
cytolytic activity against YAC-1 cells than did cells obtained from
wild-type littermates (p < 0.02).
Administration of polyICLC, a cytokine-inducing biologic response
modifier, induces a rapid increase in the total number and the
cytolytic activity of organ-associated and blood NK cells
(21). In vivo stimulation with polyICLC markedly augmented
the cytolytic activity of both wild-type and
LT-/- splenocytes. Splenocytes from
wild-type mice stimulated by the i.p. injection of polyICLC displayed
at 24 h a percentage specific cytotoxicity of 34.9% ± 2.5%
(mean ± SE) against YAC-1 target cells at the E:T ratio of 100:1,
whereas LT-/- splenocytes that were
stimulated similarly displayed cytotoxicity of only 20.4% ± 3.5% at
the same E:T ratio (p < 0.04) (Fig. 3).
Similar results were obtained for splenocytes stimulated in
vitro with polyICLC or rIL-12 (data not shown).
|
-/- mice contained 2- to
3-fold more leukocytes than that from wild-type littermates. Therefore,
it was possible that the lower cytolytic activity observed in
LT-/- mice might be the result of a lower
content of splenic NK cells. Indeed, flow cytometric analyses indicated
that the percentage of
NK1.1+CD3e- cells in
spleens from wild-type mice was 2.6% ± 0.2% (mean ± SE,
n = 6), whereas the NK level in
LT-/- mice was 1.2% ± 0.2%
(n = 6; p < 0.001 vs
LT+/+). Twenty-hour culture in vitro in the
presence of the stimulants did not significantly increase the number of
NK cells in either type of mice.
Two distinct molecular pathways, perforin/granzyme-mediated and
Fas-mediated pathways, are involved in tumor cell lysis by CTL and NK
cells (29, 30, 31, 32). NK cell-mediated, perforin-dependent
cytotoxicity is known to be crucial for the rejection of MHC class
I-negative tumor cells (33), such as B16F10 melanoma.
Sayers et al. (34) compared the contributions of these
pathways in killing of the murine renal carcinoma cell line Renca and
found that activated NK cells predominantly use the
perforin/granzyme-dependent pathways and activated T cells use
Fas/FasL-dependent pathways. To investigate the possible role of LT
in perforin regulation, immunoblot analysis was performed with
partially purified splenic NK cells. Our data indicated that perforin
protein was clearly detectable in NK cells from
LT-/- mice under both unstimulated and
polyICLC-stimulated conditions, and the expression level was only
slightly lower than that of the wild-type littermates (data not
shown).
Overall, these data suggest that the reduced in vitro cytotoxicity of
splenic NK cells from LT-/- mice primarily
resulted from a reduced frequency of these cells in the splenic
leukocyte population, although contribution of a reduced cytotoxic
potential of the individual NK cells cannot be excluded and should be
further analyzed.
LT-/- mice display a defective NK cell recruitment
to parenchymal organs
The recruitment of leukocytes into sites of infection or tumor
growth represents a critical early step in the development of an
effective host defense. Therefore, we next analyzed NK cell recruitment
to the parenchymal organs in response to exogenous stimuli. Lungs and
livers from unstimulated LT-/- mice
contained an increased number of nonparenchymal cells (data not shown),
consistent with the immunohistochemical observations reported
previously (11). Fig. 4
shows NK cell recruitment to the lung induced by polyICLC, an agent
known to augment the organ-associated NK cell number (21, 35, 36). A dramatic expansion of the
NK1.1+CD3e- population was
observed in the wild-type mice 72 h after i.p. injection of
polyICLC. The content of
NK1.1+CD3e- cells was
6.5% ± 1.5% (mean ± SE) in unstimulated mice, and this
elevated to 25.7% ± 0.9% after stimulation. Conversely, polyICLC
failed to induce a marked NK cell increase in
LT-/- mice. The content of
NK1.1+CD3e- cells was
1.3% ± 0.5% and 5.7% ± 3.2% in unstimulated and polyICLC
stimulated mice, respectively. A significant difference was found
between polyICLC-stimulated wild-type and
LT-/- mice (p =
0.026). In unstimulated conditions, the total number of NK cells per
lung in LT-/- mice was comparable with that
in wild-type littermates (Table I)
because of the increased leukocyte count in the
LT-/- mice. However, NK cell numbers in
LT-/- mice were significantly lower than in
wild-type littermates after polyICLC stimulation (Table I). No
appreciable change was found in
NK1.1+CD3e+ cell
populations after polyICLC stimulation in both strains (Fig. 4).
Similar results were obtained for liver-associated NK cells (Table I
and data not shown). These data indicate that
LT-/- mice show slower migration of NK cells
to the parenchymal organs.
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|
-/- mice
It has been demonstrated that the rapid accumulation of NK cells
in the liver induced by biologic response modifiers is mainly
associated with the rapid NK cell development from BM precursors
(35). Therefore, the impaired NK recruitment in response
to exogenous stimuli may suggest a defective production of this cell
population from BM precursors. To address the possible involvement of
LT in NK cell development, we cultured BM cells in the presence of
IL-2 and/or IL-15 and estimated the efficiency of lytic NK cell
development by cytotoxicity assay. In vitro stimulation with IL-2,
IL-15, or the combination of these cytokines markedly augmented
NK-mediated cytotoxicity in vitro in wild-type BM cells (Fig. 5, A and B), as
reported previously (37). However, exogenous IL-2 and/or
IL-15 could not efficiently induce the development of lytic NK cells in
LT-/- mice. Cytolytic activity of
LT-/- BM cells was inducible to some extent
but was much lower than that of similarly stimulated wild-type BM cells
(Fig. 5, A and B). These data suggested that
production/maturation of lytic NK cells from BM precursors is impaired
in LT-/- mice, and this impaired production
is likely due to a defective responsiveness to cytokine
stimulation.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/-
mice have impaired antitumor surveillance defenses. In particular,
syngeneic tumor cells were more metastatic and grew more rapidly in
LT-/- mice as compared with wild-type
littermates. Additional analyses indicated that
LT-/- mice were defective in NK migration to
parenchymal organs and NK cell development from BM cells induced in
vitro. Our data are consistent with the previous reports demonstrating
that mice with impaired NK activity manifest increased growth and
metastasis of experimental tumors (25, 26, 27, 28).
Because LT3 and
LT1ß2 can induce cell
death through TNF receptors and LTßR (38, 39, 40), the
effect of these molecules on the viability of inoculated tumor cells
should be considered. Although we do not have data to demonstrate
direct cytotoxic effects of LT molecules on B16F10 cells in vivo, the
results in another tumor model argue against contribution of direct
cytotoxicity. Indeed, despite the ability of Ab-LT fusion protein to
induce apoptotic death of M24met melanoma cells in vitro, M24met growth
could not be inhibited by systemic administration of this fusion
protein to scid/beige mice (41), suggesting
that LT molecules are not able to suppress the in vivo growth of tumor
cells even if they are sensitive to LT-induced apoptosis. Therefore, we
favor the hypothesis that LT contributes to the tumor rejection by
stimulating the host immune response, not by killing the tumor cells
directly.
LT-/- mice completely lack peripheral lymph
nodes; thus, it is possible that such an anatomical defect may affect
the innate and/or adaptive antitumor defense of
LT-/- mice. However, previous studies
demonstrated that BM is the major source of rapidly migrating NK cells
in response to biologic response modifiers (35).
Additionally, it was also shown that NK cell activation occurs
systemically after i.v. injection of bacterial adjuvant or polyIC, and
that the NK activity was augmented in spleens and peripheral blood more
efficiently than in peripheral lymph nodes (42). These
data suggest that peripheral lymph nodes may play a relatively minor
role in NK recruitment and in systemic NK-mediated antitumor responses.
We believe that the enhanced efficiency of experimental metastasis in
LT-/- mice is mainly due to the impaired
production of lytic NK cells from BM precursors and recruitment to the
organs and not to insufficient activation of NK cells resulting from
the lack of peripheral lymph nodes.
Similarly, it is also conceivable that the impairment of some functions
of tumor-specific CTL might contribute to the enhanced tumor growth and
metastasis. Previous studies demonstrated that transfection of MHC
class I and/or IFN-
gene to B16 cells (43) or injection
of IFN- retroviral vector to the B16 s.c. tumors (44)
successfully induced a strong anti-B16 response mediated by
CD8+ T cells. Additionally, in vitro treatment of
B16 cells with IFN resulted in an augmented expression of MHC class I
and an enhanced sensitivity to CTL (45, 46). However,
inoculation of untreated parental B16 cells failed to induce detectable
CTL activity (46). In fact, parental B16 melanoma cells
are considered to be less immunogenic because of lower MHC class I
expression on their cell surface (47). Additionally, it
has been demonstrated that in vitro induction of allogeneic CTL
response appears normal in LT-/- mice
(6). Therefore, it is unlikely that the enhanced tumor
susceptibility in LT-/- mice was largely due
to defects in Ag-specific CTL responses.
The total number of NK cells per spleen in
LT-/- mice was similar to that of wild-type
mice, but the NK cell content per organ was actually lower in the
knockout mice, although the total number of leukocytes was increased.
Because the NK1.1 cell surface molecule is expressed on C57BL/6 but not
on 129/Sv NK cells (37), the detection of NK cells with
anti-NK1.1 Ab may be complicated in C57BL/6-129/Sv mixed-background
mice. To avoid this potential problem with
LT-/- mice initially generated on mixed
C57BL/6-129/Sv background, we backcrossed these
LT-/- mice to the C57BL/6 background at
least 10 times and always used wild-type control littermates obtained
from LT+/- heterozygous breeding pairs as our
controls. Although our preliminary data suggest some down-regulated
expression of perforin protein in LT-/-
splenic NK cells (data not shown), we concluded that the lower content
of NK cells in LT-/- spleens is the main
reason for the suppressed in vitro cytotoxicity.
Importantly, our data showed that de novo production of NK cells from
BM is impaired in the knockout mice. IL-2 and IL-15 failed to augment
cytolytic NK cells in the in vitro culture of
LT-/- BM cells, and flow cytometric analyses
also indicated that
NK1.1+CD3e- cells were not
efficiently produced from cultured LT-/- BM
cells stimulated in vitro with these cytokines (data not shown).
PolyICLC is a strong inducer of several cytokines, including TNF and
IFN-, and augments NK cell development from BM precursors, migration
to the organs, and cytolytic activity (21, 35, 36). Fogler
et al. (23) showed that the VCAM-1/very late Ag-4
interaction is strongly enhanced by TNF rather than IFN- after
polyICLC treatment and is critical for the migration of newly recruited
NK cells into parenchymal organs and tumor lesions. Okahara et al.
(48) demonstrated that exogenous TNF up-regulates the
expression of VCAM-1 on vascular endothelial cells and very late Ag-4
on B16 melanoma cells and enhances the experimental lung metastasis of
B16. The exact regulatory role of LT signaling in NK development
remains to be determined. Although we cannot exclude the possibility
that LT, like TNF, plays a role in the regulation of endothelial
adhesion molecules that are necessary for NK cell migration to
parenchymal organs or tumor cell binding to the peripheral vasculature,
the lower production of this cell population from BM precursors may
significantly contribute to defective NK recruitment to the liver and
enhanced tumor growth/metastasis observed in
LT-/- mice. The failure of exogenously added
cytokines to induce NK cell development sufficiently in the culture of
LT-/- BM cells suggests an impairment in
cytokine receptor pathways. In particular, LT may control the
expression of IL-2/IL-15Rß or the signal transduction pathway
associated with this receptor system. Alternatively, the lack of LT
may result in defects in the biologic environment that is essential for
NK activation and thus may indirectly affect the NK activities.
While this manuscript was in preparation, Smyth et al.
(49) reported that TNF-deficient mice are defective in
tumor rejection in the peritoneum. However, the functional
characteristics of NK cells observed in the
LT-/- mice were quite different from those
reported for TNF-/- mice. Contrary to our
findings with the LT-deficient mice, Smyth et al. (49)
found that NK-mediated in vitro cytotoxicity was normal, but the NK
recruitment to the peritoneum in response to i.p. injection of tumor
cells was abrogated in the absence of endogenous TNF. In contrast, the
NK response to polyIC, one of the components of polyICLC, was retained
in TNF-/- mice. NK cell migration to the
peritoneal cavity could be effectively stimulated by polyIC in vivo. In
our experiments, polyICLC could induce only a small increase in the
percentage of lung NK cells in LT-/- mice.
As suggested by Smyth et al. (49), TNF may make only a
relatively minor contribution to the regulation of polyIC-stimulated NK
cell migration to the peritoneum. At variance with these findings, our
results suggest the importance of LT in polyICLC-stimulated NK cell
recruitment to parenchymal organs.
IFN- has immunomodulatory effects on several cell populations,
including NK cells. Previous reports demonstrated that excess tumor
growth was observed in IFN--deficient mice (50). Thus,
the impaired tumor control and NK activity observed in
LT-/- mice might be due to a down-regulated
expression of IFN-. However, studies using other biologic models
suggested that IFN- production is not impaired in mice with LT
deficiency (51, 52, 53). Therefore, it is unlikely that the
defective NK activity in LT-/- mice is the
result of IFN- down-regulation.
Although the specific molecular mechanisms of LT-mediated NK activation
have not been investigated in detail, the overall evidence may suggest
that signals coming through the LTßR are more important for NK cell
function than those coming through TNF receptors p55 and p75. In fact,
our preliminary data indicate that genetic inactivation of TNF receptor
p55 did not affect the efficiency of experimental lung metastasis of
B16F10 (data not shown). Considering the earlier report that surface LT
species on human LAK cells can support their cytotoxicity by
up-regulating effector-target adhesion (54), it is quite
possible that the lack of the surface LT in
LT-/- mice may affect the NK cell adhesion
required for NK recruitment and target cell lysis. To address such a
possibility, blocking strategies both in vitro and in vivo with Abs or
soluble ligands/receptors would be necessary. Alternatively, LT
signaling may regulate the expression of other classes of adhesion
molecules essential for interaction of NK cells with the endothelium,
and such expression may be low in LT-/-
mice.
Overall, our findings in this study suggest that LT-LTßR signaling is important for development and recruitment of NK cells and, therefore, ultimately in NK-mediated host defense. The molecular mechanisms for TNF- and LT-mediated regulation of nonspecific cellular immune responses should be further addressed in future studies.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Daisuke Ito, Laboratory of Molecular Immunoregulation, Division of Basic Sciences, NCI-FCRDC, Building 560, Room 31-33, Frederick, MD 21702-1201. E-mail address:
3 Address correspondence and reprint requests to Dr. Sergei A. Nedospasov, Intramural Research Support Program, SAIC Frederick, NCI-FCRDC, Building 560, Room 31-70, Frederick, MD 21702-1201. E-mail address:
4 Abbreviations used in this paper: LT, lymphotoxin; BM, bone marrow; polyIC, polyinosinic-polycytidylic acid; polyICLC, polyIC and polyL-lysine stabilized in carboxymethyl cellulose.
Received for publication February 24, 1999. Accepted for publication June 28, 1999.
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D. Elewaut, L. Brossay, S. M. Santee, O. V. Naidenko, N. Burdin, H. De Winter, J. Matsuda, C. F. Ware, H. Cheroutre, and M. Kronenberg Membrane Lymphotoxin Is Required for the Development of Different Subpopulations of NK T Cells J. Immunol., July 15, 2000; 165(2): 671 - 679. [Abstract] [Full Text] [PDF]
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 B. J. Lee, S. Santee, S. Von Gesjen, C. F. Ware, and S. R. Sarawar Lymphotoxin-alpha -Deficient Mice Can Clear a Productive Infection with Murine Gammaherpesvirus 68 but Fail To Develop Splenomegaly or Lymphocytosis J. Virol., March 15, 2000; 74(6): 2786 - 2792. [Abstract] [Full Text]
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