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Division of Developmental Immunology and
Division of Molecular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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and LTß, but the
effects of LT on T cell function have not been characterized
extensively. Upon TCR cross-linking in vitro, splenocytes from both
LT-/- and
LTß-/- mice failed to produce IL-4 and
IL-10 due to a reduction in NK T cells. Concordantly,
LT-/- and
LTß-/- mice did not respond to the
lipoglycan -galactosylceramide, which is presented by mouse CD1 to
V14+ NK T cells. Interestingly, both populations of NK T
cells, including those that are mouse CD1 dependent and
-galactosylceramide reactive and those that are not, were affected
by disruption of the LT and LTß genes. NK T cells were not
affected, however, in transgenic mice in which LT signaling is blocked,
beginning on day 3 after birth, by expression of a soluble decoy LTß
receptor. This suggests that membrane-bound LT is critical for NK T
cells early in ontogeny, but not for the homeostasis of mature
cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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ß, and a pattern of cell surface proteins
typical of activated T cells (1, 2). NK T cells are found
in spleen, liver, bone marrow, and thymus at a relatively constant
number of 
106 cells/organ (1, 3).
A striking characteristic of NK T cells is their ability to quickly
produce large amounts of cytokines, particularly IL-4 and IFN-
, upon
TCR stimulation (4). These cytokines have the potential to
affect the responses by conventional T lymphocytes and other cell
types. In fact, several reports suggest that NK T cells are important
for some forms of tumor rejection, prevention of autoimmunity,
protection against bacterial or parasitic infections, and altering the
T cell cytokine profile in response to soluble protein Ags
(5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15). Many NK T cells in mice are positively selected
by mouse CD1 (mCD1),4
a ß2-microglobulin
(ß2m)-associated protein related to the
MHC-encoded Ag-presenting molecules (16, 17). Recently,
however, it has become clear that not all mouse NK T cells are mCD1
reactive, as a significant fraction of NK T cells are found in
mCD1-/- mice (18, 19). The mCD1-dependent NK T cells are the majority in the
thymus and liver, but they are somewhat less prevalent in the spleen
and bone marrow. CD1-dependent NK T cells in the mouse are
predominantly CD4+ or
CD4-CD8- (double negative
(DN)), they have a biased TCR repertoire characterized by the
expression of a V14-J281 rearrangement with an invariant junction
(20, 21, 22, 23), and they recognize the lipoglycan
-galactosylceramide (-GalCer) presented by mCD1 (24, 25). CD1-independent NK T cells, on the contrary, mostly are
either CD8+ or DN, have a diverse TCR repertoire,
and are not -GalCer responsive (23). There is evidence
that NK T lymphocytes originate in the thymus, although data from some
studies are consistent with an extrathymic pathway for the
differentiation of some of these cells (3, 26, 27, 28, 29, 30, 31). It has
been reported that NK T cells are not dependent upon the Ras pathway
for their differentiation (32), but they are more
sensitive to the absence of Fyn than conventional T cells (33, 34), suggesting that distinct signals are required for their
development. In this study we have explored the relationship between
lymphotoxin (LT) and ß, two members of the TNF family, and the
development and function of NK T cell populations. TNF family cytokines
play diverse roles in the genesis of lymphoid organs and in the
regulation of Th1 cytokine production and chronic inflammation, but
relatively little is known concerning their effects on the development
and function of conventional T and NK T cells.
Members of the TNF family include TNF and LT, which form
biologically active homotrimers. Both TNF and LT can be secreted,
and they share the ability to bind to either TNF receptor I (60 kDa,
CD120a) or II (80 kDa, CD120b) (35). TNF and LT also can
be membrane bound. In the case of LT, this occurs through
association with another TNF family member, membrane-bound LTß, to
form the LT1ß2 heterotrimer (36). By contrast with
LT, the LT1ß2 heterotrimer signals exclusively via a specific
receptor (37), the LTß receptor (LTßR), which is
present on nonlymphoid cells (38). Although TNF is
expressed by many cell types, LT expression is restricted to activated
T and B lymphocytes and NK cells; within the CD4+
population LT is expressed by Th1 effector cells (39, 40).
TNF family cytokines and receptors often have overlapping
specificities. Consistent with this, in addition to LT1ß2, the
LTßR also interacts with LIGHT, a recently defined TNF family member.
Like many TNF family members, LIGHT binds another receptor in addition
to LTßR, the herpes virus entry mediator A (previously designated
HVEM) (41). Herpes virus entry mediator A also serves as a
third receptor for secreted LT in addition to the two TNF receptors
(41). Deficiency for a single TNF family gene (for
example, LT) therefore generally interrupts more than one possible
ligand-receptor interaction.
Studies in gene-targeted mice have indicated essential roles for LT,
LTß, and LTßR in secondary lymphoid organ structure (lymph node and
Peyer’s patches) and function, which may be linked to downstream
defects in production of T and B cell chemoattractants
(42, 43, 44, 45, 46, 47, 48).
LT-/- mice have
relatively normal T and B cell numbers and are not globally immune
deficient, although they do show reduced secondary Ab responses to some
Ags (43, 49). By contrast, here we find that LT has
profound affects on NK T cell differentiation early in ontogeny,
underscoring the unique developmental requirements of this T lymphocyte
subpopulation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-GalCer was synthesized at the Pharmaceutical Research
Laboratories, Kirin Brewery (Gunma, Japan), and was provided by Dr.
Yasuhiko Koezuka. The following mAbs were used in cytokine ELISAs:
anti-IFN- mAbs R4-6A2 and biotinylated XMG1.2, anti-IL-10
mAbs JES5-2A5 and biotinylated SXC-1, and anti-IL-4 mAbs BVD4-1D11
and biotinylated BVD6-24G2. The cytokine standards consisted of the
corresponding recombinant cytokines, IFN- (108
U/mg), IL-10 (2 x 107 U/mg), and IL-4
(107 U/mg). The following mAbs were used for
phenotypic analysis of lymphocytes: anti-CD16/32 (2.4G2),
FITC-labeled anti-TCRß (H57-597), FITC-labeled anti-Ly49G.2
(4D11), PE-labeled anti-NK1.1 (PK136), PE-labeled anti-CD44
(IM7), PE-labeled anti-CD122 (TM-b1), PE-labeled anti-CD1
(1B1), PE-labeled anti-pan-NK cells (DX5), PE-labeled
anti-Ly49C (5E6), PE-labeled anti Vß8.1-Vß8.2 TCR (MR5-2),
biotinylated anti-heat-stable Ag (M1/69), Cy-Chrome-labeled
anti-CD4 (H129.19), allophycocyanin-labeled anti-CD8
(53-6.7), and fluorochrome-labeled isotype-matched controls. All Abs
and recombinant cytokines were purchased from PharMingen (San
Diego, CA).
Mice and immunizations
C57BL/6 and (C57BL/6 x 129SV)F1
mice,
ß2m-/-
mice on the mixed C57BL/6 x 129SV background, and mice with a
disrupted LT gene
(LT-/-) on the mixed
C57BL/6 x 129SV background (43) were obtained from
The Jackson Laboratory (Bar Harbor, ME). The
LT-/- mice on an
inbred C57BL/6 background have been described previously
(50). Mice with a disrupted LTß gene (51)
on the mixed C57BL/6 x 129SV background were obtained from Dr. M.
Von Herrath (The Scripps Research Institute, La Jolla, CA) with
permission from Dr. R. Flavell (Yale University, New Haven, CT). LTßR
decoy transgenic mice on the BALB/c background were provided by Drs. R.
Ettinger and H. O. McDevitt (Stanford University, Stanford, CA)
(52, 53). These animals constitutively express a soluble
mouse LTßR human IgG1 fusion protein under the control of the human
CMV promoter. Two different lines, 1610 and 201, characterized by
different serum levels of the soluble decoy receptor, were used in this
study. The levels of soluble chimeric protein were determined by ELISA
as previously described (52). All mice were housed and
bred under specific pathogen-free conditions in the La Jolla Institute
for Allergy and Immunology vivarium. Experiments were initiated with 8-
to 12-wk-old mice. For in vivo immunizations, -GalCer was dissolved
in 0.5% polysorbate 20 (Nikko Chemicals, Tokyo, Japan) in a 0.9% NaCl
solution. Mice of both sexes were immunized both i.p. and i.v. with
either vehicle alone or 5 µg of -GalCer as previously described
(54). At the indicated time points, blood was obtained
from the retro-orbital plexus.
Primary cell cultures
For cytokine detection, suspensions containing freshly isolated
spleen cells were seeded at 1–2 x 106
cells/ml in 24-well plates, with 10 µg/ml of coated anti-CD3
(2C11) mAb, 200 ng/ml of -GalCer, or vehicle as a control.
Supernatants were harvested at the indicated time points, and cytokine
levels were detected using standard sandwich ELISAs, according to the
manufacturer’s protocol (PharMingen). Cytokine levels are expressed as
the mean ± SD of culture triplicates. To measure DNA synthesis,
cells were seeded at 4 x 105 cells/well in
96-well plates coated with 10 µg/ml anti-CD3.
[3H]Thymidine (1 µCi, 35 Ci/mmol; ICN, Costa
Mesa, CA) was added for the last 16 h to assess cell
proliferation.
Cell preparation and flow cytometry
Liver mononuclear cells were prepared as described previously
(55). Cells from thymus, spleen, and bone marrow were
prepared by conventional methods. RBC were removed from spleen cell
suspensions using a standard Ficoll gradient (Accurate Chemical &
Scientific, Westbury, NY) and from peripheral blood samples by osmotic
lysis. For surface staining, cells were suspended in buffer comprised
of PBS (pH 7.4) containing 2% BSA (w/v) and 0.02%
NaN3 (w/v). After blocking with 2.4G2
anti-FcR mAb, the cells were stained at 4°C for 20 min with
the labeled mAbs, then washed and analyzed on a FACSCalibur (Becton
Dickinson, San Jose, CA) flow cytometer. Lymphocytes were enumerated
out of the heterogeneous cell population by electronic gating, as
determined by forward and side angle light scatter.
Analysis of expression of V14-J281 and TCRß mRNA
Total RNA was extracted from various tissues of
LT-/-,
LTß-/-, and
ß2m-/-
mice as well as from wild-type and heterozygous control littermates
with an RNeasy kit (Qiagen, Valencia, CA). Specific mRNA was amplified
by RT-PCR. In brief, 5 µg of RNA was reverse transcribed using
oligo(dT) primers; the cDNAs then underwent 40 cycles of amplification
at 94°C for 45 s, 64°C for 45 s, and 72°C for 45 s
with primers specific for either V14-J281 or Cß. The sequences
used for primers were 5'-GTTGTCCGTCAGGGAGAGAA-3' for V14,
5'-CAATCAGCTGAGTCCCAGCT-3' for J281 (19, 56),
5'-CACTGATGTTCTGTGTGACA-3' for Cß-forward, and
5'-GAGGATCTGAGAAATGTGACTCCAC-3' for Cß-reverse. The amount of
template cDNA used in each reaction was normalized to the amount of
Cß mRNA amplified. Each experiment included negative controls in
which RNA was omitted from the RT mixture, and cDNA was omitted from
the PCR reaction. Agarose gels were stained with ethidium bromide and
photographed under UV illumination.
Preparation and use of -GalCer mouse CD1 dimers
The properties of the -GalCer mCD1 dimers will be described
in detail elsewhere (J. Matsuda, O. Naidenko, D. Elewaut, and M.
Kronenberg, manuscript in preparation). The production and purification
of soluble recombinant mCD1 from D. melanogaster tissue
culture cells were previously described (57). Purified
mCD1 protein was incubated for 12 h at room temperature with a
3-fold molar excess of -GalCer. Subsequently, mCD1/-GalCer
complexes were incubated with a 1.5-fold molar excess of the
biotinylated anti-CD1 mAb 2B9 (a gift from Dr. C.-R. Wang,
University of Chicago, Chicago, IL), and the mCD1 dimers were separated
from free Ab by gel filtration on Superdex 200 column (Amersham
Pharmacia, Piscataway, NJ). For staining, cells were incubated for 30
min with -GalCer mCD1 dimers, washed, incubated for 20 min with
tricolor-streptavidin (Caltag, South San Francisco, CA), washed again,
and resuspended in staining buffer for analysis. The specificity of the
dimer staining has been verified using NK T cell and control hybridomas
and lymphocytes from wild-type mice and control mice known to be
defective for NK T cells. T cell staining is dependent upon both
-GalCer and dimerization of the -GalCer/mCD1 complexes.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- T cells is caused by NK T cell
deficiency
The rapid ability to produce cytokines is a characteristic of NK T
cells. We therefore assessed the capacity of splenocytes from
LT-/- mice on the
mixed C57BL/6 x 129SV background to produce IFN-, IL-4, and
IL-10 following in vitro stimulation with plate-bound anti-CD3
mAbs. Splenocytes from
LT-/- and
LT+/+ mice produced comparable amounts of
IFN-, although in some experiments there was a reduction in IFN-
secretion at the earlier time points (Fig. 1
A). By contrast, spleen cells
from LT-/- mice showed
a drastic decrease in both IL-4 and IL-10 secretion (Fig. 1, B and C) even after 120 h of in vitro
culture (data not shown). The proliferative capacity of
anti-CD3-stimulated splenocytes from
LT-/- mice was
comparable to that of wild-type mice (Fig. 1D). The
proliferation and IFN- secretion by T lymphocytes from
LT-/- mice argues
against any global defect in T cell function in these animals despite
their lack of lymph nodes. This finding is consistent with data
published previously indicating that
LT-/- mice can reject
allogeneic grafts and can carry out other cell-mediated immune
responses (43).
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-/- and
LT+/+ mice and determined the fraction of
NK1.1+ TCRß+ cells in the
various organs by flow cytometry (Fig. 2
and Table I). The percentage of
NK1.1+ TCRß+ lymphocytes
was greatly and consistently reduced in
LT-/- vs
LT+/+ mice (Fig. 2).
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-/- mice (data
not shown). In addition, the fraction of DN thymocytes that are TCR
Vß8+ cells, the ß-chain to which the
predominant V14-J281 TCR in thymic NK T cells preferentially
pairs, also was reduced in
LT-/- compared with
wild-type mice (data not shown). Finally, to rule out any
strain-dependent effect due to the presence of varying amounts of 129
and C57BL/6 genes in the mice on the mixed genetic background, we
repeated the analysis in
LT-/- and
LT+/+ mice on an inbred C57BL/6 background.
The data in Table I
summarize the fraction of NK T cells found in
LT-/- mice on the two
different backgrounds, in the appropriate wild-type positive controls
and in negative control
ß2m-/-
mice, which should lack all NK T cells dependent upon either mCD1 or
other class I molecules. Most importantly, the data demonstrate a
consistent reduction in the percentage of NK T cells in all organs of
the LT-/- mice on both
genetic backgrounds, with the reduction in
LT-/- mice comparable
to that in
ß2m-/-
mice. Overall, the reduction in NK T cell percentage is most pronounced
in the liver and thymus. Furthermore, the effect of LT deficiency is
more pronounced on the inbred than on the mixed background, whereas the
opposite pattern would be expected if allelic differences in NK1.1
expression were responsible for the observed decrease.
The number of leukocytes is increased in
LT-/- mice, as
previously described (58, 59). We therefore also
determined the absolute numbers of NK T cells that were present in
inbred C57BL/6 LT-/-
mice and C57BL/6 controls (Fig. 3).
Statistically significant (p < 0.05, by
Student’s t test) decreases were found in every case, with
the most dramatic reductions in NK T cell number observed in liver and
thymus (Fig. 3).
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The reduced NK T cell numbers could be dependent upon the activity
of LT, LT1ß2, or both. To address this issue, we determined the
fraction of NK T cells in spleen, liver, thymus, bone marrow, and blood
of LTß-/- mice (Table I
and Fig. 2). Interestingly, a reduced percentage of NK T cells was
observed in LTß-/-
mice, similar to the findings in
LT-/- mice. Reductions
in absolute cell numbers also were observed, and as for LT
deficiency, these were most significant in liver and thymus (Fig. 3).
The expression of other NK T cell-associated markers also was decreased
on TCR intermediate cells in the
LTß-/- mice (data not
shown), and some residual NK1.1+ T cells were
present. Furthermore, similar to the results obtained in
LT-/- mice, spleen
cells from LTß-/- mice
showed reduced IL-4 and IL-10 production following CD3 cross-linking in
vitro (data not shown). These data confirm that NK1.1 allelism is not
responsible for the observed decrease in NK T cells in
LTß-/- mice, and they
are consistent with a predominant role for membrane LT1ß2 in
determining NK T cells numbers in different organs.
Reductions in mCD1-dependent and mCD1-independent NK T cell populations in LT-deficient mice
LT-/- and
LTß-/- mice have a
dramatic reduction in the numbers of NK T cells in liver and thymus.
These data suggest that the defect in NK T cells primarily affects the
mCD1-dependent population, which is enriched in these sites. A key
feature of the mCD1-dependent population is their rapid cytokine
release following TCR stimulation (18, 19); therefore, a
selective defect in mCD1-dependent NK T cells also could account for
the poor IL-4 release observed following in vitro stimulation (Fig. 1).
To determine whether any of the remaining NK T cells in LT-deficient
mice were in fact mCD1 dependent, the expression of the invariant
V14-J281 chain was analyzed by RT-PCR in spleen (Fig. 4A), liver, and thymus (data
not shown) of
ß2m-/-,
LT-/-, and
LTß-/- mice.
Surprisingly, V14-J281 mRNA could be detected in all tissues
analyzed in LT-deficient mice, although the signal was reduced compared
with that in wild-type mice. By contrast, no signal could be detected
under these conditions in mRNA from
ß2m-/-
mice (Fig. 4A).
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B) and liver (data not shown) of
LT-/- and
LTß-/- mice. The DN NK
T cells were less affected by LT deficiency.
The presence of V14J281 mRNA and the pattern of coreceptors
expressed by NK T cells, however, do not directly determine the
fraction of mCD1-dependent NK T cells. Moreover, there is some
controversy about whether CD8+ NK T cells are
solely mCD1 independent (60). We therefore stained
lymphocyte preparations with a soluble mCD1 dimer loaded with
-GalCer. The -GalCer mCD1 dimers specifically stain those T cells
that are -GalCer reactive and mCD1 restricted (J. Matsuda, O.
Naidenko, D. Elewaut, and M. Kronenberg, manuscript in preparation).
Interestingly, the numbers of -GalCer mCD1
dimer+ and -GalCer CD1
dimer- NK T cells were both statistically
significantly reduced in the spleen of
LT-/- mice (Fig. 4C; p < 0.05, by Student’s t
test). Approximately one-half of the NK1.1+
TCRß intermediate T cells in the spleen are mCD1 dimer binding
regardless of whether the cells were obtained from
LT+/+ or
LT-/- mice (Fig. 4D). Very few NK1-
TCRß+ cells stained with the dimers in either
wild-type or LT-/-
mice (Fig. 4D), consistent with the NK T cell specificity of
this reagent. Similar data were obtained in the liver, although the
relative frequency of mCD1 dimer+ cells among NK
T cells was higher (data not shown). Based upon these experiments, we
conclude that LTß influences the number of both the large
population of -GalCer-reactive lymphocytes as well as other NK T
cell populations.
LT-deficient mice do not respond to -GalCer
We assessed the immune responsiveness of the residual NK T cell
population in LT-deficient mice. The glycosphingolipid -GalCer is
presented by mCD1 to those NK T cells that express the invariant
V14+ TCR (24, 25). When freshly
isolated splenocytes from
LT-/- mice on the
mixed C57BL/6 x 129SV background were stimulated with -GalCer,
very little production of IFN- (Fig. 5A) or IL-4 (Fig. 5B) was observed. Similar results were obtained in
LT-/- mice on the
C57BL/6 background (data not shown). Moreover, the release of IFN-
upon -GalCer administration in vitro was strongly reduced in
LTß-/- mice (<2 ng/ml
in 5-day cultures; three independent experiments). Mouse CD1 levels on
APC were unaffected in all organs analyzed, and
LT-/- splenocytes are
able to present -GalCer to mouse V14/Vß8+
hybridomas as well as LT+/+ splenocytes (data
not shown). This indicates that the reduced -GalCer responses in
LT-deficient mice are not due to a reduced ability to form stimulating
lipid-mCD1 complexes at the surface of APCs.
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5- to 10-fold (Fig. 4).
Therefore, we assessed whether similar numbers of
CD4+ NK T cells of LT+/+
and LT-/- splenocytes
differed in their ability to produce IL-4 upon cross-linking with
anti-CD3 by titrating the number of spleen cells added to the
culture. Interestingly, comparable amounts of IL-4 were produced by
similar numbers of splenic CD4+ NK T cells from
LT-/- vs
LT+/+ mice (data not shown). We conclude that
the decreased responsiveness by the LT-deficient spleen cells to
-GalCer in vitro primarily is due to decreased NK T cell numbers
rather than to a functional deficiency of the remaining NK T cells.
Consistent with this conclusion, the in vitro proliferation of
LT-/- splenocytes to
-GalCer was reduced compared with that in wild-type splenocytes,
although it was higher than that observed in splenocytes from
ß2m-/-
mice (Fig. 5C).
To determine whether the decreased response to the glycosphingolipid
was also observed in vivo, cohorts of
LT-/- and
LT+/+ mice, on either the C57BL/6 or the mixed
C57BL/6 x 129SV background, were immunized with -GalCer.
Sixteen hours after immunization, serum levels of IFN- were
determined by ELISA. Whereas in vivo immunization with -GalCer
results in the release of high amounts of IFN- in serum of
LT+/+ mice, a dramatically reduced response
was observed in LT-/-
mice regardless of their genetic background (Fig. 5D).
Homeostasis of NK T cells in LT-/-
mice
In vivo administration of anti-CD3 results in a rapid
depletion of NK T cells by activation-induced cell death, followed by
repopulation of the spleen and liver from a proliferating pool of cells
in the bone marrow (61). We have found similar dynamics of
NK T cells in vivo following the administration of -GalCer
(54). To determine whether the homeostasis of NK T cells
is abnormal in LT-/-
mice, the number of NK T cells in the blood was determined before
immunization, 1 and 14 days after administration of -GalCer. One day
after immunization a significant reduction in NK T cells was observed
in wild-type mice, followed by a recovery of NK T cell numbers in the
circulation by day 14 (Fig. 6). At each
time point analyzed the fraction of circulating NK T cells in
LT-/- mice was lower
than that in LT+/+ mice (Fig. 6). A depletion
of circulating NK T cells upon -GalCer administration was also
observed in LT-/-
mice, however, with a recovery in cell numbers by day 14. These data
suggest that NK T cells can respond in vivo to -GalCer in a fashion
similar to normal mice, although their absolute number is significantly
reduced.
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The previous results suggest that LT1ß2 could be required for
NK T cell differentiation, with a perhaps lesser affect on NK T cell
function. To directly address the possibility that mature NK T cells
require LT1ß2 to function effectively, we measured NK T cell
number and function in LTßR-Fc transgenic mice. These mice express
the transgene under the control of the human CMV promoter. The
LTßR-Fc acts as a decoy receptor, thereby blocking LTßR-mediated
signaling, activated by either LT1ß2 or LIGHT. Previously
published data have demonstrated that circulating LTßR-Fc fusion
protein cannot be detected until 3 days after birth (52).
Whereas peripheral lymph node development in the LTßR-Fc transgenic
mice is normal, anatomic abnormalities affecting spleen and Peyer’s
patches are present (52), indicating that some aspects of
lymph node organogenesis can proceed normally when LT signals are
delivered only early in ontogeny.
As the transgenic mice are on the BALB/c background, which lacks the
NK1.1 marker, we determined the fraction of NK T cells by staining for
IL-2Rß (CD122) and TCRß. Two lines of LTßR-Fc transgenic mice,
expressing higher (line 1610) or lower (line 201) levels of the decoy
receptor, were analyzed. The levels of soluble chimeric protein in
individual transgenic mice from both lines were determined (data not
shown) to confirm the difference in expression levels and to detect a
possible correlation between those levels and NK T cell function. Only
mice producing sufficiently high levels of soluble chimeric protein to
affect the development of splenic architecture (Fig. 7A) and Peyer’s patches (not
shown) were included in this study, indicating that the LTßR-Fc was
functional and present at sufficient levels to alter lymphoid
organogenesis. Interestingly, the circulating fractions of
CD122+ cells expressing intermediate levels of
TCRß were comparable in the transgenic mice and the controls (data
not shown). In addition, the levels of IFN- released in serum after
-GalCer administration were comparable in both lines of LTßR-Fc
transgenic mice and wild-type mice (Fig. 7B). No correlation
between the levels of circulating LTßR-Fc fusion protein in
individual mice and NK T cell numbers or function was observed. Thus,
similar to the results obtained when the formation of lymph nodes is
analyzed, the impaired response to -GalCer in
LT-/- and
LTß-/- mice is most
likely caused by a critical role of LT1ß2 early in the ontogeny of
NK T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and LTß
deficiency are very similar, implicating a critical role for the
LT1ß2 heterotrimer and suggesting that there is no major,
nonredundant role for LT homotrimers.
Several findings suggest that the major effect of LT deficiency is on
the differentiation of NK T cells as opposed to their long-term
survival, localization, or ability to expand in response to Ag. First,
the number of NK T cells is reduced in the sites where NK T cells are
known to develop, including the thymus, as well as the bone marrow and
the liver, which are possible sites of extrathymic NK T cell
differentiation (3, 26, 27, 28, 29, 30, 31). Second, LTßR-Fc transgenic
mice have no defects in either NK T cell number or immune responses,
although the levels of LTßR-Fc measured in the sera of these animals
should saturate the two LTßR ligands (52), LT1ß2
and LIGHT, beginning a few days after birth. In agreement with
previously reported results (52), the transgenic mice
producing high levels of soluble LTßR decoy we analyzed did in fact
display a dramatically altered splenic architecture and entirely lacked
visible Peyer’s patches. The normal number and function of NK T cells
in the transgenic mice suggest, therefore, that LT1ß2 is critical
very early in ontogeny. Furthermore, LT1ß2 and LIGHT are not
likely to be required either for homeostasis of mature NK T lymphocytes
or as a trophic factor for these cells once they have matured. Third,
the developmental defect in the generation of NK T cells in
LT-/- and
LTß-/- mice is not
absolute, and the residual population is able to respond to antigenic
stimulation. This population in LT-deficient mice includes
mCD1-dependent cells, as indicated by levels of V14-J281 mRNA
greater than those observed in
ß2m-/-
mice and residual cells that stain with the -GalCer mCD1 dimers. The
mCD1-dependent NK T cells in LT-deficient mice can respond to
-GalCer, as detected by proliferation in vitro in response to the
lipid Ag, and changes in the level of circulating NK T cells following
Ag administration. Interestingly, while the decline and recovery of NK
T cells in LT-/- mice
following -GalCer treatment resemble the events that occur in
wild-type mice, the set-point or maintenance level of NK T cells in
LT-/- mice is reduced.
It should be noted that, as opposed to proliferation, the cytokine
response to -GalCer was greatly reduced in most experiments. This
reduction can be accounted for in part by correcting for the decreased
numbers of CD4+ NK T cells in LT-deficient mice.
Therefore, while we cannot rule out a partial reduction in the function
of the residual NK T cells in LT-deficient mice, particularly with
regard to cytokine secretion, the results from the LTßR-Fc transgenic
mice indicate that a putative reduction is likely to be a result of a
developmental defect, rather than a requirement for LT1ß2 by
mature NK T cells.
Several experiments were conducted to determine whether the absence of
lymphotoxin caused reduced numbers of mCD1-independent as well as
mCD1-dependent NK T cells. Many of the peripheral mCD1-dependent NK T
cells lack CD8 (18, 23), and therefore the proportion of
CD8+ NK T cells can be used to assess indirectly
the effects of LT deficiency on the mCD1-independent NK T cell
population. We found that the proportions of CD4, CD8, and DN NK T
cells are similar in the spleens of
LT-/-,
LTß-/- and wild-type
mice, concordant with a global reduction in all NK T cell populations.
Consistent with this, the number of -GalCer mCD1
dimer+ NK T cells (the great majority of which
are likely to be V14+ lymphocytes) and
-GalCer mCD1 dimer- NK T cells were both
strongly reduced in
LT-/- compared with
wild-type mice. Therefore, it is most likely that mCD1-independent as
well as mCD1-dependent NK T cells are affected by LT deficiency.
The stage of NK T cell differentiation that requires the activity of
the LTß heterotrimer is not known. Given the pattern of expression
of this receptor-ligand pair, we speculate that the critical
interaction involves LT1ß2 expression by a lymphocyte precursor
and LTßR expression by a stromal cell. Defects in the NK T cell
precursor, the stromal cell, or both are consistent with this model.
Interestingly, dysfunction of stromal cells has been reported in
spleens of LT-/- and
LTß-/- mice, as
follicular stromal cells and T zone stromal cells have markedly
depressed expression of B lymphocyte chemoattractant and secondary
lymphoid tissue chemokine compared with wild-type mice
(48).
Lantz et al. have proposed a stepwise model for the differentiation of
mCD1-dependent NK T cells in the thymus (62). This
includes primary selection via their invariant TCR -chain to confer
the IL-4-producing phenotype, followed by acquisition of NK-associated
markers, maturation, and export to the periphery. This model was based
on results obtained in mice lacking the common -chain of cytokine
receptors
(c-/-
mice), which also lack NK cells. Despite the absence of the
NK-associated markers (NK1.1, Ly49) on T cells in these mice, NK
thymocytes retain the characteristic expression of the cytokine
receptors IL-7R and IL-2Rß, they can produce normal amounts of
IL-4 upon TCR cross-linking, and like their wild type counterparts,
many of the cells are Vß8+. Moreover, normal
amounts of V14-J281 mRNA were detected in the thymus, but not the
periphery, of
c-/-
mice. Our findings suggest that the effect of LTß occurs at an
earlier stage, as thymocytes from
LT-/- and
LTß-/- mice have
reduced levels of V14-J281 mRNA and Vß8+
DN cells, and they lack IL-2Rß + cells (data
not shown).
While this manuscript was in preparation, impaired function of NK cells
in LT-/- mice was
reported (58, 59, 63), consistent with the possibility
that NK and NK T cells share a common precursor. The precise details of
the process by which LT affects NK cells remain controversial. Iizuka
et al. suggested a developmental defect caused by the lack of
LT1ß2. They found that administration of LTßR-Ig fusion protein
to pregnant C57BL/6 and
C57BL/6-Rag1-/- mice
resulted in a profoundly impaired development of splenic NK cells.
Others reported, however, that
LTß-/- mice exhibit
normal NK cell function and suggested that multiple deficiencies
underlie the NK cell inactivity in
LT-/- mice
(59). These include lower NK cell numbers in bone marrow,
the main source of NK cell progenitors, as well as a reduced NK cell
cytotoxicity caused by lower perforin expression by
LT-/- NK cells.
Moreover, the recruitment of NK cells to parenchymal organs was
reported to be defective in
LT-/- mice
(58). One of the above-cited studies on NK cells
(63) also suggested a splenic NK T cell defect in
LT-/- mice. However,
they could not differentiate between improper localization to the
spleen similar to what has been described for dendritic cells
(64) or a global deficiency caused by a developmental
block, and the ability of NK T cells to respond to specific Ags was not
tested. Thus, the results presented in our study demonstrate a
fundamental and novel role for LTß in the differentiation of
mature NK T cells. Once the NK T cells have matured, LTß is not
required for their homeostasis.
In summary, the studies reported herein indicate that LT1ß2 is
required for the development of both mCD1-dependent and
mCD1-independent NK T cells. NK T cells are reduced in all organs of
both LT-/- and
LTß-/- mice. The
remaining NK T cells in LT-deficient mice are functional, and they can
respond to Ag. In LTßR-Fc transgenic mice, normal numbers of
circulating NK T cells and -GalCer responses are found. These
findings demonstrate a crucial role early in ontogeny for LT in the
differentiation of NK T cells, emphasizing that these lymphocytes have
distinct requirements for their development.
|
Acknowledgments |
|---|
-GalCer
(KRN7000); Dr. C.-R. Wang for providing anti-CD1 mAb 2B9;
Drs. M. Huflejt and T. Prigozy for their helpful suggestions
and advice; and C. Lena for his help with breeding the mice used in the
experiments. |
Footnotes |
|---|
2 This is manuscript 343 from the La Jolla Institute for Allergy and Immunology.
3 Address correspondence and reprint requests to Drs. Mitchell Kronenberg or Carl F. Ware, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, CA 92121.
4 Abbreviations used in this paper: mCD1, mouse CD1.1; ß2m, ß2-microglobulin; DN, double negative; GalCer, galactosylceramide; IL-2Rß, IL-2 receptor ß-chain; LIGHT, lymphotoxin-like, inducible expression, and competes with HSV glycoprotein D for HVEM, a receptor involved in costimulation of T lymphocytes; LT, lymphotoxin; LTßR, LT ß receptor.
Received for publication January 31, 2000. Accepted for publication April 24, 2000.
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