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*
Department of Pathology and Committee on Immunology, and
Gwen Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; and
Department of Immunology, Mayo Clinic, Rochester, MN 55905
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A cellular ligand for herpes virus entry mediator and lymphotoxin
receptor (LIGHT),4 a newly discovered member of the TNF
superfamily, closely related to lymphotoxin, binds the lymphotoxin-
receptor (LTR) and the
herpesvirus entry mediator (HVEM) (13). In vitro studies
have shown that LIGHT may have effects on both activation and apoptosis
(14, 15, 16, 17). LIGHT is mainly expressed on lymphoid tissues,
such as thymus and spleen (13). LIGHT expression has also
been detected on activated T cells (13, 14). Therefore,
LIGHT appears to be a strong potential candidate molecule to facilitate
negative selection. To demonstrate the role of LIGHT in T cell
development in the thymus, we have generated LIGHT transgenic (Tg) mice
and manipulated negative selection in TCR Tg mice by addition of
soluble receptors blocking the action of LIGHT. Our results reveal a
critical role for LIGHT in thymic negative selection.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FTOCs were performed according to procedures previously
described (18, 19). Briefly, on gestational day 16.5
thymic lobes from
D7-TCR
-/- (D7
Tg-) or
D7+TCR-/- (D7
Tg+) mice were harvested and incubated with or
without 1 µM LemA peptide (Research Genetics, Huntsville, AL)
in the presence of control-Ig, anti-LT Ab, HVEM-Ig, or LTR-Ig
(30 µg/ml). Media, peptide, and fusion proteins were replenished
every 2 days. After 10 days, thymocytes were stained with
CyChrome-anti-CD4, PE-anti-CD8, and FITC-anti-V5 Abs (BD
PharMingen, San Diego, CA) and analyzed by FACScan (BD Biosciences,
Mountain View, CA). LTR-Ig fusion protein used in this study was
described previously (20). Anti-LT Ab was kindly
provided by Dr. J. Browning (Biogen, Cambridge, MA)
(21).
Generation of Tg mice
LIGHT cDNA was initially cloned by RT-PCR into pCDNA3.1, it then
was inserted into the AscI site of plck.E2 (a generous gift
from Dr. T. Hettmann, University of Chicago, Chicago, IL) which
contains the proximal lck promoter, human growth hormone
gene (polyadenylation site) and locus control region elements from the
human CD2 gene. An 8-kb fragment was excised by NotI and
used for subsequent microinjection performed by the University of
Chicago Cancer Research Center Transgenic Mice Facility.
PstI-digested tail DNA from mice was hybridized to a
radiolabeled 0.7-kb LIGHT-specific probe. Four positive founders were
generated; two were in the C3H background and the other two were in the
C57BL/6 background. Most mice used in this study were in the C3H
background. LIGHT protein expression was detected in thymocytes from Tg
mice by FACS with LTR-Ig (20) and anti-LIGHT Ab
(14). Tg mice and littermates were analyzed at the age of
5–8 wk unless otherwise indicated.
Flow cytometric analysis
Single cell suspensions of thymocytes and splenocytes were collected and stained with anti-CD8-PE and anti-CD4-FITC (for three-color FACS, anti-CD4-CyChrome was used) Abs (BD PharMingen, San Diego, CA) in PBS plus 0.01% NaN3 for 30 min at 4°C. After incubation with mAbs, the cells were analyzed on a FACScan (BD Biosciences, Mountain View, CA). Annexin V and propidium iodide (PI) double staining for apoptosis was performed using a commercially available kit as described by the manufacturer (BD PharMingen).
In vivo treatment protocol
Age- and sex-matched H-Y/RAG-2-/- mice
(Taconic Farms, Germantown, NY) received 3–4 i.p. injections weekly of
100 µg soluble murine LTR-Ig or PBS. Thymi were collected 7 days
after the last injection. Thymocytes and splenocytes were stained with
CyChrome-anti-CD4, PE-anti-CD8, and FITC-anti-V8 (F23.1)
Abs (BD PharMingen) and analyzed by FACScan (BD Biosciences).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Negative selection is one of the major mechanisms to induce
apoptosis of immature thymocytes, and previous studies indicate that
members of the TNF family of ligands and receptors may be involved in
negative selection; however, none of these molecules has been proven to
be essential for negative selection (4, 10, 11). Due to
the unique features of LIGHT in both activation and apoptosis, the
potential role of LIGHT in negative selection was investigated in this
study. A key aspect of negative selection is associated with the
interaction of the TCR with self-MHC/peptide complexes. However, it is
extremely difficult to distinguish the specificity of antigenic
negative selection with undefined TCR or too diversified TCR
repertoire. To overcome this problem, we chose a model system in which
D7+ TCR -/- Tg (D7
Tg+) mice express a specific single TCR (D7)
restricted by the H2-M3, a MHC class Ib molecule, which recognizes
hydrophobic N-formylated peptides (19). In our
D7 Tg model, negative selection can be mediated efficiently by the
H2-M3 molecule when antigenic peptide, LemA, is provided
(19). In vitro incubation of the thymic lobes from
D7+ TCR -/- Tg mice
with LemA peptide (one N-formylated peptide) results in a
dramatic reduction of double-positive (DP) and
CD8+ single-positive (SP) cells as compared
with the group without peptide treatment (19). The D7
Tg+ mice and D7 Tg-
littermates were both on TCR -/- background
and the CD8+ population in
D7+ TCR -/- mice is
exclusively positive for M3-LemA tetramer staining
(19).
We introduced LTR-Ig, a soluble receptor for LIGHT, into these FTOC
of D7+ TCR-/- Tg mice
and their non-Tg littermates to determine whether LIGHT plays a
critical role in negative selection. Interestingly, we found that the
addition of LTR-Ig into the FTOC system led to a significant
increase of the percentage of DP and CD8+ SP
cells in D7 Tg+ mice (Fig. 1
a). In contrast, the
percentage of these cells remained very low in the presence of control
Ig when the specific peptide was added in D7 Tg+
FTOC (Fig. 1a). In our model, negative selection mediated by
MHC (H2-M3)/peptide (LemA) was Ag specific since the addition of
LTR-Ig into FTOC had little effect on D7-
thymocytes. In D7 Tg- FTOC, there was no
significant difference in the subset distribution of thymocytes between
LTR-Ig and control-Ig treated groups (Fig. 1a). Moreover,
negative selection was not induced in the absence of LemA peptide in D7
Tg+ FTOC (Fig. 1a).
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R, LIGHT binds another receptor HVEM
(13). Using the same FTOC system, our data showed that
HVEM-Ig, binding to LIGHT in vitro, could also rescue the DP and
CD8+ SP in a way similar to LTR-Ig (Fig. 1
b). The total number of thymocytes recovered from FTOC was
determined and there was no significant difference between the
different groups in D7 Tg- mice regardless of
the addition of peptide LemA and different fusion proteins (data not
shown). In contrast, the addition of peptide (LemA) led to the decrease
of total number of thymocytes in D7 Tg+ mice due
to the strong negative selection induced by this high-affinity peptide
LemA (Fig. 1d, control-Ig group). LTR-Ig and HVEM-Ig at
least can partially block the negative selection mediated by
high-affinity peptide: DP and CD8 SP thymocytes recovered from FTOC in
LTR-Ig and HVEM-Ig groups were significantly higher than control-Ig
group (Fig. 1d). For CD4/8 DP thymocytes, there was a
40-fold increase in the LTR-Ig group (0.81 ± 0.09 x
104 vs 0.02 ± 0.01 x
104, p < 0.0001) and an 80-fold
increase in the HVEM-Ig group (1.65 ± 0.39 x
104 vs 0.02 ± 0.01 x
104, p < 0.0001) compared with
control-Ig group (Fig. 1d). In the absence of either the
cognate TCR (D7) or the deleting peptide (LemA) which induced the
strong negative selection in D7 Tg+ FTOC,
LTR-Ig and HVEM-Ig treatments had no significant effect on cell
numbers nor were the thymocyte subset percentages altered compared with
control-Ig group. These results clearly demonstrated the critical role
of LIGHT in negative selection.
LTR has another ligand called membrane lymphotoxin (mLT) which is a
heterotrimer LT12
(21, 22, 23). To determine whether mLT is involved in this
Ag-specific negative selection, we performed FTOCs in the presence of
anti-LT Ab that selectively disrupts the interaction between mLT
and LTR (21, 22, 23). The lot of anti-LT Ab that we
used in this study was capable of blocking LT activity, such as
germinal center follicular dendritic cell formation and Ab
production (data not shown). We found that anti-LT Ab was unable
to block the negative selection in D7 Tg+ FTOC in
the presence of LemA peptide, and the immature DP and SP thymocytes
were deleted due to the Ag-induced negative selection (Fig. 1c). In D7 Tg- FTOC, the addition of
peptide did not cause any significant change to the subset distribution
of thymocytes in the presence of anti-LT Ab (Fig. 1c). The control-Ig used in this set of experiments behaved
similarly as the control-Ig used previously (Fig. 1c). These
data suggested that mLT may not contribute significantly in this
negative selection model and the effect of LTR-Ig on this model
appears to be predominantly dependent on the LIGHT pathway.
Consistently, our data showed that HVEM-Ig, binding to LIGHT but not
mLT, could also block negative selection in a way similar to LTR-Ig.
Therefore, our data strongly implicate that LIGHT, as the interacting
ligand, is responsible for the blocking effects on our negative
selection model.
The blockade of LIGHT signaling in vivo reduces negative selection
H-Y TCR Tg mice have been a well-defined system for the study of
positive and negative selection of T cells. H-Y TCR Tg mice express a
receptor for the male (H-Y) Ag in the context of class I
H-2Db MHC molecules (2, 24). In
female H-Y mice, CD8+ SP T cells that express the
transgene were positively selected by the restricting
H-2Db MHC molecules. In male H-Y mice,
CD8+ SP and DP thymocytes were deleted due to the
strong interaction between TCR and MHC/peptide complexes. In periphery,
transgene-expressing T cells have either no CD8 molecules or only low
levels. By using this well-defined model for T cell development, we
also found that LIGHT may play an important role in H-Y Ag-mediated
negative selection. To eliminate the possibility of the rearrangement
of endogenous -, 
-, 
-chains that may contribute to the
selection events, the H-Y TCR Tg mice we used in the study were in
RAG2-/- background. Administration of LTR-Ig
into male H-Y mice in RAG2-/- background could
rescue DP and CD8+ SP thymocytes. There was an
increase in the percentage of DP and CD8+ SP
thymocytes in the LTR-Ig treated group compared with the control one
(Fig. 2); and the total number of DP and
CD8+ SP thymocytes was also increased in treated
groups (3.82 ± 0.58 x 106 in treated
vs 1.88 ± 0.32 x 106 in control
group, n = 5, p < 0.01). However, such
treatment in the male H-Y mice could not fully rescue the DP and CD8 SP
thymocytes as compared with the female H-Y mice in which the H-Y male
Ag was absent and the numbers of DP and CD8 SP were significantly
higher (92.31 ± 7.95 x 106,
n = 3). Nevertheless, the data indicate that the
disruption of LIGHT signaling during this short time window can block
negative selection at least partially. In periphery, the percentage of
H-Y TCR+CD8+ T cells was
increased too (Fig. 2), and an increase in the total number of
TCR+CD8+ T cells was
observed in the treated group (4.26 ± 0.56 x
106 vs 2.25 ± 0.43 x
106, n = 5, p <
0.01) suggesting that the H-Y TCR+ T cells with
high level of CD8 can be also rescued and exit the thymus. These
results indicate that the disruption of the interaction between LIGHT
and its receptors may block negative selection therefore protect
H-Y-specific DP and CD8+ SP thymocytes from
undergoing apoptosis. Using TCR Tg mice, we found that the blockade of
LIGHT signaling in vitro and in vivo prevented negative selection
induced by peptide and intrathymically expressed Ags and, therefore,
rescued thymocytes from apoptosis. Taken together, our results
establish the critical role of LIGHT playing in negative selection of T
cell development.
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To further investigate the mechanism of LIGHT-mediated T cell
development in vivo, we generated Tg mice that express LIGHT protein
under the control of the proximal lck promoter and CD2
enhancer (Fig. 3a). These
regulatory elements confer a high level of transgene expression to all
thymocyte subsets (25). Four independent Tg lines
expressing LIGHT were obtained and total RNA from wild-type (wt) and Tg
thymus and spleen was analyzed by Northern blot (Fig. 3, b
and c). Higher LIGHT protein expression was observed in the
thymocytes of Tg mice compared with wt mice when studied by flow
cytometry using LTR-Ig and anti-LIGHT Ab (Fig. 3d).
Lines 12 and 18 in a C3H background were selected for further
investigation (data not shown). Similar phenotypes were observed in
both lines; therefore, the results described in this study are not
distinguished between them.
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Investigation of LIGHT Tg animals revealed a striking gross defect
in the thymus development. The thymi from LIGHT Tg animals
(n = 6) were markedly reduced in weight (60–80%
reduction) as compared with non-Tg littermates (Fig. 4a, 60.3 ± 12.6 mg in wt
vs 25.4 ± 3.6 mg in Tg, p < 0.001).
Consistently, there was a dramatic reduction in the total number of
thymocytes in LIGHT Tg animals as compared with non-Tg littermates
(Fig. 4b, 177.5 ± 36.6 x
106 in wt vs 30.5 ± 5.6 x
106 in Tg, p < 0.0001).
Histological analysis showed that the thymic structure in Tg animals
was disorganized. The thymic cortex compartment was remarkably
decreased and the cortex/medulla segregation disappeared (Fig. 4c). Moreover, the destruction of thymic structure is a
dynamic process that is aggravated in older Tg mice (Fig. 4c).
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50% of the total
population as compared with 80% in wt mice (Fig. 5a, top). In
contrast, LIGHT Tg thymocytes showed a relative increase in the
percentage of SP cells (Fig. 5a). The remarkable reduction
of DP cells (153.8 ± 31.7 x 106 vs
15.1 ± 2.8 x 106, >90% reduction)
predominantly accounted for the reduced total number of thymocytes,
whereas the absolute number of CD4+ or
CD8+ SP thymocytes was reduced to a lesser
extent. These results indicate that there is a preferential reduction
of DP cells in LIGHT Tg mice. The reduced number of thymocytes could be
attributed to either increased exit from the thymus or enhanced
apoptosis. However, examination of splenocyte populations showed a
reduction in the percentage of CD4+ or
CD8+ T cells (Fig. 5a) in LIGHT Tg
mice. Given that there was no significant difference between wt
and Tg mice in the total number of splenocytes (79.1 ± 19.4 x
106 in wt vs 68.9 ± 19.3 x
106 in Tg, n = 6), the absolute
number of CD4+ or CD8+ T
cells in Tg spleen was reduced at the age of 5–8 wk. Thus, the reduced
number of thymocytes unlikely was due to the increased exit of T cells
from the thymus but rather the impaired T cell development within the
thymus.
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The apoptosis is a major pathway for the elimination of most
thymocytes during T cell development in the thymus. Because we did not
observe necrosis or inflammation in the thymus of Tg mice, we predicted
that the reduced number of thymocytes in the Tg mice may be associated
with increased apoptosis in the thymus. Therefore, we examined whether
there was increased apoptosis in the thymus of Tg mice. Flow cytometry
revealed a >300% increase in the percentage of apoptotic thymocytes
in LIGHT Tg mice as characterized by positive staining of annexin V, an
early-stage apoptotic marker, and PI, a marker for late-stage apoptosis
(Fig. 5b). Dramatically increased apoptosis was detected in
the DP thymocytes (13% positive for annexin V) of Tg mice vs 4% in
wt thymocytes (Fig. 5b). Consistent with annexin-V/PI
staining, a higher number of apoptotic cells was seen in LIGHT Tg
thymus as determined by TUNEL assay (TdT-mediated dUTP nick-end
labeling) (data not shown). These data demonstrate that the dramatic
reduction of CD4+CD8+ DP
cells in LIGHT Tg mice is caused by enhanced apoptosis. Our model
strongly suggests that LIGHT is sufficient to promote the apoptosis of
immature thymocytes in vivo. Our preliminary data indicated that all DP
and SP thymocytes express HVEM, one receptor for LIGHT, as determined
by Ab staining (data not shown).
The reduction in the number of DP thymocytes and the enhanced apoptosis in this population were consistent with the notion that LIGHT may increase the Ag-specific negative selection. Our preliminary data obtained from male H-Y/LIGHT double-Tg model suggested that LIGHT indeed plays a role in promoting the Ag-specific negative selection. LIGHT Tg mice were crossed with H-Y TCR Tg mice, and we found that the presence of LIGHT transgene further induced the reduction of thymus cellularity in the male double-Tg mice (19.35 ± 3.98 x 106 in H-Y+LIGHT- vs 3.37 ± 1.01 x 106 in H-Y+LIGHT+ mice, n = 4, p < 0.01). However without the complete study of its effect in the female H-Y/LIGHT mice, we could not rule out the other possibilities such as nonspecific inflammatory effects. Therefore, the impacts of LIGHT in various TCR Tg models under positive and negative selection conditions remain to be further explored.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Blocking the interaction of LIGHT with its receptors by LTR-Ig can
rescue the Ag-specific TCR bearing immature thymocytes from apoptosis.
LTR has another ligand, mLT, which is a heterotrimer
LT12 (22, 23). In our FTOCs system, we used anti-LT Ab which blocks
mLT, but not LIGHT, binding to its receptor (13, 23). Our
results show that anti-LT Ab cannot block the Ag-specific
negative selection because DP and SP cells are deleted when the
specific Ag is provided (Fig. 1). In addition, LIGHT binds another
receptor, HVEM. Using the same FTOC system, our data showed that
HVEM-Ig, binding to LIGHT, but not mLT, could also rescue the DP and SP
thymocytes in a way similar to LTR-Ig (Fig. 1). An anti-LIGHT Ab
that efficiently blocks LIGHT is not available at this time. More
importantly, our data suggested that the observed cell rescue resulted
from the blockade of negative selection rather than the loosening of
positive selection. In our FTOC experiments, in the absence of either
the cognate TCR (D7) or the deleting peptide (LemA) which induced the
strong negative selection in D7 Tg+ FTOC,
LTR-Ig and HVEM-Ig treatments did not have significant effect on the
cell numbers nor the subset percentages of the thymocytes. However, in
the presence of LemA peptide and the proper TCR DP thymocytes were
dramatically rescued. In terms of cell number, there is a 40-fold
increase in the LTR-Ig group and an 80-fold increase in the HVEM-Ig
group compared with control-Ig group. Were it the case that these
treatments affected positive selection, we would expect to see
increased cell numbers in response to treatment irrespective of the
presence of LemA or its specific TCR. These results indicated that the
addition of LTR-Ig and HVEM-Ig would rescue the thymocytes from
negative selection and demonstrated that LIGHT, as the interacting
ligand, plays a critical role in this process. Furthermore, our data
from in vivo treatment in male H-Y/RAG2-/- mice
also supported the notion that LIGHT could be an important candidate
mediating negative selection because the blockade of LIGHT signaling in
vivo increased the percentage and cell number of DP and
CD8+ SP thymocytes. The effect on negative
selection in vivo in male H-Y mice was not the same as seen in
peptide-induced FTOC may be attributed to several possible reasons: 1)
these two model systems have many biological differences, in the H-Y
model negative selection is mediated by intrathymically expressed Ag
while in the D7 model it is induced by deleting peptide; 2) the
apparent difference in the treatment may be also related to the timing
and dose of fusion protein treatment; 3) moreover, the accessibility of
fusion protein to the thymus in the whole mice may also be different
from the in vitro FTOC. Together with the data from LIGHT Tg mice, our
results obtained from the in vivo treatment of H-Y mice or FTOC treated
with two soluble receptors and anti-LT Ab strongly implicate
that LIGHT, as the interacting ligand, is responsible for the blocking
effect on our negative selection models and prove that LIGHT plays a
key role in negative selection.
Our LIGHT Tg model strongly implies that LIGHT-mediated negative
selection is attributed to the enhanced apoptosis in thymocytes,
especially cortical thymocytes. We tested whether LIGHT can directly
induce apoptosis in vitro by culturing wt thymocytes with immobilized
LIGHT protein; however, we did not find direct apoptotic effect of
LIGHT on thymocytes in 6-, 18-, and 24-h culture in vitro. Therefore,
the increased apoptosis in thymocytes of LIGHT Tg mice could be due to
more complex effects in the development of these mice. To rule out the
possibility that the thymocyte depletion seen in the LIGHT Tg mice
could be caused by some indirect mechanism of toxicity rather than as
direct consequence of LIGHT’s interaction with its receptors, we
treated LIGHT Tg mice with LTR-Ig and the administration of
LTR-Ig indeed reversed the phenotype in vivo at least partially. In
addition, the overexpression of LIGHT had no obvious effect on DN
thymocytes and reduced effect on SP thymocytes. The selective damage to
DP thymocytes would suggest that the phenotype observed is due to the
specific interaction between ligand and receptors instead of a general
toxic effect on all cell types. In the blockade experiments, the total
number of thymocytes rescued in the D7-negative FTOC
(D7- TCR-/-) did not
show any significant difference between control-Ig, LTR-Ig, and
HVEM-Ig groups (data not shown), which would support that the blocking
effect of LIGHT-soluble receptors is mediated by Ag-specific negative
selection rather than general impact on the survival of thymocytes. In
addition, the number of DN thymocytes is not altered by LTR-Ig or
HVEM-Ig treatments compared with control-Ig group, suggesting that
deletion did not occur before negative selection of
TCR+ cells. Although the key aspect of negative
selection is the interaction between TCR and self-MHC/peptide, optimal
negative selection appears to require signals in addition to an
antigenic stimulus (7, 8, 9). LIGHT-soluble receptors
LTR-Ig and HVEM-Ig can block at least partially the strong negative
selection induced by a high-affinity peptide in FTOC. It will be of
great interest to study the potential role of these rescued cells in
autoreactive disease models, which will provide deeper insights into
the communication and balance between central and peripheral
tolerance.
The mechanisms by which LIGHT induces apoptosis could be complicated:
1) LIGHT could indirectly induce apoptosis in thymocytes via LTR on
thymic stromal cells. LTR is expressed on nonhematopoietic cells
(22, 23), and its expression on different subsets of
thymic stromal cells has not been identified. It is possible that
interaction between LIGHT and LTR may regulate the balance of
thymocyte-stromal cell interaction in T cell development. 2)
Alternatively, signaling via HVEM on thymocytes by LIGHT may directly
induce apoptosis of thymocytes in the presence of TCR engagement. The
key aspect of negative selection is the avidity between TCR and
self-MHC/peptide complexes, the interaction between LIGHT and its
receptor HVEM may alter the strength of TCR signaling therefore enhance
negative selection. Previous studies have shown that LIGHT is expressed
in the thymus and activated T cells (13, 15). Furthermore,
our FACS data suggest that LIGHT is expressed on thymocytes although
the level of the expression is very low. Indeed, this is a
characteristic finding of many members of the TNF superfamily. We also
detected the expression of HVEM, which is a receptor for LIGHT, on
thymocytes by flow cytometry (data not shown). It will be useful to
determine whether the blocking effect of soluble receptors reported in
this study is mediated through LTR and/or HVEM signaling, and we
cannot exclude the possibility that additional molecular and cellular
components may be also involved in LIGHT-mediated negative
selection.
Superantigen has also been indicated as another mechanism to mediate
negative selection. We also explored superantigen model using an A/J
mouse strain (H-2k/k) that expresses an
endogenous viral superantigen, Mls-2a
(27).
V3+CD4+ and
V11+CD4+ cells, both of
which are reactive to Mls-2a, were efficiently
deleted in the thymus and the lymph nodes (data not shown). LTR-Ig
fusion protein treatment over 3 wk led to a nonsignificant increase in
the total number of
V11+CD4+ cells in the
lymph nodes (16.7 ± 2.88 x 104 in
treated vs 11.6 ± 2.56 x 104 in
control group), similar results were seen in
V3+CD4+ cells (data not
shown). These data suggest that LIGHT is not critical for clonal
deletion of T cells reactive to this endogenous viral superantigen.
In this study, we demonstrate that LIGHT plays a critical role in
negative selection by using both blockade and Tg approaches. Our
results from LIGHT Tg model implicate LIGHT as an important mediator of
thymocyte apoptosis during negative selection. Our data using H-Y TCR
Tg mice suggest that the blockage of LIGHT in vivo can rescue
anti-H-Y DP and CD8 SP+ thymocytes from
negative selection. In periphery, the percentage and total number of
H-Y TCR+CD8+ T cells was
increased too (Fig. 2), suggesting that the anti-H-Y
TCR+ T cells with high level of CD8 can be also
rescued and exit the thymus. Previous data from H-Y TCR Tg model showed
that the few CD8+ T cells that could escape from
negative selection and be present in periphery failed to respond to the
H-Y Ag stimulation (2, 24). It would be interesting to
investigate whether the cells rescued by LIGHT-soluble receptor could
respond to the Ag stimulation, therefore might cause the autoimmune
disease in this model. This study has revealed a critical role of LIGHT
in negative selection and provided an animal model to study the
essential requirement for central and peripheral tolerance.
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Footnotes |
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2 J.W. and T.C. contributed equally to this work.
3 Address correspondence and reprint requests to Drs. Yang-Xin Fu or Chyung-Ru Wang, Department of Pathology, MC3083, University of Chicago, Chicago, IL 60637. E-mail addresses: yfu@midway.uchicago.edu or cwang{at}midway.uchicago.edu
4 Abbreviations used in this paper: LIGHT, a cellular ligand for herpesvirus entry mediator and lymphotoxin receptor; LTR, lymphotoxin- receptor; HVEM, herpesvirus entry mediator; DP, double positive; FTOC, fetal thymus organ culture; SP, single positive; Tg, transgenic; wt, wild type; mLT, membrane lymphotoxin; PI, propidium iodide.
Received for publication July 5, 2001. Accepted for publication September 5, 2001.
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References |
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T cell receptors. Curr. Opin. Immunol. 9:250.[Medline]
are ligands for herpesvirus entry mediator. Immunity 8:21.[Medline]
receptor and TR2/HVEM induces apoptosis and suppresses in vivo tumor formation via gene transfer. J. Clin. Invest. 102:1142.[Medline]
receptor is necessary and sufficient for LIGHT-mediated apoptosis of tumor cells. J. Biol. Chem. 275:14307. complexes on the surface of mouse lymphocytes. J. Immunol. 159:3288.[Abstract]
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M. I. Zimmer, A. Colmone, K. Felio, H. Xu, A. Ma, and C.-R. Wang A Cell-Type Specific CD1d Expression Program Modulates Invariant NKT Cell Development and Function J. Immunol., February 1, 2006; 176(3): 1421 - 1430. [Abstract] [Full Text] [PDF]
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R. A. Anders, S. K. Subudhi, J. Wang, K. Pfeffer, and Y.-X. Fu Contribution of the Lymphotoxin {beta} Receptor to Liver Regeneration J. Immunol., July 15, 2005; 175(2): 1295 - 1300. [Abstract] [Full Text] [PDF]
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J. Wang, R. A. Anders, Y. Wang, J. R. Turner, C. Abraham, K. Pfeffer, and Y.-X. Fu The Critical Role of LIGHT in Promoting Intestinal Inflammation and Crohn's Disease J. Immunol., June 15, 2005; 174(12): 8173 - 8182. [Abstract] [Full Text] [PDF]
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G. Shi, J. Mao, G. Yu, J. Zhang, and J. Wu Tumor Vaccine Based on Cell Surface Expression of DcR3/TR6 J. Immunol., April 15, 2005; 174(8): 4727 - 4735. [Abstract] [Full Text] [PDF]
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O. Cohavy, J. Zhou, C. F. Ware, and S. R. Targan LIGHT Is Constitutively Expressed on T and NK Cells in the Human Gut and Can Be Induced by CD2-Mediated Signaling J. Immunol., January 15, 2005; 174(2): 646 - 653. [Abstract] [Full Text] [PDF]
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 M. T. Fisher, M. Nagarkatti, and P. S. Nagarkatti 2,3,7,8-Tetrachlorodibenzo-p-dioxin Enhances Negative Selection of T Cells in the Thymus but Allows Autoreactive T Cells to Escape Deletion and Migrate to the Periphery Mol. Pharmacol., January 1, 2005; 67(1): 327 - 335. [Abstract] [Full Text] [PDF]
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O. Cohavy, J. Zhou, S. W. Granger, C. F. Ware, and S. R. Targan LIGHT Expression by Mucosal T Cells May Regulate IFN-{gamma} Expression in the Intestine J. Immunol., July 1, 2004; 173(1): 251 - 258. [Abstract] [Full Text] [PDF]
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 M. T. Fisher, M. Nagarkatti, and P. S. Nagarkatti Combined Screening of Thymocytes Using Apoptosis-Specific cDNA Array and Promoter Analysis Yields Novel Gene Targets Mediating TCDD-Induced Toxicity Toxicol. Sci., March 1, 2004; 78(1): 116 - 124. [Abstract] [Full Text] [PDF]
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 T. Boehm, S. Scheu, K. Pfeffer, and C. C. Bleul Thymic Medullary Epithelial Cell Differentiation, Thymocyte Emigration, and the Control of Autoimmunity Require Lympho-Epithelial Cross Talk via LT{beta}R J. Exp. Med., September 2, 2003; 198(5): 757 - 769. [Abstract] [Full Text] [PDF]
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E. Cretney, A. P. Uldrich, S. P. Berzins, A. Strasser, D. I. Godfrey, and M. J. Smyth Normal Thymocyte Negative Selection in TRAIL-deficient Mice J. Exp. Med., August 4, 2003; 198(3): 491 - 496. [Abstract] [Full Text] [PDF]
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 J. Liu, C. S. Schmidt, F. Zhao, A. J. Okragly, A. Glasebrook, N. Fox, E. Galbreath, Q. Zhang, H. Y. Song, S. Na, et al. LIGHT-deficiency impairs CD8+ T cell expansion, but not effector function Int. Immunol., July 1, 2003; 15(7): 861 - 870. [Abstract] [Full Text] [PDF]
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J. Wang and Y.-X. Fu LIGHT (a Cellular Ligand for Herpes Virus Entry Mediator and Lymphotoxin Receptor)-Mediated Thymocyte Deletion Is Dependent on the Interaction Between TCR and MHC/Self-Peptide J. Immunol., April 15, 2003; 170(8): 3986 - 3993. [Abstract] [Full Text] [PDF]
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 R. Castellano, C. Van Lint, V. Peri, E. Veithen, Y. Morel, R. Costello, D. Olive, and Y. Collette Mechanisms Regulating Expression of the Tumor Necrosis Factor-related light Gene. ROLE OF CALCIUM-SIGNALING PATHWAY IN THE TRANSCRIPTIONAL CONTROL J. Biol. Chem., November 1, 2002; 277(45): 42841 - 42851. [Abstract] [Full Text] [PDF]
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S. Scheu, J. Alferink, T. Potzel, W. Barchet, U. Kalinke, and K. Pfeffer Targeted Disruption of LIGHT Causes Defects in Costimulatory T Cell Activation and Reveals Cooperation with Lymphotoxin {beta} in Mesenteric Lymph Node Genesis J. Exp. Med., June 17, 2002; 195(12): 1613 - 1624. [Abstract] [Full Text] [PDF]
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K. Tamada, J. Ni, G. Zhu, M. Fiscella, B. Teng, J. M. A. van Deursen, and L. Chen Cutting Edge: Selective Impairment of CD8+ T Cell Function in Mice Lacking the TNF Superfamily Member LIGHT J. Immunol., May 15, 2002; 168(10): 4832 - 4835. [Abstract] [Full Text] [PDF]
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Q. Ye, C. C. Fraser, W. Gao, L. Wang, S. J. Busfield, C. Wang, Y. Qiu, A. J. Coyle, J.-C. Gutierrez-Ramos, and W. W. Hancock Modulation of LIGHT-HVEM Costimulation Prolongs Cardiac Allograft Survival J. Exp. Med., March 18, 2002; 195(6): 795 - 800. [Abstract] [Full Text] [PDF]
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) or Tg (
) mice were collected and weighed.
b, Thymi were mechanically homogenized and a
single cell suspension was obtained. The total numbers of thymocytes
were counted; there was a significant difference
(p < 0.0001) between control (