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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
Jing Wang and Yang-Xin Fu
J Immunol 2003; 170:3986-3993; ;
doi: 10.4049/jimmunol.170.8.3986
http://www.jimmunol.org/content/170/8/3986
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Copyright © 2003 by The American Association of
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References
The Journal of Immunology
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-Peptide1
Jing Wang and Yang-Xin Fu2
T
cell development is tightly controlled by selective events
in the thymus, namely positive and negative selection (1–
6). The current model proposed that the fate of immature
thymocytes is largely determined by the interaction between their
TCR and MHC/peptide complexes expressed on thymic stromal
cells. Thymocytes with a TCR of high avidity for MHC/peptide
complexes are induced to undergo apoptosis (negative selection) to
maintain central tolerance, whereas the lower avidity binding of
TCR and MHC/peptide confers immature thymocytes to undergo
positive selection and become single-positive (SP)3 T cells that
eventually migrate into periphery and play critical roles in adaptive
immunity.
Negative selection serves as a critical mechanism to maintain
self-tolerance (7). The current idea of negative selection is largely
based on transgenic (Tg) models of various TCR genes. Among
them, H-Y Tg mouse is a well-established model that provides
experimental evidence of clonal deletion and positive selection
(8 –10). In male H-Y Tg mice, the immature thymocytes undergo
increased apoptosis due to the extremely strong interaction between MHC (H-2Db)/peptide complex and TCR, whereas in female Tg mice the interaction between MHC/peptide complex and
Department of Pathology and Committee on Immunology, University of Chicago,
Chicago, IL 60637
Received for publication August 1, 2002. Accepted for publication February 4, 2003.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported in part by National Institutes of Health Grants (HD37104
and DK58897) and Juvenile Diabetes Foundation International (1-2000-875) to
Y.-X.F.
2
Address correspondence and reprint requests to Dr. Yang-Xin Fu, Department of
Pathology, MC3083, University of Chicago, Chicago, IL 60637. E-mail:
[email protected]
3
Abbreviations used in this paper: SP, single positive; DP, double positive; HVEM,
herpes virus entry mediator; LIGHT, a cellular ligand for HVEM and lymphotoxin
receptor; LT, lymphotoxin; LT␤R, lymphotoxin ␤ receptor; NL, negative littermate;
PI, propidium iodide; Tg, transgenic; TNFR, TNF receptor; wt, wild type.
Copyright © 2003 by The American Association of Immunologists, Inc.
TCR is much weaker, which leads to the positive selection of H-Y
TCR⫹ CD8⫹ T cells. This model has served as a central system to
elucidate the mechanisms of selection events in the development
of thymocytes (11).
The existence of central tolerance implies that immature thymocytes respond differently to an encounter with Ags than do mature T cells. Optimal negative selection appears to require signals
in addition to an antigenic stimulus (12–14). Our previous study
has shown that LIGHT (a cellular ligand for herpes virus entry
mediator (HVEM) and lymphotoxin receptor) functions as a costimulatory molecule for mature T cells in periphery and causes
the activation and expansion of peripheral T cells (15, 16); however, the constitutive expression of LIGHT on thymocytes promotes the apoptosis of double positive (DP) thymocytes (17). In
addition, blockade of endogenous LIGHT in a TCR Tg model, D7
Tg mice (18), could rescue the DP and CD8 SP thymocytes dramatically in fetal thymic organ culture (17). Therefore, these results raise the possibility that LIGHT may be involved in negative
selection by regulating apoptosis process. However, it is unclear
whether the LIGHT-mediated apoptosis is associated with specific
interaction of TCR between its cognate MHC/peptide complex.
Namely, the cell death mediated by LIGHT may be caused by a
general impact on cell survival of a wide variety of immature thymocytes regardless of their TCR specificity or via clonal deletion
of specific TCR⫹ thymocytes. Thus, we crossed LIGHT transgene
into H-Y Tg system and examined thymocyte development in
double-Tg mice.
Here, we show that LIGHT-mediated deletion of thymocytes is
directly linked with the interaction between TCR and MHC/peptide. With the presence of H-Y Ag (H-Y/LIGHT male mice),
LIGHT further promotes the apoptosis of thymocytes and the reduction of total thymocyte number. The enhanced apoptosis in
H-Y/LIGHT double-Tg male mice leads to the reduction of mature
CD8⫹TCR⫹ cells in the thymus and periphery. In contrast, T cell
development is largely unchanged in H-Y/LIGHT double-Tg female mice. Therefore, our study suggests that LIGHT-mediated
0022-1767/03/$02.00
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Negative selection serves as a major mechanism to maintain self-tolerance. We previously reported that LIGHT (a cellular ligand
for herpes virus entry mediator and lymphotoxin receptor), a TNF family member, plays an important role in thymocyte development via promoting apoptosis of double-positive thymocytes. Here, we demonstrated that LIGHT-mediated deletion of thymocyte requires the strong interaction of TCR with MHC/self-peptide. Transgenic mice overexpressing LIGHT in thymocytes
were bred with a transgenic mouse line expressing a TCR recognizing the H-Y male Ag in the context of H-2b class I MHC
molecules. In male H-Y/LIGHT double-transgenic mice, more efficient negative selection of H-Y T cells occurred, and total
thymocyte number was further reduced compared with H-Y/negative littermates. In contrast, the presence of LIGHT transgene
had no evident impact on the thymocyte development of female H-Y/LIGHT double-transgenic mice. Taken together, LIGHT
plays a role in negative selection of thymocytes via inducing the apoptosis of thymocytes bearing high affinity TCR during negative
selection. The Journal of Immunology, 2003, 170: 3986 –3993.
The Journal of Immunology
apoptosis of DP is associated with clonal deletion of specific
TCR⫹ thymocytes.
Materials and Methods
Mice
LIGHT Tg mice were generated as previously described (15). The
C57BL/6 (B6) Tg line was crossed to H-Y/RAG2⫺/⫺ mice on the B6
background (Taconic Farms, Germantown, NY). LIGHT Tg mice (C3H)
were crossed to lymphotoxin ␤ receptor (LT␤R)⫺/⫺ and LT␣⫺/⫺ mice for
two generations. The age of the mice examined was between 5–10 wk.
Animal care and use were in accordance with institutional guidelines of
University of Chicago.
Flow cytometric analysis
In vivo treatment protocol
Age (5–10 wk)- and sex-matched C3H wild-type (wt; JAX) mice received
three or four i.p. injections weekly of 100 ␮g of soluble murine LT␤R-Ig
or PBS. Thymi were collected 7 days after the last injection. In some cases
C3H wt mice were sublethally irradiated (600 rad) and treated with fusion
protein as described above. Thymocytes and splenocytes were stained with
CyChrome-anti-CD4 and PE-anti-CD8 Abs (BD PharMingen) and analyzed by FACScan (BD Biosciences).
TUNEL assay
TUNEL staining for apoptosis was performed using a commercially available kit as described by the manufacturer (Oncogene Research Products,
San Diego, CA). The thymus samples were fixed with 10% formalin and
embedded in paraffin. Sections were subject to TUNEL assay (Oncogene
Research Products), followed by hematoxylin counterstaining using standard methods.
Results
LIGHT-mediated apoptosis is associated with TCR specificity
To study whether TCR engagement is required for the reduction of
thymocyte number in LIGHT Tg mice, we performed a further
study of the role of LIGHT in thymocyte development using a
conventional TCR system, H-Y Tg mice. LIGHT Tg mice were
crossed with H-Y Tg mice, and the male and female progeny were
analyzed for the development and maturation of thymocytes. In
male H-Y/LIGHT double-Tg mice, the total number of thymocytes
was significantly reduced compared with male H-Y/negative littermate (H-Y/NL) Tg mice (Fig. 1A; 19.35 ⫾ 3.98 ⫻ 106 in HY/NL mice vs 3.37 ⫾ 1.01 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 5).
Moreover, the reduction of the DP population appeared to be more
severe in H-Y/LIGHT Tg mice (24-fold reduction in DP, 8-fold in
CD8 SP, 6-fold in CD4 SP). At the same time, the female H-Y/
LIGHT double-Tg mice had a similar total number of thymocytes
as female H-Y/NL Tg mice (Fig. 1A; 102.47 ⫾ 8.83 ⫻ 106 in
H-Y/NL mice vs 82.63 ⫾ 16.1 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 5).
These data suggested that the presence of LIGHT promoted the
further reduction of thymocytes in male H-Y mice, whereas it did
not affect the thymocyte development largely in female H-Y mice.
Previous studies demonstrated that DP thymocytes undergo increased apoptosis in male H-Y Tg mice compared with NL. Therefore, we further tested whether the reduction of thymocytes in
H-Y/LIGHT double-Tg mice was caused by enhanced apoptosis.
As we expected, apoptosis was increased in male H-Y/LIGHT
double-Tg mice, as shown by annexin V and PI staining (Fig. 1B).
Consistent with annexin V/PI staining, a greater number of apoptotic cells was seen in male H-Y/LIGHT Tg thymus as determined by TUNEL assay (Fig. 1C). Thus, LIGHT could further
promote apoptosis in the presence of a strong interaction between
MHC/peptide and TCR, while LIGHT seems to have no significant
impact on thymocyte development in the absence of such interaction, suggesting that LIGHT-mediated apoptosis is linked with
TCR specificity.
Perturbed thymocyte development in male H-Y/LIGHT doubleTg mice and unaffected thymocyte development in female
H-Y/LIGHT double-Tg mice
We examined the different populations of thymocytes by FACS. In
male H-Y/LIGHT double-Tg mice, the percentages of DP and
CD8 SP were further reduced in addition to the reduction of total
thymocyte number (Fig. 2A). In contrast, DP and CD8 SP populations did not show any significant change between female HY/NL and H-Y/LIGHT groups (Fig. 2A). We also detected the
TCR expression (F23.1 Ab against H-Y V␤-chain) in thymocyte
populations; the percentage of H-Y␤⫹CD8⫹ thymocytes was decreased in male H-Y/LIGHT double-Tg mice (Fig. 2B; 8.70% in
H-Y/LIGHT mice vs 18.58% in H-Y/NL mice). In addition, the
absolute number of H-Y␤⫹CD8⫹ thymocytes was more severely
reduced (3.60 ⫾ 0.74 ⫻ 106 in H-Y/NL mice vs 0.29 ⫾ 0.09 ⫻
106 in H-Y/LIGHT mice) due to the reduction of total thymocyte
number in H-Y/LIGHT Tg mice. However, the percentage of the
H-Y␤⫹CD8⫺ population appeared to be unchanged (Fig. 2B,
male; 73.83% in H-Y/LIGHT mice vs 74.12% in H-Y/NL mice).
A previous study demonstrated that DN thymocytes were also
forced to express H-Y TCR on their surface (19). T3.70 Ab (clonotypic Ab against Tg TCR ␣-chain) was used for triple staining
of CD8/CD4/T3.70. Similar results were obtained in male Tg mice
using T3.70 Ab compared with F23.1 Ab as shown in Fig. 2B.
Here, we showed that the presence of LIGHT transgene caused a
further reduction of H-Y⫹CD8⫹, suggesting that thymocytes with
higher avidity binding were selectively deleted to a larger extent.
Interestingly, the percentage of H-Y TCR⫺ thymocytes was enhanced in male H-Y/LIGHT double-Tg mice (Fig. 2B; CD8/F23.1,
16.38% in H-Y/LIGHT mice vs 5.42% in H-Y/NL mice), indicating that the deletion of this population (H-Y TCR⫺) was not as
severe as that of the H-Y TCR⫹ population.
Despite all the changes in male H-Y/LIGHT Tg mice, there was
no obvious difference in the thymocytes of female H-Y/LIGHT Tg
mice (Fig. 2B, female), which further confirmed the unaffected
thymocyte development in this mouse model. In female H-Y Tg
mice, the percentage of Tg TCR␣⫹ thymocytes was lower than
that of Tg TCR␤⫹ thymocytes, suggesting rearrangements of endogenous ␣-chains (Fig. 2B, female).
In the periphery the phenotypes were consistent with those observed in the thymus of male H-Y/LIGHT mice. The percentage of
CD8⫹ was decreased in H-Y/LIGHT double-Tg mice compared
with H-Y/NL Tg mice (Fig. 2C, male; 6.98% in H-Y/LIGHT mice
vs 14.65% in H-Y/NL mice); moreover, the intensity of CD8 staining in both H-Y/LIGHT and H-Y/NL mice was reduced compared
with that in wt B6 mice, as shown by the mean fluorescence intensity (Fig. 2D), indicating the down-regulation of CD8 coreceptor expression in the periphery. However, the percentage of CD4⫹
was increased in male H-Y/LIGHT Tg mice (Fig. 2C, male); this
was confirmed with CD4/CD8/T3.70 staining (Fig. 2E). Consistently, all populations were unchanged in the spleen of female
H-Y/LIGHT Tg mice compared with female H-Y/NL mice (Fig.
2C, female).
TCR expression of splenocytes was also assessed by FACS. The
frequency of H-Y␤⫹CD8⫹ T cells was reduced in male H-Y/
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Single-cell suspensions of thymocytes and splenocytes were collected and
stained with PE-anti-CD8, FITC-anti-CD4 (for three-color FACS, CyChrome-anti-CD4 was used), FITC-anti-V␤8.1 (F23.1), and PE-anti-CD3
Abs (BD PharMingen, San Diego, CA) in PBS plus 0.01% NaN3 for 30
min at 4°C. FITC-labeled T3.70 Ab was obtained from eBioscience (San
Diego, CA). After incubation with Abs, the cells were washed and analyzed on a FACScan (BD Biosciences, Mountain View, CA). Anti-LIGHT
Ab was described previously (17). Annexin V and propidium iodide (PI)
double staining for apoptosis was performed using a commercially available kit as described by the manufacturer (BD PharMingen).
3987
3988
LIGHT-MEDIATED SELECTION REQUIRES TCR ENGAGEMENT
LIGHT Tg mice (Fig. 2E, male; CD8/F23.1, 7.10% in H-Y/LIGHT
mice vs 15.16% in H-Y/NL mice); in addition, the percentage of
H-Y␤⫹CD8⫺ T cells was decreased (Fig. 2E, male; CD8/F23.1,
4.95% in H-Y/LIGHT mice vs 8.87% in H-Y/NL mice). Due to the
strong negative selection pressure, CD8 SP cells down-regulate
their coreceptor CD8, which leads to the generation of
F23.1⫹CD8⫺ cells (19). To further confirm the results, we performed triple staining for CD4/CD8/T3.70. As shown in Fig. 2E,
similar results were seen using T3.70 Ab compared with F23.1 Ab.
In addition, the percentage of CD4⫹T3.70⫺ cells is higher in H-Y/
LIGHT Tg mice than in H-Y/NL mice (Fig. 2E; CD4/T3.70,
11.35% in H-Y/LIGHT mice vs 5.31% in H-Y/NL). Again, there
was no significant difference in the splenocytes of female H-Y/
LIGHT Tg mice compared with H-Y/NL Tg mice (Fig. 2E, female). However, FASC staining for splenocytes from female H-Y
mice showed that the percentage of Tg TCR␣⫹ (T3.70⫹) cells is
much lower than that of Tg TCR␤⫹(F23.1⫹) cells, suggesting re-
arrangements of endogenous ␣-chains in female H-Y mice (Fig.
2E, female).
Taken together, our results suggest that the presence of the
LIGHT transgene caused severe deletion of thymocytes bearing
higher avidity TCR on their surface, and such deletion was directly
associated with TCR specificity; furthermore, the perturbation of
thymocyte development affected the distribution of peripheral T
cells.
Preferential reduction of H-Y⫹ thymocytes in male H-Y/LIGHT
double-Tg mice
A previous study (20) has shown that the H-Y ␣␤ transgene induced strong allelic exclusion of endogenous TCR genes; however, endogenous TCR genes could still undergo rearrangement,
but endogenous T cells constitute only a small fraction of thymocytes. Therefore, we examined whether the LIGHT transgene had
a differential impact on H-Y transgenic T cells defined as
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FIGURE 1. LIGHT-mediated reduction of thymocyte number was directly linked with TCR specificity. A, Severe reduction of the total number of
thymocytes in male H-Y/LIGHT Tg mice compared with H-Y/NL. Thymocytes were extracted, and total cell number was counted (19.35 ⫾ 3.98 ⫻ 106
in H-Y/NL mice vs 3.37 ⫾ 1.01 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 5). A largely unaffected total number of thymocytes was seen in female H-Y/LIGHT
Tg mice compared with H-Y/NL mice (102.47 ⫾ 8.83 ⫻ 106 in H-Y/NL mice vs 82.63 ⫾ 16.1 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 5). B, Enhanced apoptosis
was observed in male H-Y/LIGHT Tg mice. The number in the frame indicates the frequency of cells in the defined region. Annexin V and PI staining
was performed as previously described. C, An increased number of apoptotic cells (brown staining as indicated by arrowhead) was seen in male H-Y/LIGHT
Tg mice. Thymic samples were subject to TUNEL staining. The number of apoptotic cells was counted under a microscope (magnification, ⫻400; PHF,
per high power field).
The Journal of Immunology
3989
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FIGURE 2. The distribution of different thymocyte populations was affected by the LIGHT transgene in a TCR signaling-dependent manner. A, FACS
profile of thymocytes for CD4 and CD8 staining. In male H-Y/LIGHT Tg mice, the percentages of DP and CD8 SP were reduced compared with that of
H-Y/NL. In contrast, the thymocyte population was largely unchanged by the LIGHT transgene in female H-Y/LIGHT Tg mice. B, FACS profile of
thymocytes for CD8 and H-Y TCR staining. H-Y⫹CD8⫹ thymocytes were significantly decreased in male H-Y/LIGHT Tg mice compared with H-Y/NL
determined by CD8/F23.1 and CD8/T3.70 staining. The percentage of endogenous T cells (H-Y⫺) was significantly increased in male H-Y/LIGHT Tg mice.
No significant difference was observed between female H-Y/LIGHT Tg mice and H-Y/NL. C, FACS profile of splenocytes for CD4 and CD8 staining.
CD8⫹ T cells were reduced significantly, while the percentage of CD4⫹ T cells was moderately elevated in male H-Y/LIGHT Tg mice. The total numbers
of splenocytes were counted for male (62.35 ⫾ 18.38 ⫻ 106 in H-Y/NL mice vs 45.93 ⫾ 18.37 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 7) and female (77.64 ⫾
13.8 ⫻ 106 in H-Y/NL mice vs 34.0 ⫾ 5.93 ⫻ 106 in H-Y/LIGHT mice; n ⫽ 7) mice. D, The mean fluorescence intensity of CD8 expression on peripheral
T cells was down-regulated in both H-Y/LIGHT and H-Y/NL mice compared with wt B6 mice. E, FACS profile of splenocytes for CD8 and H-Y TCR
staining determined by both F23.1 and T3.70 Abs. The percentages of CD8⫹H-Y⫹ and CD8⫺H-Y⫹ were also reduced consistently with thymocyte data.
In contrast, the distribution of peripheral T cells and TCR expression were unaffected in female H-Y/LIGHT Tg mice in which the interaction between TCR
and MHC/self-peptide was relatively weak. Representative data are shown, and the number in the frame indicates the frequency of cells in the defined
regions.
3990
LIGHT-MEDIATED SELECTION REQUIRES TCR ENGAGEMENT
FIGURE 3. The LIGHT transgene preferentially
deleted H-Y TCR⫹ thymocytes. A, FACS profile of
thymocytes for CD3 and H-Y TCR staining. The endogenous thymocytes defined as CD3⫹H-Y␤⫺ were
significantly enhanced in male H-Y/LIGHT Tg mice
compared with H-Y/NL mice. The total number of
thymocytes was counted (n ⫽ 5). B, The reduction of
H-Y⫹ thymocytes was more severe than that of H-Y⫺
thymocytes. C, The percentage of H-Y⫹ T cells in
male H-Y/LIGHT Tg mice was also reduced in the
periphery. In contrast, the endogenous T cells constituted a much larger proportion of peripheral T cells in
H-Y/LIGHT Tg mice compared with H-Y/NL mice.
The total numbers of splenocytes were counted (n ⫽
7). Representative data are shown, and the number in
the frame indicates the frequency of cells in the defined regions.
LIGHT⫹ thymocytes in male H-Y/NL mice was significantly
higher than that in wt mice (Fig. 4B). The percentage of LIGHTpositive thymocytes in H-Y/NL female mice was similar to that in
wt mice, since there was no enhanced negative selection in HY/NL female mice (data not shown). For male H-Y/NL mice,
LIGHT-positive thymocytes reside in the H-Y␤⫹ population, as
determined by LIGHT/F23.1 or LIGHT/CD3 FACS staining,
which is subject to enhanced negative selection. For wt mice,
LIGHT-positive thymocytes reside in the CD3medium/high population, as determined by LIGHT/CD3 FACS staining. Thus, our results suggested that the level of LIGHT expression might be associated with the extent of apoptosis in negative selection of
thymocytes, at least in the Tg system.
LIGHT plays an important role in regulating the T cell
development of non-Tg mice
Our results obtained from the LIGHT Tg and H-Y Tg models
demonstrated the critical role of LIGHT in negative selection. To
Up-regulation of LIGHT is associated with apoptosis of H-Y⫹
thymocytes
We tested the impact of the LIGHT transgene in negative selection, and we examined whether the expression of LIGHT will be
up-regulated when thymocytes undergo enhanced apoptosis. Since
male H-Y/NL thymocytes underwent strong negative selection, we
stained thymocytes for the expression of LIGHT by FACS. Our
data showed that H-Y/NL thymocytes indeed up-regulated LIGHT
compared with wt thymocytes (Fig. 4A). The percentage of
FIGURE 4. Up-regulation of LIGHT expression was associated with
enhanced negative selection. A, LIGHT expression on thymocytes was detected by anti-LIGHT Abs. Male H-Y/NL (H-Y/LIGHT⫺) thymocytes displayed much higher LIGHT expression on their surface than wt B6 thymocytes. The total number of thymocytes was counted (n ⫽ 5). B, The
percentage of LIGHT⫹ thymocytes was significantly elevated in male HY/NL Tg mice (H-Y/LIGHT ⫺; n ⫽ 4).
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H-Y␤⫹CD3⫹ and endogenous T cells defined as H-Y␤⫺CD3⫹.
Interestingly, we found that the percentage of endogenous T cells
(H-Y␤⫺CD3⫹) was increased in H-Y/LIGHT Tg mice compared
with H-Y/NL Tg mice and constituted a larger proportion of thymocytes (Fig. 3A). A FACS profile of wt B6 thymocytes was included as a control in which the CD3⫹ population constituted
⬃40%, including CD3high and CD3medium populations (Fig. 3A).
In light of the reduction of total thymocytes (19.35 ⫾ 3.98 ⫻ 106
in H-Y/NL mice vs 3.37 ⫾ 1.01 ⫻ 106 in H-Y/LIGHT mice),
H-Y⫹ transgenic thymocytes appeared to undergo more efficient
deletion than endogenous thymocytes (H-Y⫺); the reduction rate
of H-Y⫹ thymocytes was consistently much greater than that of
H-Y⫺ thymocytes (Fig. 3B; 88% in H-Y⫹ mice vs 47% in H-Y⫺
mice). These data suggested that the presence of the LIGHT transgene preferentially led to the deletion of H-Y⫹ thymocytes. Such
changes in thymocyte population recurred in the periphery; the
percentage of endogenous T cells (H-Y␤⫺CD3⫹) was also higher
in the splenocytes of H-Y/LIGHT mice than in those of H-Y/NL
mice (Fig. 3C; 8.72% in H-Y/LIGHT mice vs 3.93% in H-Y/NL
mice), probably attributable to the LIGHT-mediated deletion of
H-Y⫹ thymocytes. Similar results were obtained using T3.70 Ab
compared with F23.1 Ab. Among the F23.1⫹ cells, the majority of
them were also T3.70⫹ (⬎90%). It appears that the Tg TCR␤⫹
cells in male H-Y mice contain very few cells with rearrangements
of endogenous ␣-chains (Fig. 2). The pan TCR ␤-chain (using Ab
against all V␤-chain) and ␥␦TCR expression were examined for
the CD3⫹F23.1⫺ population, and the percentage of TCR␤⫹ was
essentially equal to that of CD3⫹ thymocytes in this population.
␥␦TCR constitutes an extremely minor population (⬍0.2%).
Taken together, our results suggested that the presence of the
LIGHT transgene preferentially promoted the deletion of H-Y⫹
thymocytes, for which TCR showed an extremely strong interaction with MHC/self-peptide, although the LIGHT transgene could
also cause the deletion of endogenous T cells.
The Journal of Immunology
3991
FIGURE 5. LIGHT is involved in the thymocyte development of non-Tg mice. A, C3H mice
were treated i.p. with LT␤R-Ig three times, and
thymi were collected 7 days after the last treatment.
B, C3H mice were sublethally irradiated and treated
with LT␤R-Ig as described in Materials and Methods. Thymocyte number was significantly enhanced
in the treated group compared with controls.
LIGHT-mediated selection is LT␤R dependent
LIGHT binds two receptors, HVEM and LT␤R, in vitro (21). To
test which receptor was involved in LIGHT-mediated phenotypes
in thymus, LIGHT Tg mice were crossed to the LT␤R⫺/⫺ or
LT␣⫺/⫺ background, and the thymocyte population was examined. There was no significant difference in the total thymocyte
number of wt/LT␤R⫹/⫺, wt/LT␤R⫺/⫺, and LIGHT/LT␤R⫺/⫺
mice (Fig. 6A). In addition, the thymocyte FACS profile was comparable between the different groups (data not shown). Consistent
with previous data, the thymocyte number in Tg/LT␤R⫹/⫹ mice
was dramatically reduced (Fig. 6A). These data suggested that the
presence of LT␤R was required for thymic phenotypes in LIGHT
Tg mice. Furthermore, the LIGHT transgene could still lead to a
reduction of thymocyte number in LT␣⫺/⫺ mice (LIGHT/
LT␣⫺/⫺) that lack both soluble LT␣3 and membrane LT␣1␤2 (Fig.
6B). Thus, our data indicated that the signaling via LT␤R by
LIGHT might have an impact on thymocyte development, which
could function independently of LT.
Discussion
In the present study we showed that LIGHT, a member of the TNF
family, plays an important role in negative selection, which requires the strong engagement of TCR and MHC/self-peptide. Negative selection is considered to be one of the major mechanisms to
delete potentially autoreactive T cells. However, the mechanism of
thymocyte apoptosis remains unclear. Our previous results demonstrated that overexpression of LIGHT on thymocytes promotes
the apoptosis of DP thymocytes (17). Shaikh et al. (22) also
showed that constitutive expression of LIGHT on thymocytes led
to thymic atrophy. However, whether LIGHT-mediated apoptosis
of DP thymocytes is associated with the interaction of TCR and
MHC/peptide is unresolved. Here, our data suggested that up-regulation of LIGHT on thymocytes promotes the deletion of H-Y
TCR⫹ population preferentially; thus, LIGHT-mediated apoptosis
of DP thymocytes is tightly linked with TCR specificity.
TCR signaling alone is not sufficient to induce apoptosis of thymocytes; therefore, other molecules have been suggested in the
process of thymocyte deletion, including TNF/TNF receptor
(TNFR) family members, costimulation molecules, coreceptors,
and adhesion molecules (4). However, the role of TNF/TNFR family members remains unclear. TNFR⫺/⫺ mice displayed abnormal
negative selection in certain models, but not in others (23). CD30
has been indicated to play a role in negative selection (24); however, this idea has been challenged by another study showing that
CD30 is not essential for negative selection (25). Thus, it has been
recently proposed that more than one member of the TNF/TNFR
family is required for negative selection in the thymus (23). The
involvement of costimulation molecules in negative selection is
similarly controversial, CD28⫺/⫺ mice did not show evident defects in negative selection (26), although one study (27) proposed
that the dependence on CD28 for the efficient deletion of autoreactive thymocytes is determined by the concentration, affinity/
avidity, and length of exposure to the deleting ligand; and clonal
deletion induced by endogenous viral superantigen was significantly affected by the targeted mutation of CD28 (28). In addition,
one recent study (29) showed that blockade of B7-1/B7-2 in vivo
FIGURE 6. LIGHT-mediated reduction of thymocytes is dependent on LT␤R. A, Thymocyte number in
wt/LT␤R⫹/⫺, wt/LT␤R⫺/⫺, Tg/LT␤R⫺/⫺, and Tg/
LT␤R⫹/⫹ mice was counted (n ⫽ 5). Data are the
mean ⫾ SEM. B, Thymocyte number in wt/LT␣⫹/⫺,
wt/LT␣⫺/⫺, Tg/LT␣⫺/⫺, and Tg/LT␣⫹/⫹ mice was
counted (n ⫽ 6). Data are the mean ⫾ SEM.
Downloaded from http://www.jimmunol.org/ by guest on July 31, 2017
determine whether LIGHT plays an important role in regulating
normal T cell development, the interaction between LIGHT and its
receptors was disrupted by the administration of LT␤R-Ig in vivo
to non-Tg mice. Various strains of mice were treated once a week
with LT␤R-Ig for 3 wk and analyzed 1 wk later. An enlargement
of thymic size and increased thymic weight were observed in the
group of treated mice, but not in the untreated group. The weight
of thymi in the treated group was increased 50% compared with
that in the untreated one (Fig. 5A; 87.6 ⫾ 14.9 mg in treated group
vs 58.9 ⫾ 8.4 mg in untreated group; n ⫽ 11).
Since the developing thymus may be more sensitive to fusion
protein treatment than the thymus at steady state, we irradiated wt
mice and treated mice with LT␤R-Ig in the same way as described
above. The total number of thymocytes showed a 400 –500% increase in the treated group compared with the untreated group (Fig.
5B; 52.7 ⫾ 21.4 ⫻ 106 in treated group vs 11.8 ⫾ 1.3 ⫻ 106 in
controls; p ⬍ 0.001). Thus, LIGHT is not only involved in the
negative selection of Tg models, but also plays an important role
in normal T cell development.
3992
LIGHT-MEDIATED SELECTION REQUIRES TCR ENGAGEMENT
mocyte deletion is not attributed to peripheral activation of T cells
in LIGHT Tg mice, since the mice we used for the thymocyte
development study were young and showed no signs of activation
in peripheral T cells (data not shown). Although LIGHT⫺/⫺ mice
did not show any obvious defect in the thymus (35), the process of
negative selection induced by various Ags, such as viral superantigen and anti-CD3, has not been reported. The TCR Tg system
may be needed to visualize Ag-specific selection events.
Previous studies suggest that up-regulation of LIGHT in peripheral T cells promotes mature T cell activation and enhances autoimmunity (15, 22). Blockade of LIGHT activity ameliorates insulin-dependent diabetes mellitus and graft-vs-host disease,
indicating the essential role of LIGHT in T cell-mediated diseases
(15, 16). Consistently, LIGHT⫺/⫺ mice showed significant defects
in the function of cytotoxicity in CD8 cells (35, 36), and targeted
mutation of LIGHT prolonged the survival of cardiac transplantation (37). In the current study we showed that LIGHT caused a
deletion of immature thymocytes bearing high affinity TCR.
Therefore, LIGHT, as a costimulatory molecule, basically can
cause the different responses for immature vs mature T cells. Upregulation of LIGHT promotes the deletion of potentially autoreactive T cells in thymic selection, but activates mature T cells in
the periphery leading to autoimmune diseases. Thus, our results
indicate that immature vs mature T cells can indeed respond differently to the same stimulus, and LIGHT plays a role in the negative selection of thymocytes by inducing the apoptosis of thymocytes bearing high affinity TCR.
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during fetal development substantially inhibits the clonal deletion
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the periphery, resulting in fatal multiorgan inflammation.
LIGHT belongs to the TNF family and functions as a costimulation molecule for T cell activation and proliferation; therefore,
we investigated the role of LIGHT in negative selection. Our previous data indicated that LIGHT plays a critical role in T cell
development (17); however, such an impact of LIGHT could be
attributed to various possibilities, such as the survival of thymocytes. In the present study we demonstrated that LIGHT-induced
apoptosis preferentially affected TCR⫹ cells in the context of a
strong interaction between TCR and MHC/peptide. In the absence
of such engagement of TCR, the impact of LIGHT up-regulation
was rather insignificant. Therefore, the death of DP thymocytes
induced by LIGHT is closely related to TCR signaling.
The LIGHT transgene also has an impact on the development of
DN thymocytes in male H-Y/LIGHT double-Tg mice. The reduction of DN thymocytes in male H-Y/LIGHT Tg mice is probably
due to the following reasons. 1) In the H-Y Tg system the transgene was expressed by DN thymocytes prematurely, while in a
physiological situation DN thymocytes do not express rearranged
␣␤TCR, which is normally expressed at the DP stage. Due to the
forced expression of H-Y transgenic TCR on DN thymocytes,
these cells may be sensitive to the regulation by other factors. 2)
Our data suggest that the simultaneous presence of LIGHT transgene and the strong interaction between H-Y TCR and self-MHC/
peptide on these DN thymocytes also enhance the deletion of this
population. Thus, the phenotypes reported here are attributed to the
difference in both populations, although the DP thymocyte population seems to be more significantly affected by the LIGHT transgene. The presence of the LIGHT transgene affects the development of both DP and DN populations in male H-Y/LIGHT Tg
mice, since they both express H-Y TCR. However, the presence of
the LIGHT transgene does not have a significant impact on DN
thymocytes in female H-Y/LIGHT Tg mice; thus, LIGHT-mediated
thymocyte deletion is dependent on the strong interaction between
TCR and self-MHC/peptide. Our previous data from LIGHT Tg mice
suggest that the presence of the LIGHT transgene does not significantly affect DN thymocytes in a non-Tg TCR system (17).
The mechanism of LIGHT-mediated apoptosis is still unclear.
Our data suggest that LT␤R is required for thymocyte deletion in
LIGHT Tg mice. LIGHT can bind to three receptors: HVEM,
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