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CUTTING EDGE |
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI 48109
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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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The Fas:Fas ligand pathway is the principle mediator of activation-induced cell death (AICD),3 which is critical for the clearance of T cells that proliferated in response to an infection (7). Resting T cells do not have detectable Fas ligand on the cell surface. Upon activation, T cells express both Fas ligand and Fas receptor on the cell surface. The interaction between Fas ligand and the Fas receptor on activated T cells results in apoptosis of the Fas receptor bearing T cells limiting their expansion. Consequently, the immune response is down-regulated.
In T cells, NFAT is important for the activation of the Fas ligand
promoter (8). There are two NFAT binding sites within the
Fas ligand promoter region of which NFAT has been shown to activate
transcription (8). Activated T cells from mice that lack
both NFAT1 and NFAT4, two of the four NFAT family members, have
severely reduced levels of Fas ligand transcripts and, consequently,
display a resistance to apoptosis (9). Part of the
NFAT-dependant transcription maybe mediated indirectly through the NFAT
activation of early growth response 2 (Egr-2) and 3
(Egr-3), which are also important for inducible expression of the
Fas ligand gene (10, 11). Other transcription
factors, Sp1 and NF-
B, are also likely to play a role in regulating
Fas ligand gene transcription (12, 13).
Class II transactivator (CIITA) was initially described as a critical transcriptional factor regulating multiple genes involved in Ag presentation (14, 15). Although the mode of CIITA action is not well understood, interactions of CIITA with DNA binding proteins (16), the coactivator cAMP response element binding protein (CBP)/p300 (17, 18, 19), and the basal transcriptional machinery (20) are likely to be required for function. Recently, we also demonstrated that CIITA down-regulates IL-4 gene transcription during Th1 T cell development (21). The repression of IL-4 transcription by CIITA is partly due to a competition between CIITA and NFAT, an essential IL-4 transcription factor, for binding to CBP/p300 (17). As NFAT has been shown to be important for Fas ligand gene transcription, we tested whether Fas ligand expression is regulated by CIITA. Here we show that CIITA represses Fas ligand gene transcription and prevents CD4 T cell death mediated by the Fas:Fas ligand pathway upon activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs recognizing Fas ligand (MFL3) and I-E (14-4-4S) were purchased from PharMingen (San Diego, CA). 2C11 (anti-mouse TCR) and TIB105 (anti-CD8) were purified from hybridoma supernatant. All flow cytometry was analyzed using Becton Dickinson (Mountain View, CA) FACScan. Con A, proteinase K, ionomycin, and PMA were purchased from Sigma (St. Louis, MO). Human recombinant IL-2 was provided by the National Cancer Institute (Bethesda, MD). The luciferase reporter driven by the 486-bp Fas ligand promoter, and the 3x NFAT promoter, human CIITA, and NFAT2, were described (8, 17). Antisense CIITA was generated by reversing the cDNA encoding human CIITA and cloning into the same expression vector as the sense CIITA gene.
Cell lines and mice
The 68-41 Th1 cell hybridoma (22), the 002 T cell hybridoma (23), the D10 Th2 clone (24), the Jurkat T cell line, and the primary cells isolated from C57BL/6 mice were all maintained in Click’s medium supplemented with 10% FBS, 2 mM glutamine, 100 µg/ml penicillin and streptomycin, and 10-5 M 2-ME. The A20 mouse B cell line was maintained in RPMI 1640 medium with the same supplements as above. C57BL/6 mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained under specific pathogen-free conditions at the University of Michigan animal facility.
Transfections
To generate stable transfectants of T cells expressing CIITA,
1 x 107 cells were mixed with 30 µg of
DNA and then electroporated (300 V, 960 µF) using the Gene Pulser
(Bio-Rad, Richmond, CA). Cells were selected in medium containing 1
mg/ml G418 (Geneticin; Life Technologies, Grand Island, NY). Transient
transfection of 68-41 cells were performed with 5 x
106 cells, 5 µg of the reporter plasmid, 10
µg of CIITA or NFAT2, and 0.5 µg of the 
-galactosidase driven by
the CMV promoter. After electroporation (250 V, 960 µF) cells were
stimulated with PMA (25 ng/ml) and ionomycin (1.5 µM) overnight.
Luciferase and -galactosidase activity was measured as previously
described (17). Transfections into primary splenocytes
were conducted as follows. Total splenocytes from C57BL/6 mice were
prepared and activated overnight with Con A (3 µg/ml) and IL-2 (400
U/ml). Next day, 1 x 107 cells were mixed
with 10 µg of the reporter plasmid, 20 µg CIITA, anti-sense
CIITA, or NFAT2 plasmid, and 3 µg of CMV--gal in 0.25 ml Click’s
medium. Cells were electroporated (250 V, 960 µF), rested 2 h,
followed by stimulation with PMA (25 ng/ml) and ionomycin (1.5 µM)
for 5 h. Protein extracts were made and analyzed for luciferase
and -gal activity as described above. All transfections contained
the same amount of total DNA by the addition of the empty expression
vector.
RNA extraction and PCR
Total cytoplasmic RNA preparation, cDNA synthesis, and PCR were
conducted as described (21). The following primers were
used in the PCR: Fas ligand (forward, 5'-GGACCACAACACAAATCTGTG-3';
reverse, 5'-GGTCAGCACTGGTAAGATTGA-3') and 
-actin (forward,
5'-CACCCTGTGCTGCTCACCGAGGCC-3'; reverse,
5'-CCACACAGAGTACTTGCGCTCAGG-3').
DNA laddering
Control (2 x 106) or
CIITA-expressing 002 cells were treated overnight with plate-bound
anti-CD3 (5 µg/ml), PMA (25 ng/ml) and ionomycin (1.5 µM), or
3300 rad -irradiation. The cells and debris were harvested and
digested with proteinase K overnight (10 µg/ml, 0.4% SDS, 68 mM
EDTA, in 1x PBS). The next day, NaCl was added to 1.45 M, and DNA was
precipitated from the supernatant using ethanol. The DNA was digested
with RNase A for 30 min, run on a 1% TBE (90 mM Tris, 90 mM boric
acid, and 2 mM EDTA) agarose gel, and visualized using UV light.
Thymidine release assay for cell death
Target cells (5 x 105) were labeled with 5 µCi [3H]thymidine (NEN, Boston, MA) for 5 h in 1 ml of RPMI 1640 medium, washed, and resuspended at 1 x 104 cells per 100 µl on 96-well plates coated with anti-CD3 (5 µg/ml). Killer T cells were added to the labeled target cells in a series of 3-fold dilutions and incubated overnight. The plates were harvested and counted. The fragmented DNA of apoptotic cells passes through the filter, whereas the DNA of live cells is trapped on the filter. The percentage of relative DNA fragmentation was determined as follows: (the counts in the absence of killer cells - the counts in the presence of killer cells) divided by (the counts in the absence of killer cells) x 100. Each data point represents the average of triplicate. At least three independent experiments were performed. The anti-Fas ligand (MFL3) or the control Ab (anti-CD8-TIB105) were used at 10 µg/ml.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether CIITA represses transcription from the Fas
ligand promoter, we transfected the 486-bp Fas ligand promoter-driven
luciferase (8) with or without CIITA to the 68-41 Th1
hybridoma, then stimulated it with PMA and ionomycin to induce promoter
activity. This promoter region has been shown to be active in T
cells after stimulation (8). Cotransfection of CIITA
resulted in a >5-fold reduction in luciferase activity (Fig. 1
A, compare lanes 1
and 2). Cotransfection of NFAT2 enhanced luciferase
activity, and this activity was also repressed by CIITA (Fig. 1A, compare lanes 3 and 4).
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B, lanes 1 and 2). Transfection of
anti-sense CIITA had no inhibitory effects on Fas ligand
transcription (Fig. 1B, lane 3). To further examine the role
of CIITA in the NFAT-mediated activation of the Fas ligand promoter, we
tested the luciferase construct driven by the minimal IL-2
promoter with a trimerized distal Fas ligand NFAT site (3x
NFAT) (8). As previously shown (8), the
trimerized distal NFAT site was sufficient for NFAT-mediated activation
of the minimal IL-2 promoter (Fig. 1C, lanes 1 and
3). This activation was also repressed by CIITA (Fig. 1C, lanes 2 and 4). CIITA inhibits induction of endogenous Fas ligand expression
We next wanted to examine the effect of CIITA on endogenous Fas
ligand expression. To do this, we stably transfected CIITA to three T
cell lines, the 68-41 Th1 hybridoma (22), the 002 T cell
hybridoma (23), and the D10 Th2 cell clone
(24). As shown previously (21, 23), T cells
transfected with CIITA expressed MHC class II (Fig. 2A). The levels of TCR and Fas
receptor on the cell surface were comparable between the control and
the CIITA-expressing cells (data not shown). To analyze Fas ligand
expression, we stimulated T cells with plate-bound anti-CD3 Ab. The
levels of Fas ligand on the cell surface were significantly up
regulated after stimulation from the control T cells (Fig. 2B). However, T cells transfected with CIITA showed minimal
detectable expression of Fas ligand. These data show that CIITA
prevents the up-regulation of endogenous Fas ligand expression in
activated T cells.
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C). However, the level of Fas ligand transcripts in the
CIITA-expressing cells were drastically reduced. Similar data were also
obtained with the D10 cells (data not shown). Taken together, CIITA
represses the induction of endogenous Fas ligand transcription that in
turn prevents cell surface expression of Fas ligand upon
activation. CIITA protects T cells from AICD but not stress-induced cell death
T cell hybridomas typically undergo AICD when stimulated via the
TCR (25, 26). The Fas:Fas ligand pathway has been shown to
be the prime mediator of AICD. If CIITA represses the activation of
Fas ligand expression, CIITA-expressing T cells could be resistant to
AICD. To test this, the control and CIITA-expressing 002 cells were
stimulated with anti-CD3 Abs. We first compared the number of live
cells determined by trypan blue exclusion assay. As shown in Fig. 3A, <10% of the control 002
cells were alive after 24 h of activation. In contrast, >80% of
the CIITA-expressing cells were alive, indicating that they were
protected from AICD. If the cell death is due to Fas:Fas ligand
interactions, an Ab that blocks Fas ligand interaction with the Fas
receptor should prevent the cell death. When Ab recognizing Fas ligand
was added to the control cells, they were rescued from the cell death
(Fig. 3A, lane 3). These data strongly suggest that the
difference in cell death seen between the control and CIITA-expressing
T cell hybridoma is most likely due to differences in the level of Fas
ligand expression.
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A, lane 4). The expression of Fas ligand is
also induced by stress such as -irradiation or UV exposure, but this
induction does not require NFAT or Egr-2/3 (27). If the
inhibition of Fas ligand expression by CIITA is through NFAT,
stress-induced cell death should not be affected by CIITA.
CIITA-expressing cells died upon -irradiation (Fig. 3A, lane
5), and they expressed a comparable level of Fas ligand (Fig. 3B).
We further confirmed apoptotic cell death by measuring the level of DNA
laddering, a hallmark of apoptosis. Extensive DNA laddering was
seen with the control but not the CIITA-expressing cells upon
stimulation with either anti-CD3 or PMA and ionomycin (Fig. 3C, lanes 3–6). Gamma-irradiation resulted in similar DNA
laddering in both control and CIITA-expressing 002 cells (Fig. 3C, lanes 7 and 8). Collectively, our data
indicate that CIITA prevents cell death by inhibiting NFAT-mediated
induction of Fas ligand expression.
CIITA-expressing cells have reduced killing ability
Because CIITA represses Fas ligand expression, CIITA-expressing T cells would be less effective at killing Fas-bearing target cells. To test this, we measured the relative percentage of fragmented DNA of target cells using a thymidine release assay. The 002 hybridoma or D10 Th2 cells were used as killer cells. The targets were A20 mouse B cells or Jurkat human T cells, which constitutively express the Fas receptor and are capable of undergoing Fas-mediated cell death (28, 29). To measure the amount of fragmented DNA produced by the target cells but not the killer cells, target cells were labeled with [3H]thymidine. The killer and target cells were cocultured overnight in the presence of mouse anti-CD3 Abs that activates the killer but not the target cells.
DNA fragmentation of the target cells was detected when they were
cultured with the control but not CIITA-expressing killer cells (Fig. 4, A–C). This demonstrates
the inability of CIITA-expressing T cells to kill target cells.
Coculturing of killer cells with the target cells in the absence of
anti-CD3 did not result in DNA fragmentation (data not shown). The
killing was mediated through Fas:Fas ligand interactions because the
anti-Fas ligand Ab but not the control Ab inhibited DNA
fragmentation of target cells (Fig. 4D). These data indicate
that CIITA-expressing T cells are less effective at killing target
cells via the Fas:Fas ligand pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The endogenous CIITA gene is transcribed in unstimulated 002
and 68-41 cells (T.S.G. and C.-H.C., data not shown). However, upon TCR
activation, CIITA transcripts are no longer detectable in the 002 or
reduced in the 68-41 Th1 hybridoma (data not shown). It seems that
down-regulation of CIITA transcription precedes Fas ligand
transcription. Interestingly, CIITA transcripts can be detected in
unstimulated naive CD4 T cells but disappears upon TCR activation
unless cells receive IFN- signaling during the Th1 differentiation
process (Ref. 21 ; T.S.G. and C.-H.C., unpublished data).
Hence, the role of CIITA in Th1 cells could be not only to suppress Th2
type cytokine expression but also to delay or protect these cells from
AICD during the differentiation process, which would allow a wider
effector window before succumbing to cell death. Further analysis of
the kinetics of CIITA and Fas ligand expression during T cell
differentiation and reactivation may provide evidence for this
hypothesis. As the data presented was generated in vitro, further
investigation into the role that CIITA has in Fas ligand transcription
in vivo needs to be undertaken.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Cheong-Hee Chang, Department of Microbiology and Immunology, Room 6606 MSII, University of Michigan Medical School, Ann Arbor, MI 48109-0620.
3 Abbreviations used in this paper: AICD, activation-induced cell death; CIITA, class II transactivator; Egr-2/3, early growth response 2/3; CBP, cAMP response element binding protein.
Received for publication November 14, 2000. Accepted for publication January 5, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) or CIITA (•)-expressing 002 T cells were incubated
overnight with [3H]thymidine-labeled A20 B
cells (A) or Jurkat T cells (B) in the presence
of plate-bound anti-CD3. C, D10 T cells expressing CIITA
(•), unlike the control cells (