Inhibitory Role of IFN-γ-Inducible Lysosomal
Thiol Reductase in T Cell Activation
This information is current as
of June 17, 2017.
Igor Barjaktarevic, Ayman Rahman, Sasa Radoja, Branka
Bogunovic, Alison Vollmer, Stanislav Vukmanovic and
Maja Maric
J Immunol 2006; 177:4369-4375; ;
doi: 10.4049/jimmunol.177.7.4369
http://www.jimmunol.org/content/177/7/4369
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References
The Journal of Immunology
Inhibitory Role of IFN-␥-Inducible Lysosomal Thiol Reductase
in T Cell Activation1
Igor Barjaktarević,* Ayman Rahman,† Sasa Radoja,* Branka Bogunović,† Alison Vollmer,*
Stanislav Vukmanović,* and Maja Marić2†
T
he thiol reductase family of proteins encompasses a variety of enzymes (protein disulfide isomerase, thioredoxin, ErP57) capable of reducing disulfide bonds at
various intracellular locations but exclusively at neutral pH (1).
IFN-␥-inducible lysosomal thiol reductase (GILT)3 is a unique
member of this family because it is located in acidic environment
of endosomal compartment and its optimal enzymatic activity is at
pH 4.5–5.5 (2– 4). GILT is synthesized as a proenzyme and is
processed into mature form by proteolytic removal of N- and Cterminal peptides. The protein has an approximate molecular mass
of 28 –30 kDa and was therefore initially named IP-30 (2). GILT
is constitutively expressed in professional Ag-processing cells but
is inducible in other cell types by inflammatory cytokines such as
IFN-␥, TNF-␣, and IL-1␤ (5). Using GILT⫺/⫺ mice as a model,
we have shown that GILT is involved in the first steps of Ag
processing of proteins containing disulfide bonds (6). This is to
date the only known function of GILT. GILT is an essential component of presentation of peptides from proteins rich in disulfide
bonds (6). The presence or absence of GILT can affect immune
responses to viral (7) or tumor Ags (8).
GILT is expressed in mouse tissues rich in APCs, such as lymph
nodes, spleen, and lungs (6), but is also present in other tissues
(e.g., kidney) that contain much fewer APCs. The presence of
GILT in these tissues could reflect expression in cells other than
the APCs. Further, GILT is secreted from cells and can be found
in culture supernatants (3, 6) and mouse serum (M. Marić, unpub*Center for Cancer and Immunology Research, Children’s Research Institute, Children’s National Medical Center, Washington, DC 20010; and †Department of Microbiology and Immunology, Georgetown University Medical Center, Washington, DC
20057
Received for publication April 25, 2006. Accepted for publication Juy 14, 2006.
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 by American Cancer Society Grant RSG-05-204-01-LIB
(to M.M.) and National Institutes of Health Grants AI48837 and AI41573 (to S.V.).
2
Address correspondence and reprint requests to Maja Marić, Department of
Microbiology and Immunology, Georgetown University Medical Center, 3900
Reservoire Road NW, Med/Dent C301, Washington, DC 20057. E-mail address:
[email protected]
Abbreviations used in this paper: GILT, IFN-␥-inducible lysosomal thiol reductase;
WT, wild type; KO, knockout; ROS, reactive oxygen species.
3
Copyright © 2006 by The American Association of Immunologists, Inc.
lished observations). These expression patterns raise a possibility
of GILT function nonrelated to MHC class II processing. In support of this notion, proteins with different degrees of homology to
human and mouse GILT were found in Caenorhabditis elegans
and Arabidopsis thaliana (9). In fact, more detailed search of deposited sequences revealed presence of GILT homologs in a wide
array of species, including but not limited to Danio rerio, Drosophila melanogaster, Xenopus tropicalis, Gallus gallus, Canis familiaris, Bos taurus, Oriza sativa, and Triticum aestivum (M.
Marić, unpublished observations). Because some of these organisms do not express MHC class II, the findings of GILT homologs
suggest that GILT may have an additional function, possibly evolutionarily older than involvement in Ag processing. Manipulation
of redox potential in T cells results in modulation of T cell activation (10, 11). We therefore decided to test whether GILT is
expressed in T cells and whether it might have a role in T cell
activation. We demonstrate that mouse GILT is expressed constitutively in T lymphocytes, and that it reduces the impact of TCR
engagement-mediated activation.
Materials and Methods
Mice, Abs, and reagents
C57BL/6 and BALB/c mice were purchased from Taconic Farms and
RAG1-deficient mice were purchased from The Jackson Laboratory.
GILT-deficient mice (6) were backcrossed to a C57BL/6 background for
10 generations. All mice were used at 6 –12 wk of age and were age and
sex matched for individual experiments. Purified hamster anti-mouse CD3␧
mAb, PerCP- or PE-conjugated anti-mouse CD8␣, APC-conjugated antimouse CD4, FITC-conjugated anti-mouse TCR␤ (H57-597), PE-conjugated anti-mouse CD69, PE-conjugated anti-mouse CD25, and FITC-conjugated anti-mouse CD107a (LAMP-1) were purchased from BD
Pharmingen. Alexa Fluor 594-conjugated F(ab⬘)2 of goat anti-rabbit IgG
and CFSE dye were purchased from Molecular Probes. Generation of
mouse GILT-specific antiserum (TITO) was described previously (6). Con
A was purchased from Sigma-Aldrich.
Flow cytometry
Spleen cells taken directly ex vivo or cultured, or purified T cells or T cell
subsets were stained using above listed directly conjugated mAbs. Staining
was performed by incubating 1 ⫻ 106 cells for 30 min on ice with 1/50
dilution of Ab or a mixture of Abs. Cells were then washed, fixed in 1%
paraformaldehyde, and analyzed using dual laser FACSCalibur (BD
Biosciences).
0022-1767/06/$02.00
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IFN-␥-inducible lysosomal thiol reductase (GILT) is a unique thiol reductase with optimal enzymatic activity at low pH. GILT
plays a crucial role in unfolding the antigenic proteins in preparation for their proteolytic cleavage and presentation of resulting
peptides by MHC class II. In this study, we demonstrate that GILT is expressed in T lymphocytes and that it has an APCnonrelated role in the regulation of T cell activation. Surprisingly, comparison of wild-type and GILT-deficient T cell activation
in vitro revealed stronger responsiveness in the absence of GILT. The effect of GILT in reducing the proliferative and cytotoxic
responses was endogenous to T cells and resulted from decreased sensitivity at the individual cell level. Therefore, a molecule with
primarily lysosomal localization suppresses T cell activation, a process characterized by signal transmission from plasma membrane to cytoplasm and nucleus. The Journal of Immunology, 2006, 177: 4369 – 4375.
4370
Cell purification
Spleen cells were labeled and isolated according to the manufacturer’s
instructions. Briefly, the cells were labeled with biotinylated Abs against
CD8␣, CD4, or pan T cell Ab mixture microbeads (Miltenyi Biotec). Labeled cells were positively (or negatively in case of pan T cell kit) selected
on MACS columns (Miltenyi Biotec). Cell purity was analyzed with the
appropriate FITC-, PE-, or allophycocyanin-conjugated Abs at FACS. The
purity of the cells was generally 92–97%, as determined by flow cytometry.
Proliferation assays
Primary stimulation cultures
Spleen cells from WT or GILT-deficient mice were cultured for various
times (as indicated in the figure legends) in 24-well (2 ⫻ 106 cells/well in
2 ml of complete medium) or 6-well plates (4 ⫻ 106 cells in 4 ml of
medium) in the presence of various concentrations of purified anti-CD3
Ab. The cultured cells were either analyzed for expression of activation
markers or used as effectors in anti-CD3-redirected lysis assay (12). For
stimulation of alloreactive CTLs, WT or GILT-deficient splenocytes (5 ⫻
106 per well) were cultured for 5 days with 5 ⫻ 106 irradiated (2500 rad)
allogeneic (BALB/c) splenocytes in 24-well culture plates.
PBS without Ca2⫹-Mg2⫹, dried, and stored. Purified CD8⫹ T cells were
resuspended in PBS at 1 ⫻ 106/ml, and 200 – 400 ␮l of cell suspension
were added on the coverslips placed in a 24-well plate. After 20 min at
room temperature, 500 ␮l of BD Cytofix (BD Biosciences) solution was
added gently down the walls of the well and incubated for 15 min at room
temperature. After two washes with PBS, 500 ␮l of 0.1% Triton X-100
were added and incubated for 1 min at room temperature. The wells were
washed three times in 1 ml of PBS, 5% FCS, with the last wash lasting at
least 30 min at 4°C. A 1/300 dilution of 200 ␮l of TITO (rabbit anti-mouse
GILT) in PBS (1% BSA) was placed over the coverslips for 30 min at room
temperature. After three washes with PBS-1% BSA, 200 ␮l of 1/200 Alexa
Fluor 594 goat anti-rabbit IgG were added. After three more washes in
PBS, the coverslips were air dried for 5–20 min. Dried coverslips were
mounted on slides, previously washed in 70% ethanol and dried, using a
drop of mounting solution from ProLong Antifade Kit (Molecular Probes).
Slides were dried, stored in the dark, and analyzed the next day by confocal
microscopy analysis using a Zeiss LSM-510 META microscope (Carl
Zeiss).
Results
GILT is expressed in T lymphocytes
To test whether GILT is expressed in T lymphocytes, CD4⫹ and
CD8⫹ T cells were purified and lysed, and lysates were analyzed
by Western blot under both reducing and nonreducing conditions
(Fig. 1A). A band of appropriate molecular mass was detected by
anti-GILT antiserum. The characteristics of GILT from CD4⫹,
CD8⫹ T cells, and lymphoblastoid B cell line A20 were similar,
except that the molecular mass of T cell form appeared slightly
lower. To test whether this difference may be caused by alternative
splicing, we amplified and sequenced GILT mRNA isolated from
T cells. No differences were found relative to the deposited sequence (data not shown), suggesting that differences in the protein
size are due to posttranslational modification(s). Intracellular staining and immunofluorescence microscopy of purified T cells determined the subcellular localization of mouse GILT in T cells. Staining of T cells in the absence or presence of GILT-specific Abs
revealed a specific punctuate staining pattern characteristic of lysosomal localization (data not shown). To test this more rigorously, costaining of GILT (red) and lysosomal marker LAMP-2
Chromium release assay
Target cells (1 ⫻ 106) were labeled with 51Cr for 60 min and plated at
104/well in 96-well round-bottom plates. Effector populations were added
at different ratios, and the plates were incubated at 37°C for 4 h. The release
of 51Cr in the supernatant was determined by scintillation counting. Maximal release from target cells was determined by treatment of cells with 1%
Triton X-100; spontaneous release was determined from cultures of labeled
target cells incubated with medium only. Specific lysis was determined
according to the formula: [(experimental release ⫺ spontaneous release)/
(maximal release ⫺ spontaneous release)] ⫻ 100.
Western blotting
A20 cells or purified CD4⫹ or CD8⫹ T cells (3 ⫻ 106) were lysed in 150
␮l of Tris-saline, 1% Triton X-100 containing protease inhibitor mixture.
20 ␮l of lysates were separated by SDS-PAGE under reducing or nonreducing conditions. Proteins from the gels were electrophoretically transferred to Immobilon P membranes (Millipore). Membranes were incubated
in Blotto buffer (PBS, 5% milk, and 0.1% Tween 20) at room temperature
or overnight at 4°C to block nonspecific binding to the membrane. The
blots were then incubated for 2 h at room temperature with 1/300 dilution
of TITO (anti-mGILT polyclonal rabbit serum) in Blotto buffer, followed
by secondary incubation with 1/2500 dilution of HRP-conjugated goat antirabbit Ig (Jackson ImmunoResearch Laboratories) in the blocking solution
(PBS, 1% BSA, and 0.1% Tween 20). The blots were washed extensively
after each incubation using PBS containing 0.1% Tween 20. The blots were
incubated with SuperSignal CL-HRP substrate working solution (Pierce)
and exposed for 10 s to 1 min to Kodak Biomax MR film to visualize the
specific bands.
Intracellular immunofluorescence
Glass coverslips (Fisher Scientific) were coated overnight at 37°C with 1
mg/ml poly-L-lysine (Sigma-Aldrich), washed three times with Dulbecco’s
FIGURE 1. Expression of GILT by T cells. A, Lysates from purified
CD4⫹ and CD8⫹ cells were subjected to SDS-PAGE under reducing (R)
or nonreducing (N) conditions, transferred, and probed using GILT-specific polyclonal Ab. The A20 cell line was used as a positive control. B,
Spleen cells from C57BL/6 mice were activated with anti-CD3 Ab for 4
days. CD8⫹ cells were purified and stained using rabbit anti-mouse GILT,
followed by Alexa Fluor 594 (red)-conjugated anti-rabbit Ig and CD107aFITC (green) conjugated for LAMP and analyzed by confocal microscopy.
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Cells were cultured in complete RPMI 1640 supplemented with 10% heatinactivated FCS, 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml
streptomycin, 5 ⫻ 10⫺5 M 2-ME, 1 mM sodium pyruvate (Invitrogen Life
Technologies) at 37°C, 5% CO2. For direct proliferation assays, spleen
cells (5 ⫻ 105/well) were incubated for 72 h in flat-bottom 96-well plates
in the absence or presence of desired concentrations of anti-CD3 mAb or
Con A. During last 16 h of culture, cells were pulsed with 0.5 ␮Ci of
[3H]TdR (ICN Biomedicals) overnight, and thymidine incorporation was
subsequently measured on a beta scintillation counter 1450 MicroBeta
(Wallac). Mixed cell proliferation assays were performed in an identical
manner with 5 ⫻ 104 purified wild-type (WT) or GILT⫺/⫺ T cells and 7 ⫻
104 of RAG1-deficient spleen cells per well. To analyze CFSE dilution as
a function of proliferation, purified CD4⫹ WT or GILT⫺/⫺ cells were
labeled with CFSE in the following manner: 10 ⫻ 106 cells in 250 ␮l of
PBS (0.1% BSA) and 250 ␮l of 10 ␮M CFSE in PBS (0.1% BSA) were
prewarmed to 37°C. The two samples were mixed and incubated for 10 min
at 37°C. Labeling was stopped by addition of 5 ml of ice-cold RPMI (10%
FCS) and 5 min of incubation on ice. After a washing, 6 ⫻ 105 of labeled
cells were cultured with 1.4 ⫻ 106 RAG1-deficient spleen cells in wells of
24-well plates in the presence of 0.2 ␮g/ml purified anti-CD3 Ab. After
96 h of culture, cells were stained with APC-conjugated anti-CD4 Ab and
analyzed by flow cytometry.
GILT INHIBITS T CELL ACTIVATION
The Journal of Immunology
(green) was performed, which revealed largely overlapping localization of the two molecules (Fig. 1B). Taken together, these data
demonstrate that GILT is expressed in T cells and that its pattern
of localization is similar to that found in professional APCs (6).
Enhanced T cell responses in the absence of GILT
To test whether GILT affects the function of T cells, WT and
GILT-deficient spleen cells were activated with anti-CD3 Ab or
Con A. The proliferative response of T cells was assessed by
[3H]TdR incorporation. Surprisingly, we found moderately, but
consistently superior proliferation of GILT-deficient T cells (Fig.
2, A–D). The most significant differences in proliferative responses
were found in cell cultures activated with lower concentrations of
mitogen. To test the effect of GILT expression on the cytotoxic
activity of effector cells, GILT knockout (KO) and WT splenocytes were activated with anti-CD3 Ab. Four days later, the cytolytic activity of effector T cells was tested in anti-CD3 redirected
assay using 51Cr-labeled P815 target cells. Similar to proliferation
assays, GILT-KO T cells showed consistently higher cytotoxic activity compared with WT cells (Fig. 2, E and F). Interestingly, the
concentration of anti-CD3 Ab in the activation cultures determined
the degree of difference in cytotoxic activity between KO and WT
T cells: the lower concentrations of anti-CD3 Ab led to the most
significant differences in cytotoxicity. Therefore, these data suggest that the presence of GILT reduces the extent of T cell responses. There were no significant differences before T cell activation between GILT-deficient and WT spleens in the relative or
absolute numbers of total T cells, in CD4/CD8 subset distribution,
in the proportions of naive and/or memory T cells as determined
by CD62L and CD44 staining, or in the proportion of
CD4⫹CD25⫹ cells (data not shown).
GILT-deficient T cells are inherently superior
To determine whether the functional differences between GILT
KO and WT T cells are endogenous to T cells, we purified T cells
from KO and WT mice and activated them with anti-CD3 Ab in
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FIGURE 2. Stronger T cell responses in the absence
of GILT. Proliferation of GILT KO and WT spleen cells
to anti-CD3 Ab (A and B) and Con A (C and D). Values
are the means and SDs of triplicate cultures from the
experiments with multiple individually tested animals
(A and C) as well as the means and SDs of mean representative values of proliferation rates in response to
titrated mitogen (B and D). Results are representative of
at least five individual experiments. Statistically significant differences at 0.001 ⬍ p ⬍ 0.01 (ⴱ) or p ⬍ 0.001
(ⴱⴱ) levels are indicated. Cytotoxic activities of GILT
KO and WT spleen cells activated for 4 days with concentrations of 1 ␮g/ml (E) and 0.08 ␮g/ml (F) of antiCD3 Ab. Equal numbers of activated KO and WT effectors were added at the indicated ratios to 51Cr-labeled
P815 target cells in the presence of soluble anti-CD3 Ab
(1 ␮g/ml). Results are mean values of two KO and two
WT mice. The findings are representative of four
experiments.
4371
4372
the presence of spleen cells from RAG1⫺/⫺ mice. As shown in the
Fig. 3A, GILT-KO T cells exhibited superior proliferation rates
compared with GILT-positive cells. To address the same question
for cytotoxic cell function, we performed MLR assay. Spleen cells
of GILT-KO and WT (H-2b) mice were stimulated with irradiated
BALB/c (H-2d) spleen cells (GILT-positive). The cytotoxicity of
effector cells was measured in chromium release assays using P815
(H-2d) or EL4 (H-2b) cells as targets. The results shown in Fig. 3B
demonstrate more potent cytotoxic response of GILT-deficient effector cells. Therefore, we conclude that GILT expression in T
cells reduces their responses to TCR engagement.
GILT INHIBITS T CELL ACTIVATION
GILT-deficient T cells display higher levels of CD69 on
activation in vitro
GILT-KO T cells undergo more cell cycles
FIGURE 3. The superior function of GILT-deficient T cells is inherent.
Proliferation of GILT KO and WT purified T lymphocytes to anti-CD3 in
the presence of RAG1⫺/⫺ spleen cells (A). T cells from spleens from KO
and WT mice were purified with pan T cell Ab mixture microbeads and
negatively selected with MACS columns. Cells were pulsed with [3H]TdR,
and incorporation was measured by scintillation counting. Values are the
means and SDs of triplicate cultures. The assays with no anti-CD3 Ab, no
T cells, or no RAG⫺/⫺ spleen cells were shown as negative controls. Cytotoxic activity of GILT KO and WT spleen cells was measured by MLR
assays (B). Responder spleen cells (C57BL/6; H-2b) were cocultured in 1:1
ratio with irradiated allogeneic stimulator cells (BALB/c; H-2d) for 5 days.
Equal numbers of activated KO and WT effectors were added in the indicated ratios to 51Cr-labeled allogeneic (P815) or syngeneic (EL4) target
cells. Values are means of duplicate cultures.
If both GILT⫺/⫺ and WT T cells are activated in their entirety, and
yet their proliferative responses are different, then the numbers of
cycles that each population of T cells undergo must be different.
To test this possibility, we labeled purified CD4⫹ cells with CFSE
and stimulated with anti-CD3 Ab in the presence of RAG2⫺/⫺
APCs. Four days later, the cell cycle-dependent dilution of CFSE
dye was analyzed by flow cytometry (Fig. 6). This analysis demonstrated that all cells in either sample went through at least one
cycle, consistent with the CD25 induction and CD3 down-modulation data. Furthermore, larger proportion of GILT-deficient T
cells passed through four or more cycles, whereas fewer T cells
relative to the WT passed through a single cycle. Therefore, antiCD3 stimulation of GILT⫺/⫺ T cells results in more cell divisions
than in the WT T cells.
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Differences between WT and GILT-deficient T cell responses
could be due to: 1) different proportions of activated T cells; 2)
different kinetics of activation; or 3) stronger activation on a single
cell level. To distinguish between these possibilities, we analyzed
CD69 induction in anti-CD3-activated CD4⫹ and CD8⫹ T cells.
The CD69 molecule is a type II C-type lectin receptor expressed
on a small proportion of mature T cells (13–15). CD69 expression
is associated with activation and is a good indicator of sensitivity
of T cells (16). The levels of CD69 induced in GILT-deficient T
cells were significantly higher, especially at later time points (Fig.
4, A–D). Even very low concentrations of anti-CD3 Abs efficiently
up-regulated CD69 in GILT-deficient T cells (Fig. 4, E and F). The
frequency of CD69⫹ cells were at all time points only mildly
higher in GILT-deficient than in WT CD4⫹ or CD8⫹ cells and
reached the levels of ⬃90% positive cells in both types of T cells
(Fig. 4, G and H). However, at any time point, there was a minor
fraction of apparently CD69⫺ T cells. If these cells were truly
nonresponsive to TCR engagement, their proportion among WT
and GILT⫺/⫺ T cells could explain the difference in the potency of
functional responses. Alternatively, it is possible that all T cells
were activated but that some either expressed CD69 levels undetectable by flow cytometry or reversed CD69 expression before it
was detectable on all T cells.
To address this issue, we tested other consequences of T cell
activation: CD25 up-regulation; and CD3 down-modulation.
CD25 is also known as the ␣-chain of the IL-2 receptor and is
induced in T cells within several hours, with peak around 24 h
postactivation (17). TCR down-regulation has been shown to correlate with TCR occupancy (18), but unlike CD69 and CD25 induction, TCR down-regulation appears to be independent of TCR
signal transduction (19, 20). All T cells in either WT or GILT⫺/⫺
cultures up-regulated CD25 24 h after activation and down-modulated CD3 6 h postactivation (Fig. 5). The latter finding was not
due to simple blocking with the anti-CD3 Ab from the stimulation
culture, given that CD3 was highly detectable 2 h after activation.
CD3 partially reappeared in both types of T cells after 24 h (data
not shown). Collectively, these results suggest that the dynamics of
activation is similar in GILT KO and WT T cells and that the
superior function of the former is due to stronger rather than faster
activation of all T cells. Also, the activation of GILT⫺/⫺ T cells is
stronger on an individual cell basis and becomes evident at relatively later stages of activation.
The Journal of Immunology
4373
Discussion
In this study, we demonstrate that GILT is constitutively expressed
in T cells and that it has a negative effect on the process of T cell
activation. GILT was detected in T cells at mRNA and protein
levels, and the subcellular pattern of localization was indistinguishable from that previously demonstrated for APCs. GILT-deficient T cells were inherently superior to WT T cells in proliferation and cytotoxic function in response to TCR engagement.
Activation-induced CD69 levels and the number of cell cycles
were higher in the absence of GILT, suggesting stronger activation
of individual T cells in the absence of GILT. Taken together, these
findings suggest that GILT could have a broader impact on the
immune system than initially suspected. This should not entirely
come as a surprise, given that GILT comprises a family of proteins
present in evolutionary distant species (from Paramecium and
Arabidopsis thaliana to C. familiaris and Homo sapiens), indicating a possibility that GILT may have additional role(s) besides
involvement in Ag processing.
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FIGURE 4. Higher levels of CD69 induced in GILTdeficient T cells. Spleen cells from GILT KO and WT
mice were activated with 0.4 ␮g/ml anti-CD3 Ab (unless otherwise indicated), and CD69 expression on gated
CD4⫹ (A, C, E, and G) or CD8⫹ (B, D, F, and H) was
analyzed by flow cytometry. A and B, Representative
histograms displaying levels of CD69 on GILT KO and
WT gated CD4⫹ or CD8⫹ cells after 24 h of stimulation. CD4⫹ or CD8⫹ cells stained with control PE-conjugated control hamster IgG1 Ab are also shown. C and
D, Mean fluorescence intensities of CD69 staining obtained at indicated time points after activation with antiCD3 Ab. E and F, Mean fluorescence intensities of
CD69 staining obtained 24 h after activation by indicated concentrations of anti-CD3 Ab. G and H, Percentage of CD69⫹ cells at indicated time points after activation with anti-CD3 Ab. Values are the means of
duplicate cultures obtained in one of the three experiments with similar outcome.
To analyze the role of GILT in T lymphocytes, we compared the
function of GILT- deficient and WT T cells. We found that proliferation and cytotoxic responses of GILT-deficient T cells were
consistently superior to those of WT T cells. This was independent
of whether anti-CD3 Abs, Con A, or alloantigens were used for stimulation. In none of these experimental settings could the influence of
GILT on MHC class II processing and presentation explain the experimental outcome. In fact, experiments with purified GILT-deficient
or wild-type T cells showed that differences in the levels of T cell
responses were maintained even when WT APCs were used for both
types of T cells. Therefore, superior stimulation is an inherent quality
of GILT-deficient T cells. Furthermore, our results indicate that
GILT-deficient T cells are more sensitive to lower concentrations of
TCR ligands and that their response is stronger on per cell basis.
The effect of GILT on T cell activation is surprising given its
subcellular localization. We showed that GILT in T cells is mostly
colocalized with LAMP-2. This finding is consistent with previously published data showing localization and the role of GILT in
4374
GILT INHIBITS T CELL ACTIVATION
FIGURE 5. CD25 induction and CD3 down-modulation in activated T
cells. Spleen cells were activated using anti-CD3 Ab; collected after the
indicated periods of time; stained using anti-CD4, anti-CD8, anti-CD25,
and anti-CD3 mAbs; and analyzed by flow cytometry. Shown are histograms of CD3 (A) or CD25 (B) expression on gated CD4 or CD8 cells.
the MHC class II processing pathway (6), localized entirely inside
vacuoles (3). Then, how can an internal lysosomal enzyme affect
T cell activation that starts at the cell surface (TCR engagement,
formation of immunological synapse), is followed by cytoplasmic
events (transmission of signals through the TCR signaling cascade) and terminates in the nucleus (gene transcription, DNA replication)? This question is intricately linked to the mechanism of
action of GILT; hence these two questions will be discussed
jointly.
The most obvious mechanism of GILT action is its thiol reductase activity, which could be exerted in the extracellular space,
inside lysosomes, or in other intracellular locations. Maintenance
of disulfide bonds is important for the preservation of tertiary
and/or quaternary structure and the function of secreted and cell
surface molecules (21). For example, the role of thioredoxin in
cleaving the disulfide bond that forms the D2 domain of CD4 is
well established (22). Thioredoxin is constitutively secreted by
plasma cells and from other cells in response to stress (23), and
could therefore gain access to extracellular portion of CD4. In an
analogous manner, GILT is secreted in the supernatant of cell lines
(3, 6) and can be found in mouse sera (M. Marić, unpublished
observations). Therefore, GILT could theoretically gain access and
alter the structure of T cell surface protein(s) involved in T cell
activation. Although such an action is unlikely during our in vitro
assay because spleen cells were washed immediately before stimulation, it is possible that presence or absence of GILT in vivo has
preconditioned T cells for less or more potent response, respectively. Another possibility is that cell surface GILT substrates are
gaining access to GILT in lysosomes. Given that cell surface molecules are accessible to GILT during membrane recycling, the simplest hypothesis for the effect of GILT on T cell activation would
involve a role of GILT in reducing the disulfide bridges of a fraction of the T cell surface molecule(s) involved in T cell activation.
Ig reduction following receptor-mediated internalization from the
cell surface provides an example of this mechanism (4).
The identity of putative target(s) of GILT is unknown, but TCR/
CD3 complex is the most obvious candidate from the perspective
of T cell activation. TCR␣and TCR␤ chains are held together by
a disulfide bond (24, 25), and there are also Ig-like domains in each
of the chains that could be the targets of thiol reductase activity.
Staining of WT and GILT-deficient T cells with anti-TCR␤ and
anti-CD3 mAbs did not reveal any significant differences (data not
shown; see Fig. 5). Assuming that binding of these Abs is sensitive
to the presence of disulfide bonds, we would conclude that structure of TCR/CD3 complex is not affected by GILT. It is possible,
however, that no single target is responsible for the observed effect
of GILT, and that functional differences between WT and GILTdeficient T cells are a result of individually small, but cumulative
effects on different protein species.
Another set of potential direct or indirect targets for GILT are
reactive oxygen species (ROS) generated early after T cell activation (26). ROS are thought to have an important role in the activation process, at least partly by transiently inactivating protein
tyrosine phosphatase Src homology region 2 domain-containing
phosphatase 1 (27). Manipulation of redox potential in T cells
results in modulation of T cell activation (10, 11). Perhaps GILT
has a role in elimination of activation-induced ROS; in the absence
of GILT, ROS might have a protracted half-life, leading to stronger activation of GILT-deficient T cells. The contact between the
GILT and the ROS could be achieved either by diffusion of the
latter to lysosomes or, indirectly, by an effect of GILT on molecule(s) that can transport (passively or actively) across the lysosomal membrane.
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FIGURE 6. GILT-deficient T cells undergo more cycles of proliferation. Purified GILT-KO or WT CD4⫹ cells were stained with CFSE and
activated for 4 days with anti-CD3 Ab (0.2 ␮g/ml) in the presence of
RAG⫺/⫺ spleen cells. Dilution of CFSE dye was analyzed using flow cytometry. Values are percentages of cells contained within each defined gate
representing one (M1), two (M2), three (M3), four (M4), or five or more
(M5) cycles of proliferation.
The Journal of Immunology
Acknowledgments
We thank Dr. Tarik F. Haydar for help with confocal microscopy and
Daniela Papini for help with flow cytometry.
Disclosures
The authors have no financial conflict of interest.
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GILT may have another function not related to the thiol reduction. Analysis of primary sequence of GILT using Web-based
bioinformatics tools suggests several motifs involved in intracellular signaling through phosphorylation. Some of these motifs are
found in highly conserved parts of GILT, suggesting their importance throughout the evolution. Indeed, our preliminary data suggest that tyrosine residue(s) on at least fraction of GILT molecules
in different cell lines is (are) constitutively phosphorylated (data
not shown). If GILT functions in signal transmission, we would
have to postulate that a small fraction of the total cellular GILT
undetectable by immunofluorescent microscopy either leaks from
lysosomes or is misdirected and never reaches lysosomes. Study is
under way to determine specifically which tyrosine(s) are phosphorylated and whether phosphorylation status changes the intracellular localization of GILT.
Inflammatory cytokines such as IFN-␥, TNF-␣ and IL-1␤ can
induce or up-regulate the expression of GILT (5). Given the observed role of GILT in T cells activation reported here, the upregulation of GILT in T cells infiltrating sites of inflammation is
expected to down-modulate T cell function locally. Indeed, it is
known that T cells infiltrating tumors are frequently functionally
defective (28), and levels of GILT mRNA are higher in metastatic
than in nonmetastatic forms of medulloblastoma (29), breast carcinoma (30), or diffuse large B cell lymphoma (31). Thus, it is
tempting to speculate that regulation of GILT expression may be a
mechanism of tuning the immune and inflammatory responses.
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