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*
Department of Molecular Genetics, Graduate School of Medicine, and
Department of Surgery, Institute of Pulmonary Cancer Research, School of Medicine, Chiba University, Chiba, Japan;
Department of Biochemistry, Juntendo University School of Medicine, Tokyo, Japan; and
§
Division of Molecular Membrane Biology, Cancer Research Institute, Kanazawa University, Ishikawa, Japan
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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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10-fold higher affinity for
the ligands than CD28 (11), and, thus, a low level of
CTLA-4 expression on the cell surface can readily compete with CD28 for
ligand binding, resulting in inhibition of T cell activation. Third,
the surface expression of CTLA-4 is strictly regulated: CTLA-4 is
expressed transiently upon T cell activation, whereas CD28 is expressed
constitutively (1, 2). Recent studies have revealed that CTLA-4 is localized mainly in intracellular granules, and such granules containing CTLA-4 were partially overlapped with perforin-containing secretory granules and also with endocytic compartments in CD8+T cells. It has been further revealed that trafficking of CTLA-4 is dynamically regulated between intracellular stores and the cell surface (12). However, because CD4+ T cells do not possess any typical secretory granule, the intracellular localization and trafficking of CTLA-4 in CD4+ Th cells remain unclear. Although CTLA-4 was originally identified as a CTL-specific molecule, CTLA-4 is also expressed in CD4+ T cells and is suggested to play essential roles in Th1/Th2 development, cytokine production, and tolerance induction (13, 14, 15, 16, 17). Indeed, CTLA-4-deficient mice revealed strong selection toward Th2 development, indicating a crucial role of CTLA-4 in Th1 development and function (18).
We have previously demonstrated that CTLA-4 binds to the µ2 subunit of the clathrin-associated adaptor protein complex AP-2 through the tyrosine-based motif containing GVYVKM within the cytoplasmic domain of CTLA-4, and that tyrosine phosphorylation of the motif prevents the binding to AP-2 and probably keeps CTLA-4 on the cell surface (19, 20, 21, 22). We have suggested from these data that AP-2-mediated endocytosis of CTLA-4 plays an important role in the regulation of the surface expression of CTLA-4. Indeed, tyrosine mutants of CTLA-4, which failed to bind to µ2, exhibited a higher expression of cell surface CTLA-4 than wild-type (WT)3 CTLA-4 on resting T cells (23). However, we observed that a mutant CTLA-4 lacking most of the cytoplasmic tail exhibited much higher expression of the cell surface CTLA-4, and, in addition, the surface expression of the mutant CTLA-4 was further up-regulated upon T cell activation. These observations suggest that mechanism(s) other than AP-2-mediated endocytosis may be involved in the regulation of the surface expression of CTLA-4. More importantly, the mechanism to induce high expression of CTLA-4 from the intracellular pool to the cell surface has not yet been clarified.
Here, we have studied the intracellular localization and trafficking of CTLA-4 in CD4+ T cells transfected with various mutants of CTLA-4. We found that intracellular CTLA-4 is mainly localized in lysosomes, and these lysosomes are secreted upon T cell activation. Therefore, our results demonstrate that CD4+ Th cells enhance the surface expression of CTLA-4 upon activation by the mechanism of the activation-induced secretion of lysosomes. From these aspects, we will discuss the dynamic regulation of CTLA-4 expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A murine Th1-type T cell clone, 23-1-8 (24, 25), was cultured as previously described (23). 23-1-8 cells were transfected by retrovirus-mediated gene transfer with WT CTLA-4 and its mutants, and infected T cells were used as bulk population and not as a clone as previously described (23).
Antibodies
Anti-CTLA-4 mAb, UC10, was kindly provided by Dr. J. Bluestone
(University of Chicago, Chicago, IL), and biotinylated UC10 was
prepared by standard technique. Alexa488 (green
fluorescence)-conjugated UC10 was prepared using an Alexa488 Protein
Labeling Kit (Molecular Probes, Eugene, OR). Rabbit anti-lysosomal
glycoprotein (lgp) 110 Ab is directed to the peptide of the C region of
the rat lgp110, and rabbit anti-cathepsin D Ab is prepared against
rat cathepsin D. Rabbit anti-rat lgp85 was provided by Dr. M.
Himeno (Kyushu University, Fukuoka, Japan). Biotinylated anti-mouse
Fas ligand (FasL) mAb, K10, was provided by Dr. K. Okumura (Juntendo
University, Tokyo, Japan). Rabbit anti-human Rab5a Ab was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). FITC-conjugated goat
anti-armenian hamster IgG Ab and Cy3-conjugated goat
anti-rabbit IgG Ab were purchased from Jackson ImmunoResearch
Laboratories (West Grove, PA). FITC-goat anti-rabbit IgG Ab,
streptavidin-PE, and streptavidin-Cy3 were purchased from ICN
Pharmaceuticals (Aurora, OH), PharMingen (San Diego, CA), and
Sigma-Aldrich (St. Louis, MO), respectively. Anti-TCRß (H57-597),
anti-CD3
(145-2C11), and anti-CD3
(H146-968) mAbs were
used for T cell activation. H57-597 and H146-968 were kindly provided
by Dr. R. Kubo (La Jolla Institute for Allergy and Immunology, San
Diego, CA) and 145-2C11 by Dr. J. Bluestone.
Immunostaining and confocal laser scanning microscopy
Cells were cultured on coverslips overnight, and fixed in 4% (w/v) EM grade paraformaldehyde (Nacalai Tesque, Kyoto, Japan) for 15 min at room temperature. The coverslips were then washed twice in PBS for 5 min, inverted in 30 µl of first Ab solution that was diluted with 0.1% saponin PBS for 45 min at room temperature, washed twice in PBS for 5 min, and second Ab was applied while shielded from light. After 45 min incubation at room temperature, and coverslips were washed and mounted onto slides with PermaFluor Aqueous Mounting Medium (Shandon, Pittsburgh, PA) and examined with a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss, Jena, Germany). For standard acquisition, we used a double dichroic mirror for the excitation beam, a band pass 505- to 550-nm barrier filter for FITC or Alexa488, and a long pass barrier filter >585 nm for Cy3 detection.
Flow cytometry
T cells were stained with first Ab for 45 min followed by appropriate second Ab for 45 min. All steps were performed at 4°C in a buffer containing 0.1% BSA and 0.05% sodium azide. Cells were analyzed by FACScan (Becton Dickinson, Mountain View, CA) with the CellQuest software.
T cell stimulation
For plate coating, 1 µg/ml anti-TCRß mAb (H57-597),
anti-CD3 mAb (145-2C11), and anti-CD3 mAb (H146-968) as
control were incubated on 24-well plates at 4°C overnight. Wells were
washed with PBS four times. WT CTLA-4-transfected T cells were added at
2 x 106/well in 600 µl to complete RPMI
1640 in the presence of 5 µg/ml murine IL-2 and incubated at 37°C
with 5% CO2 for 24 h. For detection of
protease activity, the culture medium was collected and the
supernatants after centrifugation at 11,000 x g for 5
min were used. For normal T cell stimulation,
CD4+ T cells were isolated from spleen cells of
C57BL/6 mice by depleting CD8+ cells and surface
Ig+ cells using anti-CD8 mAb and
anti-mouse Ig Ab and magnetic beads. After
CD4+ T cells were stimulated with Con A with
irradiated C57BL/6 splenocytes for 2 days, T cells were separated by
lympholight M and subjected for restimulation.
Assay for ß-hexosaminidase (ß-hex) activity
Fifty microliters of supernatants were preincubated for 5 min at 37°C with 150 µl of 0.2 M sodium acetate buffer (pH 4), and further incubated with 100 µl of 3 mM 4-methyl-umbelliferyl-N-acetyl-ß-D-glucosaminide. The reaction was stopped by addition of 2 ml of 0.1 M glycine (pH 10.5). Fluorescence was measured with an F-2000 spectrophotometer (Hitachi, Tokyo, Japan) at excitation 360 nm/emission 450 nm. The culture medium of nonstimulated T cells was also measured and subtracted as background from each sample.
Assay for cathepsin D activity
Fifty microliters of the culture medium were incubated for 10
min at 37°C with 80 µl of 250 µM (7-methoxycoumarin-4-yl)
acetyl-GKPILFFRLK (DNP) 
-NH2 in 0.5% DMSO and
10 ml of 50 mM sodium acetate buffer. The reaction was stopped by
addition of 1 ml of 5% trichloroacetic acid, and fluorescence was
measured at excitation 328 nm/emission 393 nm. The background level of
cathepsin D was measured similarly to the assay for ß-hex.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In a previous study (23), we generated four different
forms of mutant CTLA-4 as illustrated in Fig. 1
A: three mutants with the
alteration of either or both tyrosines in the cytoplasmic domain of
CTLA-4 to glycine(s) (Y165G, Y182G, Y165/182G), and one deletion mutant
lacking most of the cytoplasmic tail except for the membrane-proximal 7
amino acids (
CP7). These mutants were transfected into a murine
CD4+Th1 clone, 23-1-8, to analyze the structural
requirement for surface expression and signal transduction. In the
previous analysis (23), the expression of CTLA-4 on the
cell surface of these mutant CTLA-4-expressing T cell clones
demonstrated that Y165 mutation induces high expression of the surface
CTLA-4, whereas WT stayed very low. It was noted that further
mutation/deletion over the Y165 mutation induced even higher expression
of the surface CTLA-4, namely, Y165G < Y165/182G <
CP7.
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B). CTLA-4 was hardly
detected in T cells transfected with the vector alone, but a large
amount of CTLA-4 was observed within intracellular vesicles in T cells
transfected with WT CTLA-4. Interestingly, although Y165G mutant, which
failed to bind with AP-2, exhibited a high expression of surface CTLA-4
by flow cytometry, confocal microscopy showed only weak expression of
CTLA-4 on the cell surface, and most CTLA-4 was still present in the
intracellular vesicles, similar to WT (Fig. 1B). In
addition, the intracellular vesicles containing CTLA-4 were
significantly reduced in T cells expressing Y165/182G mutant CTLA-4,
whereas the cell surface became bright. In distinct contrast, these
granules were greatly diminished in T cells transfected with CP7.
Although brightly stained and large vesicles were prominent in WT and
mutant CTLA-4 transfectants, it was noted that weakly stained and
smaller vesicles as well as a perinuclear compartment were also stained
with the Ab (Fig. 1B). These data suggest that intracellular
CTLA-4 was located mainly in large granules but was also distributed in
some other organelles. Localization of CTLA-4 in lysosome
To define the characteristics of CTLA-4-containing vesicles in
CD4+ T cells, we performed double staining for
both CTLA-4 and several marker proteins that are well-defined and known
to be specific for various organelles (Fig. 2). It has been suggested that CTLA-4 is
partially colocalized with perforin (as in secretory granules) and
transferin receptor (as in endocytic compartments) in
CD8+ T cells (12). However, neither
perforin nor typical secretory granules are present in
CD4+ T cells. Because it has been reported that
FasL was localized in the perforin+ lytic
granules in CD8+ T cells (26) and
FasL is induced even in CD4+ T cells upon
activation, colocalization of CTLA-4 and FasL in these Th1 clones was
examined after stimulation with Con A for 2–6 days. As shown in Fig. 2A, FasL was mainly present in the intracellular large
vesicles and colocalized with CTLA-4 (Fig. 2A).
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B). In contrast, anti-lgp110 Ab stained
intracellular large vesicles that were mostly colocalized with the
large type of CTLA-4-containing vesicles (Fig. 2C). Because
the preform of cathepsin D immediately changes into the active form and
hardly detected, anti-cathepsin D has been thought to stain only
the active form of cathepsin D, which is specific for lysosomes.
Therefore, we stained for cathepsin D to discriminate late endosomes
and lysosomes. The staining pattern of WT CTLA-4-transfected Th1 cells
for cathepsin D was similar to that of lgp110 (Fig. 2D).
From these data, we concluded that most of the intracellular large
vesicles containing CTLA-4 in CD4+ Th cells were
lysosomes. Degradation of intracellular CTLA-4 in lysosome
Because we found that intracellular CTLA-4 is accumulated mainly
in lysosomes, we next tried to analyze the physiological significance
of lysosomal localization of CTLA-4, whether CTLA-4 is accumulated in
lysosomes for degradation or storage. To address this question, we
treated T cells with cyclohexamide (CHX) to inhibit new synthesis of
CTLA-4 and examined whether ready-made CTLA-4 could be stably
maintained in lysosomes. The amount of intracellular CTLA-4 was
significantly decreased during the 2-h treatment with CHX, and
remarkably diminished after 4 h (Fig. 3A). This observation suggests
that intracellular CTLA-4 was degraded in lysosomes in a relatively
short period in the absence of any stimulation. Indeed, when we treated
cells with several reagents, which are known to inhibit lysosomal
degradation, such as NH4Cl, methylamine, and
bafilomycin, the cell surface expression (Fig. 3B) as well
as intracellular expression (data not shown) of CTLA-4 was augmented
with any of these reagents. In contrast, these treatments did not
induce any surface expression of lysosomal proteins, such as lgp85
(data not shown).
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Activation-induced secretion of lysosomes in CD4+ T cells
Lysosome is the acidic organelle containing a characteristic set
of acid hydrolases, and is responsible for the degradation of
internalized proteins from the endocytic pathways. However, it has been
described that CTLs possess "secretory lysosomes" containing
perforin and granzyme, which behave as secretory granules
(27). Activation-induced extracellular release of
hydrolytic enzymes toward the target cells is one of the mechanisms for
target cytotoxicity. Such secretory lysosomes have been found widely in
hemopoietic lineages, such as CTLs, NK cells, and granulocytes, but
have not been analyzed in CD4+ T cells. To
analyze the secretory lysosomes in CD4+ T cells,
we examined whether CD4+ T cells secrete
lysosomes upon physiological stimulation by using a lysosomal marker
protein, lgp85, together with CTLA-4. Upon stimulation by cross-linking
with anti-TCR or anti-CD3 mAb, the surface expressions of
both CTLA-4 and lgp85 were increased (Fig. 4), suggesting that CTLA-4-containing
lysosomes were secreted and lysosomal membrane proteins appeared on the
cell surface upon TCR stimulation.
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mAb on a coverslip. As shown in Fig. 5, the number of lysosomal granules was
dramatically reduced upon stimulation, and the remaining granules were
found to be localized to the proximal region of Ab-coated plates. These
data indicate that lysosomes containing CTLA-4 indeed moved toward the
plasma membrane and were secreted upon TCR stimulation.
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A, TCR stimulation
with anti-TCR or anti-CD3 mAb induced ß-hex release into the
supernatant. Considering that these so-called lysosomal markers
(lgp110, lgp85, and CD63, etc.) are present on both lysosomes and late
endosomes and these granules also contain ß-hex, the surface
expression of lgp110/lgp85 and the secretion of ß-hex does not
necessarily reflect to lysosomal secretion. Therefore, we measured the
activity of cathepsin D in the culture medium to confirm the lysosomal
secretion, because the active form of cathepsin D is exclusively
present in lysosomes. Enzymatic activity of cathepsin D was detected in
the culture supernatants by TCR cross-linking (Fig. 6B).
Thus, secretion of both ß-hex and cathepsin D confirms lysosomal
secretion upon TCR stimulation.
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, C and D) with a high dose of
Ca2+ ionophore, suggesting similar mechanism to
the degranulation in mast cells.
Furthermore, we analyzed whether the regulation of CTLA-4 expression by
lysosomal secretion is observed in normal T cells. Resting T cells do
not possess CTLA-4 transcript, and CTLA-4 mRNA and protein are produced
upon stimulation. CTLA-4-transfected Th clones, which express CTLA-4
within the cells, represent the cells equivalent to preactivated T
cells. CD4+ splenic T cells were isolated and
stimulated with Con A for 2 days to induce CTLA-4 within the cells as
well as a low level of the cell surface expression. In the normal T
cells, we confirmed that the most CTLA-4-containing vesicles are also
stained with anti-lgp110 Ab similar to WT CTLA-4-transfected T cell
clones (Fig. 7A). Thereafter,
the preactivated T cells were restimulated by TCR cross-linking, and
the surface expressions of CTLA-4 and lgp85 were analyzed. Similar to
the results obtained on Th1 clones, stimulation of splenic
CD4+ T cells induced enhancement of the cell
surface expression of both CTLA-4 and lgp85 (Fig. 7B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we first analyzed the relationship between surface and
intracellular CTLA-4. As previously shown (23), the
majority of CTLA-4 is localized in intracellular large granules.
Despite our expectation that the majority of Y165G mutant would be
expressed on the cell surface, we surprisingly found that even Y165G
mutant T cells accumulate intracellular large granules similarly to
WT-CTLA-4; although the large vesicles containing CTLA-4 were reduced,
they still remained to a significant degree in these Y165G-expressing
cells. These data suggest that there is a mechanism other than the
tyrosine motif/AP-2 system for internalizing and accumulating CTLA-4 in
the large granules. In contrast, we found that large granules had
almost disappeared in CP7 transfectants. These results suggest a
tyrosine-independent sorting signal within the cytoplasmic domain of
CTLA-4 for targeting to lysosomes in addition to the tyrosine signals
responsible for endocytosis through AP-2-binding. This feature of
CTLA-4 resembles the regulation of FasL; the cytoplasmic tail of FasL
may contain specific signal(s) for targeting to the lytic granules
(26). Furthermore, considering that Y165G and Y165/182G
mutant cells still possess CTLA-4-containing large granules within the
cells, and that because the tyrosine phosphorylation of Y165, a
physiologically resembling form of Y165G mutation, results in
dissociation from AP-2 and induction of high expression of cell surface
CTLA-4, these data suggest that the majority of the CTLA-4 protein is
localized inside the cell even under the condition of maximal CTLA-4
expression on the surface of activated T cells (12, 28).
Confocal microscopic analysis revealed that CTLA-4 is localized in two distinct compartments, a majority in large granules and a minority in small vesicles. Because these small vesicles containing CTLA-4 were partially overlapped with Rab5a, a part of CTLA-4 was also localized in early endosomes, which is consistent with the endocytosis of CTLA-4. Although it has been generally believed that CD4+ T cells do not possess typical secretory granules, unlike CTL, NK cells, or granulocytes, we found that the large granules containing CTLA-4 were stained with various lysosome-specific markers, particularly for lgp110 and the active form of cathepsin D, and that they were secreted upon TCR stimulation. It has been described that "secretory lysosomes" are present in hemopoietic lineage cells and, further, in the case of CTLs, cell activation induces extracellular release of hydrolytic enzymes present within the lysosomes toward the target cells, which might be one of the cytotoxic mechanism (29). Indeed, it has been shown that CTLA-4 is partly colocalized with lytic granules and endocytic compartments in CD8+ T cells (12).
Similar to these previous observations in CD8+ T cells, we observed that CTLA-4-expressing CD4+ T cells induced the cell surface expression of lgp85 upon T cell activation similarly to FasL. It was recently shown that the surface expression of CD63 was increased by Ca2+ ionophore treatment in T cells (26). However, because these marker proteins such as lgp110, lgp85, and CD63 are present in both lysosomes and late endosomes, it would be premature to conclude definitively that the increased expression of these markers reflects lysosomal secretion. In this study, therefore, we measured the activity of cathepsin D released into the culture medium because the active form of cathepsin D is exclusively present in lysosomes. The activity of cathepsin D was detected in the culture medium, in parallel with the increase of lysosomal markers and CTLA-4 on the cell surface upon TCR stimulation. Therefore, these observations demonstrated for the first time to our knowledge that the activation-induced secretion of lysosomes can be induced in CD4+ T cells. This may also partly explain the cytotoxic mechanism by CD4+ Th cells. These vesicles in CD4+ T cells contain CTLA-4 and are secreted upon T cell activation. Furthermore, we observed that CTLA-4-containing large granules moved to the proximity of the cell surface upon TCR engagement, which is consistent with the previous observation that CTLA-4 is localized toward the TCR engagement sites upon stimulation (12). The importance of the secretory granules for CTLA-4 expression has been recently suggested by the analysis of CD8+ T cells from patients with Chediak-Higashi syndrome (30). These cells failed to express CTLA-4 on the cell surface and resulted in lymphoproliferative disorder, whereas it is accumulated in the perforin+ giant secretory granules, suggesting that trafficking from the secretory granule is crucial for the surface expression of CTLA-4.
Most of our experiments were performed on various CTLA-4-transfected CD4+ Th clones that represent preactivated phenotype of T cells because these cells already accumulate intracellular CTLA-4 in lysosomes. In contrast, resting T cells do not have CTLA-4 within the cells. Upon TCR stimulation, CTLA-4 transcripts and then protein are synthesized and accumulated in intracellular lysosomes. Therefore, TCR stimulation of preexisting CTLA-4-containing lysosomes to induce secretion in Th clones is equivalent to restimulation of Con A-stimulated naive splenic T cells. We clearly demonstrated that lysosomal secretion was induced under such physiological condition.
It was recently reported that CTLA-4 also interacted with AP-1, which regulates trafficking from the Golgi to the lysosomal compartments (31). A part of CTLA-4 may be transported from the Golgi directly to lysosomes without targeting to the cell surface, although the exact contribution of each pathway needs to be determined. CTLA-4 transported to lysosomes either directly from the Golgi or by endocytosis from the cell surface was degraded in a short period. Because inhibition of this degradation resulted in an increase in the surface expression of CTLA-4, degradation of CTLA-4 in lysosomes appears to be one of the regulatory mechanisms for the surface expression of CTLA-4. In addition to the down-regulation by degradation in lysosomes, lysosomal targeting of CTLA-4 has another advantage, in that activation-induced secretion of lysosomes can be utilized for regulating the surface expression of CTLA-4. Therefore, in combination with the previously described AP-2-mediated endocytosis of the cell surface CTLA-4, two additional mechanisms for regulating the cell surface expression of CTLA-4, degradation and lysosomal secretion, have been uncovered in this study. Furthermore, we previously found that tyrosine-phosphorylation of Y165 prevents AP-2-mediated endocytosis and keeps CTLA-4 on the cell surface (21). Because the level of tyrosine phosphorylation of CTLA-4 was very low, it is possible that tyrosine phosphorylation is induced only on the cell surface. Together with our recent finding that negative signals through CTLA-4 can be delivered in the absence of these tyrosines (23), phosphorylation may serve primarily to maintain CTLA-4 on the cell surface for signal transduction 32 .
The molecular mechanism of lysosomal secretion is largely unknown. Similar to degranulation in mast cells and basophilic cells upon stimulation with Ca2+ ionophore, CTLA-4-containing lysosomes were also secreted upon Ca2+ ionophore stimulation within as quick as 10 min, as demonstrated by secretion of cathepsin D. Similarly, the surface expression of CTLA-4 and lgp85 were significantly increased upon Ca2+ ionophore stimulation during such short period. However, it takes more than 10 h to reach the maximum level of the surface expression of CTLA-4 and lgp85 in CD4+ T cells. Therefore, it might be possible that some unique mechanism is involved to induce lysosomal secretion in addition to the increase of intracellular Ca2+ concentration.
As a summary, our current results together with previous observations allow us to make a new proposal concerning the dynamics of the sorting/expression regulation of CTLA-4. CTLA-4 may be mainly targeted to lysosomes and accumulated during the early stage of T cell activation. Although some proportion of CTLA-4 may be delivered to the cell surface, CTLA-4 present on the cell surface is immediately endocytosed by association with AP-2, keeping cell-surface CTLA-4 at a low level. CTLA-4 in lysosomes would be degraded in the absence of further stimulation, whereas CTLA-4-containing lysosomes may move toward the site of TCR engagement and be secreted upon stimulation, resulting in an increase of the cell surface CTLA-4. CTLA-4 would be kept on the cell surface upon tyrosine phosphorylation, possibly be induced by ligand binding, whereas it would be endocytosed by AP-2 without phosphorylation. Endocytosed CTLA-4 must be targeted to lysosomes through early endosomes. This scenario looks appealing, but we still have to clarify the role of ligand binding, signaling for moving/secreting lysosomes, the contribution of recycling, and also the mechanism of negative signaling.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takashi Saito, Department of Molecular Genetics, Chiba University Graduate School of Medicine, 1-8-1 Inohana, Chuo-ku, Chiba 260-8670, Japan.
3 Abbreviations used in this paper: WT, wild type; ß-hex, ß-hexosaminidase; CHX, cycloheximide; lgp, lysosomal glycoprotein; FasL, Fas ligand.
Received for publication April 12, 2000. Accepted for publication July 31, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/CD3 complex, but not CD28, interact with clathrin adaptor complexes AP-1 and AP-2. J. Immunol. 163:1868.This article has been cited by other articles:
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 Y. Koguchi, T. J. Thauland, M. K. Slifka, and D. C. Parker Preformed CD40 ligand exists in secretory lysosomes in effector and memory CD4+ T cells and is quickly expressed on the cell surface in an antigen-specific manner Blood, October 1, 2007; 110(7): 2520 - 2527. [Abstract] [Full Text] [PDF]
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K. I. Mead, Y. Zheng, C. N. Manzotti, L. C. A. Perry, M. K. P. Liu, F. Burke, D. J. Powner, M. J. O. Wakelam, and D. M. Sansom Exocytosis of CTLA-4 Is Dependent on Phospholipase D and ADP Ribosylation Factor-1 and Stimulated during Activation of Regulatory T Cells J. Immunol., April 15, 2005; 174(8): 4803 - 4811. [Abstract] [Full Text] [PDF]
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F. Maszyna, H. Hoff, D. Kunkel, A. Radbruch, and M. C. Brunner-Weinzierl Diversity of Clonal T Cell Proliferation Is Mediated by Differential Expression of CD152 (CTLA-4) on the Cell Surface of Activated Individual T Lymphocytes J. Immunol., October 1, 2003; 171(7): 3459 - 3466. [Abstract] [Full Text] [PDF]
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 C. C. Wreden, J. Johnson, C. Tran, R. P. Seal, D. R. Copenhagen, R. J. Reimer, and R. H. Edwards The H+-Coupled Electrogenic Lysosomal Amino Acid Transporter LYAAT1 Localizes to the Axon and Plasma Membrane of Hippocampal Neurons J. Neurosci., February 15, 2003; 23(4): 1265 - 1275. [Abstract] [Full Text] [PDF]
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 S. Chikuma and J. A. Bluestone CTLA-4: Acting at the Synapse Mol. Interv., July 1, 2002; 2(4): 205 - 208. [Abstract] [Full Text] [PDF]
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 K. E. Lukong, V. Seyrantepe, K. Landry, S. Trudel, A. Ahmad, W. A. Gahl, S. Lefrancois, C. R. Morales, and A. V. Pshezhetsky Intracellular Distribution of Lysosomal Sialidase Is Controlled by the Internalization Signal in Its Cytoplasmic Tail J. Biol. Chem., November 30, 2001; 276(49): 46172 - 46181. [Abstract] [Full Text] [PDF]
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