Augmentation of CTLA-4 expression by wortmannin: involvement of lysosomal sorting properties of CTLA-4

Shinji Oki, Takao Kohsaka and Miyuki Azuma

Department of Immunology, National Children's Medical Research Center, 3-35-31 Taishido, Setagaya-Ku, Tokyo 154-8509, Japan

Correspondence to: M . Azuma


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CTLA-4 (CD152) is transiently induced on the cell surface of activated T cells and expression is limited at a low level. In this study, we investigated the possibility that phosphatidylinositol 3 kinase (PI 3-K) and other related PI kinases associated with the cytoplasmic domain of CTLA-4 are involved in intracellular trafficking and sorting of CTLA-4 protein. Treatment with micromolar concentrations of wortmannin (WN) for >4 h enhanced both cell-surface and intracellular CTLA-4 without affecting its transcriptional activities in a murine mastocytoma cell line transfected with the human CTLA-4 gene and normal activated CD4+ T cells. However, a more specific PI 3-K inhibitor, LY294002, failed to affect CTLA-4 expression, indicating that the action of WN is independent of conventional PI 3-K activities. WN down-regulated specific association of CTLA-4 with adaptor proteins and its endocytosis. The fact that lysosomotropic agents, ammonium chloride and monensin, enhanced CTLA-4 expression suggests that WN may also block lysosomal sorting and consequent degradation of CTLA-4. Co-localization of CTLA-4 and lysosome-associated membrane protein-1 detected by immunofluorescence microscopy indicates the actual lysosomal sorting of CTLA-4. Our data suggest the existence of WN-sensitive enzymes, which promote lysosomal sorting of CTLA-4. In addition to rapid endocytosis by clathrin-associated adaptor complex, a prompt sorting of CTLA-4 to lysosomes may be one of the regulatory mechanisms for managing CTLA-4 signals in intracellular trafficking pathways.

Keywords: CTLA-4 expression, endocytosis, lysosomal trafficking, post-translational regulation


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
CTLA-4 (CD152) and CD28 are T cell surface molecules that share extensive structure and sequence similarity, and compete for common ligands, CD80 (1) and CD86 (2) on antigen-presenting cells. In contrast to CD28 which provides a critical co-stimulatory signal for T cell activation, CTLA-4 functions as a negative regulator of T cell activation (37), and may play an important role for the induction and maintenance of peripheral T cell tolerance (8,9). Despite the important role of CTLA-4 in regulation of T cell activation, cell-surface expression is strictly regulated at both transcriptional (10) and post-transcriptional levels. CTLA-4 is transiently expressed only after T cell stimulation (11) and their cell-surface expression level is limited by the intracellular localization motif (YXXM) in the cytoplasmic tail of CTLA-4 which can function to prevent the cell-surface accumulation of translated CTLA-4 in activated T cells (12,13). Although this intracellular accumulation was initially believed to be due to retention of CTLA-4 in the trans-Golgi compartment, recent reports demonstrated that unphosphorylated tyrosine of the intracellular localization motif interacted with AP50, the medium chain of the clathrin-associated coated pit adaptor complex AP-2 which causes rapid internalization of CTLA-4 from the cell surface (1416). Interestingly, the SH2-containing tyrosine phosphatase (SHP)-2 binds to the phosphorylated tyrosine in the same motif, resulting in induction of signal transduction (17). This evidence indicates that both surface expression and signal transduction could be controlled by the phosphorylation state of the single tyrosine residue in the cytoplasmic tail. The association of CTLA-4 with SHP-2 appears to be responsible for delivering the negative signal in T cell activation (17). In addition to SHP-2, phosphatidylinositol 3 kinase (PI 3-K) subunit p85 has been shown to be capable of binding to the phosphorylated YXXM motif, although the functional importance of PI 3-K in CTLA-4 signals has not been identified (18). In contrast, examination of the CD28 cytoplasmic tail showed the presence of a putative binding motif (YMXM) for the SH2 domains of PI 3-K and CD28 binding to PI 3-K is likely to be of major importance to CD28-mediated T cell activation (1921).

In the case of growth factor receptors, PI 3-K and their 3-phosphoinositide products were initially identified as components of intracellular signaling pathways for endocytosis of cell-surface receptors (22,23). Recent studies have implicated a new role for PI 3-K and 3-phosphoinositides in intracellular protein sorting at specific steps of the trans-Golgi network (TGN)–endosomal–pre-lysosomal system based on the findings from yeast systems and a mammalian PI 3-K (24,25). The involvement of PI 3-K in endocytosis and intracellular protein sorting of CTLA-4 after the ligand-mediated internalization through clathrin-coated pits and vesicles has not been investigated. In this study, we investigate a possible link between intracellular trafficking of CTLA-4 protein and PI kinases using a murine P815 cell line transduced with human CTLA-4 gene and activated normal human T cells expressing CTLA-4.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antibodies and other reagents
Monoclonal anti-CTLA-4 (11D4, 10A8, mouse IgG1) antibody was kindly provided by Dr P. S. Linsley (Bristol-Myers Squibb, Seattle, WA). Biotinylated-anti-CTLA-4 mAb (BNI3, mouse IgG2a) and FITC-conjugated anti-mouse lysosome-associated membrane protein (LAMP)-1 (CD107a) mAb (1D4B, rat IgG2a) were obtained from PharMingen (San Diego, CA). Rhodamine Red -X-conjugated streptavidin was purchased from Molecular Probes (Eugene, OR). Polyclonal goat anti-CTLA-4 antibody and monoclonal anti-adaptin ß were from Santa Cruz Laboratories (Santa Cruz, CA) and Transduction Laboratories (Lexington, KY) respectively. Wortmannin (WN), LY294002 and monensin were obtained from Sigma (St Louis, MO).

Vectors and transfection
Full-length human CTLA-4 cDNA was obtained from anti-CD3-activated peripheral blood mononuclear cells (PBMC) by using RT-PCR with the combination of sense primer 5'-GATCCTCGAGATGGCTTGCCTTGGATTTCA-3' and antisense primer 5'-GATCGCGGCCGCTCAATTGATGGGAATAAA-3', and was cloned into the XhoI- and NotI-digested BCMGShygro expression vector containing a hygromycin resistance gene (26). Nucleotide sequences of the obtained cDNA were confirmed by dideoxy method employing 373A Autosequencer (Perkin-Elmer, Rockville, MD). Human CD28 cDNA (generously provided by L. L. Lanier; DNAX, Palo Alto, CA) was subcloned into the pBJ expression vector containing a neomycin resistance gene (27).

P815, a murine mastocytoma cell line, was obtained from ATCC (Rockville, MD) and cultured in RPMI 1640 supplemented with L-glutamine, gentamycin and 10% FCS. P815 cells were transfected with 15 µg hCTLA-4/BCMGShygro plasmid by electroporation as described previously (28). After drug selection with 0.3 mg/ml of hygromycin B (Wako Pure Chemical Industries, Osaka, Japan), CTLA-4-transfected cells were cloned and selected for the highest cell-surface expression of CTLA-4 by flow cytometry. P815 cells expressing a high level of CTLA-4 (CTLA4-P815) were further co-transfected with 15 µg of human CD28-pBJ plasmid and were selected in culture medium containing 0.5 mg/ml G418 (Wako). After drug selection, cells expressing both CTLA-4 and CD28 were cloned, and stable surface expression of CTLA-4 and CD28 was confirmed by flow cytometry.

Cell-surface and intracellular staining for flow cytometry and fluorescence microscopy
Cell-surface staining was performed at 4°C in staining buffer (PBS containing 1% FCS and 0.1% sodium azide). For staining of intracellular CTLA-4, cells were fixed for 20 min at 4°C in fixation buffer (4% EM grade paraformaldehyde/PBS). After washing with staining buffer, cells were stained with either phycoerythrin (PE)-conjugated anti-CTLA-4 or PE-conjugated mouse IgG2a control mAb for 30 min at 4°C in 50 µl of permeabilization buffer (PBS containing 1% FCS, 0.1% sodium azide and 0.1% saponin). After washing, stained cells were analyzed on a FACScan or FACSort (Becton Dickinson, San Jose, CA) supported by CellQuest software. For measurement of incorporated anti-CTLA-4 mAb as an endocytic activity, WN-treated or untreated cells were incubated with PE-conjugated anti-CTLA-4 mAb at 37°C. Every 30 min, aliquots (200 µl) of stained cells were transferred to new tubes with 4 ml of pre-chilled acidic elution buffer (50 mM glycine–HCl, 150 mM NaCl, pH 3.0) to remove surface-bound mAb. After washing by centrifugation, cells were analyzed for fluorescence intensity by flow cytometry.

For demonstrating intracellular localization by immunofluorescence microscope, WN-treated or non-treated CTLA-4-P815 cells were fixed with fixation buffer, washed with staining buffer and then incubated with biotinylated anti-CTLA-4 mAb for 30 min in permeabilization buffer as described in intracellular staining. Cells were washed extensively and then incubated with FITC-conjugated anti-mouse LAMP-1 mAb and Rhodamine Red-X-conjugated streptavidin for 30 min. After washing, fluorescent images were obtained using a confocal laser scanning microscope (GB-200; Olympus, Tokyo, Japan) equipped with a triple line Kr–Ar laser with excitation at 488 nm and detection at 500–530 nm bandpass for FITC and >590 nm for Rhodamine Red-X. Two optical sections were overlaid to provide two-color images. Data were presented using Photoshop software (Adobe, Mountain View, CA).

Detection of CTLA-4 transcription by RT-PCR
Total RNA was extracted from CTLA-4-P815 cells treated with WN for the indicated period using Trizol (Gibco/BRL, Rockville, MD). First-strand cDNA was synthesized using oligo(dT) primer and Superscript II reverse transcriptase (Gibco/BRL) from 4 µg of RNA sample. For PCR, serially diluted cDNA products were amplified in PCR reaction buffer containing 1.25 µM each of 5' and 3' primers, 200 µM dNTP and 1 U of Taq DNA polymerase (Takara Shuzo, Shiga, Japan). The primer sequences for CTLA-4 were 5'-ATGGCTTGCCTTGGATTTCAG-3' (5' primer) and 5'-TCAATTGATGGGAATAAAATA-3' (3' primer) and those for ß-actin were 5'-GAGGGAAAT- CGTGCGTGACATCAA-3' (5' primer), and 5'-GGAACCGCTCGTTGCCAATAGTGA-3' (3' primer). PCR was performed on a DNA thermal cycler (Perkin-Elmer) for 40 cycles (94°C for 1 min, 55°C for 1 min and 72°C for 2 min) followed by a 15 min extension at 72°C. The PCR products were electrophoresed on 1% agarose gel for CTLA-4 and 2.5% for ß-actin. The gel was stained with ethidium bromide and photographed under UV light.

Immunoprecipitation and immunoblotting
The WN-treated cells were solubilized in NP-40 lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 1 mM Na3VO4, 2 mM PMSF, 10 µg/ml of leupeptin and 1 µg/ml of aprotinin). The clarified lysates were precleared with protein A-conjugated Sepharose beads (Pharmacia Biotech K.K., Tokyo, Japan) for 60 min at 4°C and then immunoprecipitated for 2 h with anti-CTLA-4 mAb-coupled Protein A–Sepharose beads. After washing and elution with Laemmli's SDS sample buffer, precipitates were separated by SDS–PAGE and proteins were transferred to PVDF membranes. After blocking with 5% skim milk in PBS containing 0.05% Tween 20 (T-PBS), blots were probed with the indicated antibodies, followed by the addition of horseradish peroxidase-conjugated secondary antibody and developed with ECL (Amersham, Little Chalfont, UK). In the case of reprobing, the blots were incubated for 30 min at 50°C in a stripping solution containing 62.5 mM Tris–HCl (pH 6.7), 100 mM 2-mercaptoethanol and 2% SDS, followed by washing in T-PBS.

Isolation of peripheral blood CD4+ T cells
Peripheral blood from healthy adult donors was obtained from the Japanese Central Red Cross Blood Center (Tokyo). PBMC were obtained by density gradient centrifugation. Adherent cells were depleted by using adherence to plastic and then CD4+ T cells were isolated by positive selection using anti-CD4 coated Dynabeads (M-450) and DetachaBeads (Dynal, Oslo, Norway), according to the manufacturer's recommendation. Isolated cells were analyzed by flow cytometry and purity >97% was confirmed. CD4+ T cells were stimulated with a combination of immobilized anti-CD3 (OKT3, 5 µg/ml) and soluble anti-CD28 (2 µg/ml) mAb for 5 days. Then cells were washed and incubated with culture medium containing the indicated concentrations of WN or vehicle control at 37°C.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Enhancement of cell-surface CTLA-4 expression on P815 transfectants by WN treatment
PI 3-K is known to be associated with cytoplasmic tails of CD28 and CTLA-4. To investigate a possible link between CD28/CTLA-4 signals and regulation of surface expression by the PI 3-K, we have examined the effects of WN, which is a popular inhibitor for PI 3-K and related PI kinases, on cell-surface expression of CTLA-4. We established a stable P815 transfectant expressing both CD28 and CTLA-4. The transfectant was treated with serially titrated amounts (0.001–10 µM) of WN for 18 h, and the change in cell-surface expression of CTLA-4 and CD28 was assessed by flow cytometry. As shown in Fig. 1Go(A and B), CTLA-4 expression was significantly enhanced at >1 µM of WN, while CD28 expression was hardly affected. CTLA-4 expression at the highest concentration of 10 µM increased to >20-fold in the fluorescence intensity. As the long-term treatment with high concentrations of WN caused a significant reduction in cell viability, we have determined the optimal incubation time without affecting cell viability. The augmentation of cell-surface CTLA-4 expression was obvious after 6–8 h and cell viability was kept >90% even at high concentrations (2 and 10 µM) of WN (Fig. 2Go). We, therefore, selected treatment for 6–8 h in the following experiments.



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1. Effect of WN on cell-surface expression of CTLA-4 and CD28. P815 transfectants expressing both CD28 and CTLA-4 were cultured with the indicated amounts of WN for 18 h, and then stained with either FITC-conjugated anti-CTLA-4 or anti-CD28 mAb. Viable cells were analyzed by flow cytometry using PI exclusion. (A) Filled histograms and open histograms represent expressions of CTLA-4 and CD28 respectively. Histograms of cells stained with FITC-conjugated mouse IgG1 control mAb are superimposed (dotted line). (B) Cell-surface expression of CTLA-4 (solid column) and CD28 (open column) is presented by mean fluorescence intensity (MFI). Open circles represent percent cell viability. Percent cell viability was calculated as: (total counts – the count for PI+ cells)/total countsx100. Similar results were obtained in three independent experiments.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2. Kinetic change of surface CTLA-4 expression by WN treatment. CTLA-4-P815 cells were cultured in the presence of 2 (A) and 10 (B) µM of WN for the indicated period, and then cells were stained with FITC-conjugated anti-CTLA-4 mAb. Samples were analyzed as described in Fig. 1Go. Data at each incubation time are presented as MFI (solid columns) and percent cell viability (open circles). Similar results were obtained in two independent experiments.

 
To investigate whether up-regulation of surface CTLA-4 expression resulted from the change in a transcriptional level, we performed a semi-quantitative analysis for CTLA-4 mRNA by using RT-PCR. We did not observe a clear change in CTLA-4 transcriptions by 10 µM of WN treatment (Fig. 3Go). This result suggests that the augmentation of cell-surface expression by WN treatment may be due to post-transcriptional modification at a protein level.



View larger version (51K):
[in this window]
[in a new window]
 
Fig. 3. Effect of WN on transcriptional activity of CTLA-4. CTLA-4-P815 cells were treated with 10 µM of WN for indicated periods and then total RNAs were isolated. RT-PCR for CTLA-4 and ß-actin was performed as described in Methods. The sizes of amplified products were 672 and 152 bp for CTLA-4 and ß-actin respectively.

 
Augmentation of cell-surface expression of CTLA-4 is independent of p85-p110 PI 3-K
Although WN was originally discovered as an inhibitor of myosin light chain kinase (29), it was revealed that WN possessed a relative wide target spectrum for several enzymes including p85-p110 PI 3-K. Therefore we next used a more specific inhibitor for p85-p110 PI 3-K, LY294002. Unexpectedly, LY294002 did not affect cell-surface CTLA-4 expression when we increased the concentrations of LY294002 up to 100 µM, which was an amount optimal to inhibit PI 3-K activity (30) (Fig. 4AGo). The inhibitory activity of LY294002 used in our assay was confirmed by an efficient inhibition in the CD28-dependent cytotoxicity by YT cells, which is known to require PI 3-K activation (31,32) (Fig. 4BGo).



View larger version (17K):
[in this window]
[in a new window]
 
Fig. 4. Effect of LY294002 on cell-surface expression of CTLA-4. (A) CTLA-4-P815 cells treated with LY294002 for 8 h were stained with FITC-conjugated anti-CTLA-4 mAb. Samples were analyzed by flow cytometry and data are presented as described in Fig. 2Go. Similar results were obtained in two independent experiments. (B) Inhibitory activity of LY294002 was assessed by the inhibitory effect in CD28-dependent cytotoxicity of YT cells. YT cells were incubated with 51Cr-labeled JY cells in the presence of the titrated amount of LY294002 (6.25–100 µM) for 4 h and cytotoxicity was measured by a standard 51Cr-release assay. E:T ratio was 25:1.

 
WN treatment down-regulates specific association of adaptor protein to CTLA-4 and inhibits endocytosis
We next investigated the correlation between cell-surface CTLA-4 expression and down-regulation of endocytosis by WN treatment. CTLA-4-P815 cells treated with a various concentration of WN for 6 h were reacted with fluorochrome-conjugated anti-CTLA-4 mAb for the indicated time and then cells were washed with acidic buffer to elute surface-bound mAb. The internalized fluorescence was measured by flow cytometry. As shown in Fig. 5Go(A), treatment with WN partially inhibited endocytosis of CTLA-4 even at lower concentrations (0.1–0.3 µM) than that required for an effective cell-surface induction of CTLA-4. The experiments in using 4 and 8 h treatment gave similar results (data not shown). We next assessed the protein amounts of adaptin ß which bind to CTLA-4 in WN-treated or untreated CTLA-4-P815 cells. Similar to the results of internalization, adaptin complexes associated with CTLA-4 were decreased even at the lower concentration (0.4 µM) of WN, while the amount of CTLA-4 protein itself was not significantly changed by the treatment (Fig. 5BGo). These data suggest that the inhibition of endocytosis is only a part of the reason for the up-regulation of cell-surface CTLA-4 by WN.



View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5. Internalization of CTLA-4 and association of adaptor complexes to CTLA-4 by WN treatment. (A) CTLA-4-P815 cells were treated with indicated amounts of WN for 6 h. Treated cells were stained with PE-conjugated anti-CTLA-4 mAb at 37°C for an indicated period and then immediately washed with pre-chilled acidic buffer. Samples were analyzed by flow cytometry as described above. To confirm the complete elution for surface-bound mAb, the aliquots of untreated or WN-treated CTLA-4-P815 cells were stained with PE-conjugated anti-CTLA-4 at 4°C for 20 min, transferred to acidic elution buffer and then analyzed for the surface-bound mAb. After washing, MFI for WN-untreated cells was reduced from 27.0 to 4.3 and MFI for WN-treated cells were reduced from 23.0–49.9 to 4.5–6.4. (B) CTLA-4-P815 cells treated with the indicated amounts of WN for 8 h were lysed and immunoprecipitated with anti-CTLA-4 mAb. Each anti-CTLA-4 precipitate from 2x106 cells was electrophoresed and immunoblotted with anti-adaptin ß antibody. Whole-cell lysate from CTLA-4-P815 cells was used as positive control. The blot was stripped and reprobed with anti-CTLA-4 antibody. Arrow indicates the position of adaptin ß and CTLA-4.

 
WN impairs lysosomal degradation of CTLA-4
To further clarify the mechanisms for up-regulation of CTLA-4 expression by WN, both cytoplasmic CTLA-4 and cell-surface CTLA-4 were comparatively analyzed by flow cytometry. As shown in Fig. 6Go, the amount of intracellular CTLA-4 was increased in proportion to cell-surface expression of CTLA-4 by WN treatment, suggesting the impaired degradation of CTLA-4 by WN. We, therefore, examined the effect of two lysosomotropic agents, ammonium chloride and monensin, both of which alter vacuolar pH and impair lysosomal degradation of proteins, on intracellular and cell-surface CTLA-4. Intracellular and cell-surface amount of CTLA-4 were significantly augmented by either treatment in a dose-dependent manner (Fig. 7Go). These results suggest that WN may act on degradation of proteins in lysosomes, presumably by inducing the mis-sorting of CTLA-4.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6. The change of cell-surface and intracellular expression of CTLA-4 by WN treatment. CTLA-4-P815 cells were treated with 2 (open column) or 10 (solid column) µM of WN for the indicated period, and then stained for cell-surface and cytoplasmic expression of CTLA-4 using PE-conjugated anti-CTLA-4 mAb as described in Methods. Samples were analyzed by flow cytometry. Data are presented as MFI. Similar results were obtained in three independent experiments.

 


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 7. Effect of lysosomotropic reagents on cell-surface and intracellular expression of CTLA-4. CTLA-4-P815 cells were treated with either ammonium chloride or monensin for 24 h, and cell-surface (open column) and cytoplasmic (solid column) staining was performed with PE-conjugated anti-CTLA-4 mAb as described in Methods. Samples were analyzed by flow cytometry and data are presented as MFI. Similar results were obtained in two independent experiments.

 
To directly detect the subcellular localization of CTLA-4, we examined the distribution of CTLA-4 using a confocal microscope. LAMP-1 was used as a major glycoprotein in the lysosome membrane (33). As shown in Fig. 8Go, the punctate distributions for LAMP-1 (Fig. 8a and dGo) and CTLA-4 (Fig. 8b and eGo) in the cytoplasm have been shown in red and green in the individual images respectively. Co-localization of two proteins, LAMP-1 and CTLA-4, is shown in yellow in the merged images (Fig. 8c and fGo). The total amount of CTLA-4 expression seems to be augmented in the WN-treated cells. These results indicate that CTLA-4 could be actually distributed in lysosomal compartments.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 8. Subcellular distribution of CTLA-4. CTLA-4-P815 cells were incubated in the presence or absence of WN (10 µM) for 6 h. The untreated (a–c) or WN-treated (d–f) cells were then fixed, permeabilized, stained and analyzed by confocal microscope. The cells were either optically sectioned in 1.6 µm slices. LAMP-1 was visualized using FITC-conjugated anti-LAMP-1 mAb (a and d), and CTLA-4 molecules were visualized (red fluorescence) using biotinylated anti-CTLA-4 mAb and Rhodamine Red-X-labeled streptavidin (b and e). Yellow, co-distribution of the two markers (c and f).

 
WN induces CTLA-4 expression in normal CD4+ T cells
To investigate whether the phenomenon observed in P815 transfectants is applicable to normal T lymphocytes, we confirmed the effects of WN on expression of CTLA-4 in CD4+ T cells. To induce CTLA-4 expression, purified CD4+ T cells were stimulated with a combination of immobilized anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) mAb for 5 days, and then incubated in the presence or absence of WN for 16 h. As shown in Fig. 8Go, both cell-surface and intracellular CTLA-4 on activated CD4+ T cells were augmented by WN treatment. Consistent with the results observed in P815 cells, cell-surface expression of CD28 on human activated CD4+ T cells was hardly affected (data not shown). Lysosomotropic ammonium chloride and monensin also increased CTLA-4 expression in both the cytoplasmic and cell surface, as observed in CTLA-4-P815 transfectants (data not shown). Our results indicate that the impaired degradation in lysosomes contributes the up-regulation of CTLA-4 in normal T lymphocytes.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this study, we demonstrated that treatment with micromolar concentrations of WN for >4 h induced both surface and intracellular CTLA-4 protein without affecting its transcriptional activities in a murine mastocytoma cell line transfected with human CTLA-4 gene and normal activated CD4+ T cells. Unlike CTLA-4, CD28 expression on P815 cells (Fig. 1Go) and activated CD4+ T cells (data not shown) was not affected by WN treatment. Recently, a possible involvement of PI 3-K in endocytosis of CD28 and its co-stimulatory activities has been suggested from the synchronized enhancement between CD28–PI 3-K complexes and endocytosis (34). However, no direct evidence for the correlation between enzymatic activity of PI 3-K and endocytosis of CD28 was provided. Although additional studies will be required to clarify this issue, our results consistently showed no effect of WN treatment on CD28 expression.

Here, we have investigated the regulatory mechanism of CTLA-4 expression using murine P815 cells transfected with the human CTLA-4 gene in most of our experiments. At present, we cannot completely negate the possibility that these observations are the specific events in this particular system. However, we confirmed the observation that WN treatment enhanced CTLA-4 expression in three other different types of cells including mouse T lymphoma EL-4 cells stimulated with PMA and ionomycin, human YT cells transfected with human CTLA-4 (data not shown), and human activated CD4+ T cells (Fig. 9Go). We, therefore, believe that this phenomenon observed in our P815 system might be applicable to normal mouse and human T cells.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 9. Up-regulation of CTLA-4 expression by WN in CD4+ T cells. Purified CD4+ T cells were stimulated with a combination of immobilized anti-CD3 (5 µg/ml) and soluble anti-CD28 (2 µg/ml) mAb for 5 days. After washing, activated CD4+ T cells were cultured in the presence or absence of WN for 16 h. For determination of cell-surface CTLA-4, samples were stained with FITC-conjugated anti-CD4 mAb and PE-conjugated anti-CTLA-4 mAb. Viability of CD4+ T cells >90% in each sample was confirmed by using PI exclusion. For detection of cytoplasmic CTLA-4, samples were stained with FITC-conjugated anti-CD4 mAb, fixed and reacted with PE-conjugated anti-CTLA-4 mAb in permeabilization buffer. Expression of CTLA-4 is presented by histograms (left panels, bold lines) and MFI (right panel). Histograms of cells stained with the isotype-matched control antibody are superimposed (plain lines). Data are presented as the mean ± SEM of MFI from six individuals. Statistical significance was determined by Student's t-test. *P < 0.05, **P < 0.01. Similar results were obtained in three independent experiments (data not shown).

 
The target molecules of WN in this event are unlikely to be a conventional p85-p110 PI 3-K, since the concentration and the duration required for CTLA-4 induction are clearly distinct from those required for inhibition of a conventional PI 3-K. Although two structurally distinct inhibitors, WN and LY294002, have been known to block the lipid kinase activity of several PI 3-K isoforms, recent reports have shown that some PI 4-K isoforms (35,36), phospholipase A2 (37) and phospholipase D (38) may be sensitive to WN. The fact that the treatment with LY294002 in the micromolar range had no effect on CTLA-4 expression further supports that the WN-sensitive target in this event may not be p85-p110 PI 3-K but other PI 3-K or WN-sensitive PI 4-K isoforms, although a possible involvement of some other enzymes is not excluded. Recently, the requirement of PI 3-K for the formation of constitutive transport vesicles from the TGN has been demonstrated and the dose of WN required for augmenting cell-surface expression of CTLA-4 is similar to that required for inhibition of a PI 3-K isoform for the formation of TGN-derived exocytic transport vesicles (39). Further studies will be required to clarify the identification of this PI 3-K isoform with WN-sensitive target enzymes for intracellular trafficking of CTLA-4.

PI 3-K and its products are known to have important roles in the intracellular trafficking from one compartment to another. We first examined whether WN acts on endocytosis from the cell surface. A partial reduction in internalization and the loss of CTLA-4-associated adaptor complexes were observed even at 10- to 20-fold less concentration than that required for induction of CTLA-4. Therefore, the augmentation of CTLA-4 expression by high doses of WN is unlikely to be mediated by endocytosis. Furthermore, a comparative analysis for CTLA-4 expression in cell-surface and cytoplasmic levels revealed the parallel augmentation of CTLA-4 protein by WN treatment. These results suggest that the high dose of WN-sensitive enzymes may not contribute to endocytosis of CTLA-4 from the cell surface and prompted us to investigate other intracellular trafficking events.

Martys et al. (40) reported the presence of WN-sensitive enzymes at three distinct steps in intracellular trafficking pathways using Chinese hamster ovary cells: (i) internalization, (ii) transit from early endosomes to the recycling and degradative compartments, and (iii) transit from the recycling compartment back to the cell surface. Among these three steps, WN-sensitive enzymes involved in sorting newly synthesized lysosomal enzymes are distinct from enzymes required for endocytosis and recycling. Compared with the amount required to inhibit a conventional PI 3-K activity, >20-fold higher dose of WN is required to induce mis-sorting of the lysosomal enzyme cathepsin D to the secretary pathway. Our results that lysosomotropic agents, ammonium chloride and monensin enhanced CTLA-4 expression at both the cell-surface and cytoplasmic levels further support the notion that WN may act on lysosomal sorting and degradation of CTLA-4 resulting in a late accumulation of CTLA-4 protein. The simultaneous localization of CTLA-4 and a lysosomal marker, LAMP-1, in CTLA-4-P815 cells detected by immunofluorescence detection indicates the actual lysosomal sorting of CTLA-4.

In this study, we examined the change in association of adaptor protein, presumably AP-2 complexes, to CTLA-4 by WN. In addition to AP-2, which mainly acts on transit from the cell surface to early endosomes in the plasma membrane, two other AP complexes in intracellular trafficking have been reported. AP-1 may act on TGN–endosome sorting (41) and AP-3 may act on endosome–lysosome sorting (42,43). Additional studies will be required to identify the involvement of these adaptor protein complexes in WN-mediated CTLA-4 induction.

Recent studies have demonstrated that PI 3-K is specifically activated by CD28 and mediates proximal events in the CD28-mediated signaling pathway (20,21,44). On the contrary, it has been reported that WN partially blocks CD28-induced tyrosine phosphorylation of the putative p110 catalytic subunit of PI 3-K but did not block both CD28-induced association of the p85 binding subunit of PI 3-K with CD28 and CD28-mediated co-stimulation for proliferation and IL-2 production in murine T cells and Jurkat cells (4548). Taub et al. (49) reported antigen-specific T cell tolerance by the treatment of human T cells with WN and the amelioration of murine graft-versus-host disease by the treatment of allogeneic donor lymphocytes with WN. Due to the multiple activities of WN on various signaling pathways, it is difficult to determine whether CTLA-4 protein induced by WN is concerned with the induction of T cell tolerance in these system. It is possible that controversial reports on CD28 signals and PI 3-K using WN (44,46,47,50) may partly result from the CTLA-4 action induced by WN. Recent accumulating observations suggest that CTLA-4 may play a crucial role for induction of peripheral T cell tolerance in vivo (8,9). For managing normal immune responses, CTLA-4 signals may be required to be strictly regulated by multiple pathways. To retain a low expression level of surface CTLA-4, it seems that a prompt sorting of CTLA-4 to lysosomes is one of the important regulatory mechanisms in intracellular levels, in addition to clathrin-mediated rapid endocytosis. In this study, we first demonstrate the existence of WN-sensitive enzymes, which promote lysosomal sorting of CTLA-4 protein. Further studies are required to identify the molecules responsible for lysosomal sorting of CTLA-4 and to clarify a direct contribution of this event for CTLA-4-mediated immune regulation.


    Acknowledgments
 
We thank Dr Peter S. Linsley for generously providing anti-CTLA-4 mAb and Dr Lewis. L. Lanier for providing human CD28 cDNA. We also thank Dr Kazuhiro Ohmi for technical assistance in confocal microscopic analysis. This work was supported, in part, by CREST of Japan Science and Technology Corp. and by grants from the Ministry of Health, Japan.


    Abbreviations
 
LAMPlysosome-associated membrane protein
MFImean fluorescence intensity
PBMCperipheral blood mononuclear cells
PEphycoerythrin
PIpropidium iodide
PI 3-Kphosphatidylinositol 3-kinase
SHPSH2-containing tyrosine phosphatase
TGNtrans-Golgi network
WNwortmannin

    Notes
 
Transmitting editor: K. Okumura Back

Received 1 June 1998, accepted 3 June 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Linsley, P. S., Clark, E. A. and Ledbetter, J. A. 1990. T-cell antigen CD28 mediates adhesion with B cells by interacting with activation antigen B7/BB-1. Proc. Natl Acad. Sci. USA 87:5031.[Abstract]
  2. Azuma, M., Ito, D., Yagita, H., Okumura, K., Phillips, J. H., Lanier, L. L. and Somoza, C. 1993. B70 antigen is a second ligand for CTLA-4 and CD28. Nature 366:76.[ISI][Medline]
  3. Krummel, M. F. and Allison, J. P. 1995. CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. J. Exp. Med. 182:459.[Abstract]
  4. Krummel, M. F. and Allison, J. P. 1996. CTLA-4 engagement inhibits IL-2 accumulation and cell cycle progression upon activation of resting T cells. J. Exp. Med. 183:2533.[Abstract]
  5. Walunas, T. L., Lenschow, D. J., Bakker, C. Y., Linsley, P. S., Freeman, G. J., Green, J. M., Thompson, C. B. and Bluestone, J. A. 1994. CTLA-4 can function as a negative regulator of T cell activation. Immunity 1:405.[ISI][Medline]
  6. Walunas, T. L., Bakker, C. Y. and Bluestone, J. A. 1996. CTLA-4 ligation blocks CD28-dependent T cell activation. J. Exp. Med. 183:2541.[Abstract]
  7. Thompson, C. B. and Allison, J. P. 1997. The emerging role of CTLA-4 as an immune attenuator. Immunity 7:445.[ISI][Medline]
  8. Perez, V. L., Van, P. L., Biuckians, A., Zheng, X. X., Strom, T. B. and Abbas, A. K. 1997. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 6:411.[ISI][Medline]
  9. Bluestone, J. A. 1997. Is CTLA-4 a master switch for peripheral T cell tolerance? J. Immunol. 158:1989.[Abstract]
  10. Perkins, D., Wang, Z., Donovan, C., He, H., Mark, D., Guan, G., Wang, Y., Walunas, T., Bluestone, J., Listman, J., et al. 1996. Regulation of CTLA-4 expression during T cell activation. J. Immunol. 156:4154.[Abstract]
  11. Lindsten, T., Lee, K. P., Harris, E. S., Petryniak, B., Craighead, N., Reynolds, P. J., Lombard, D. B., Freeman, G. J., Nadler, L. M., Gray, G. S., et al. 1993. Characterization of CTLA-4 structure and expression on human T cells. J. Immunol. 151:3489.[Abstract/Free Full Text]
  12. Leung, H. T., Bradshaw, J., Cleaveland, J. S. and Linsley, P. S. 1995. Cytotoxic T lymphocyte-associated molecule-4, a high-avidity receptor for CD80 and CD86, contains an intracellular localization motif in its cytoplasmic tail. J. Biol. Chem. 270:25107.[Abstract/Free Full Text]
  13. Linsley, P. S., Bradshaw, J., Greene, J., Peach, R., Bennett, K. L. and Mittler, R. S. 1996. Intracellular trafficking of CTLA-4 and focal localization towards sites of TCR engagement. Immunity 4:535.[ISI][Medline]
  14. Chuang, E., Alegre, M. L., Duckett, C. S., Noel, P. J., Vander, H. M. and Thompson, C. B. 1997. Interaction of CTLA-4 with the clathrin-associated protein AP50 results in ligand-independent endocytosis that limits cell surface expression. J. Immunol. 159:144.[Abstract]
  15. Shiratori, T., Miyatake, S., Ohno, H., Nakaseko, C., Isono, K., Bonifacino, J. S. and Saito, T. 1997. Tyrosine phosphorylation controls internalization of CTLA-4 by regulating its interaction with clathrin-associated adaptor complex AP- 2. Immunity 6:583.[ISI][Medline]
  16. Zhang, Y. and Allison, J. P. 1997. Interaction of CTLA-4 with AP50, a clathrin-coated pit adaptor protein. Proc. Natl Acad. Sci. USA 94:9273.[Abstract/Free Full Text]
  17. Marengere, L. E., Waterhouse, P., Duncan, G. S., Mittrucker, H. W., Feng, G. S. and Mak, T. W. 1996. Regulation of T cell receptor signaling by tyrosine phosphatase SYP association with CTLA-4. Science 272:1170.[Abstract]
  18. Schneider, H., Prasad, K. V., Shoelson, S. E. and Rudd, C. E. 1995. CTLA-4 binding to the lipid kinase phosphatidylinositol 3-kinase in T cells. J. Exp. Med. 181:351.[Abstract]
  19. Prasad, K. V., Cai, Y. C., Raab, M., Duckworth, B., Cantley, L., Shoelson, S. E. and Rudd, C. E. 1994. T-cell antigen CD28 interacts with the lipid kinase phosphatidylinositol 3-kinase by a cytoplasmic Tyr(P)-Met-Xaa-Met motif. Proc. Natl Acad. Sci. U SA 91:2834.[Abstract]
  20. Pages, F., Ragueneau, M., Rottapel, R., Truneh, A., Nunes, J., Imbert, J. and Olive, D. 1994. Binding of phosphatidylinositol-3-OH kinase to CD28 is required for T-cell signalling. Nature 369:327.[ISI][Medline]
  21. Cai, Y. C., Cefai, D., Schneider, H., Raab, M., Nabavi, N. and Rudd, C. E. 1995. Selective CD28pYMNM mutations implicate phosphatidylinositol 3-kinase in CD86–CD28-mediated costimulation. Immunity 3:417.[ISI][Medline]
  22. Joly, M., Kazlauskas, A., Fay, F. S. and Corvera, S. 1994. Disruption of PDGF receptor trafficking by mutation of its PI-3 kinase binding sites. Science 263:684.[ISI][Medline]
  23. Joly, M., Kazlauskas, A. and Corvera, S. 1995. Phosphatidylinositol 3-kinase activity is required at a postendocytic step in platelet-derived growth factor receptor trafficking. J. Biol. Chem. 270:13225.[Abstract/Free Full Text]
  24. Schu, P. V., Takegawa, K., Fry, M. J., Stack, J. H., Waterfield, M. D. and Emr, S. D. 1993. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260:88.[ISI][Medline]
  25. Shepherd, P. R., Nave, B. T. and O'Rahilly, S. 1996. The role of phosphoinositide 3-kinase in insulin signalling. J. Mol. Endocrinol. 17:175.[Free Full Text]
  26. Karasuyama, H. and Melchers, F. 1988. Establishment of mouse cell lines which constitutively secrete large quantities of interleukin 2, 3, 4 or 5, using modified cDNA expression vectors. Eur. J. Immunol. 18:97.[ISI][Medline]
  27. Lin, A. Y., Devaux, B., Green, A., Sagerstrom, C., Elliott, J. F. and Davis, M. M. 1990. Expression of T cell antigen receptor heterodimers in a lipid-linked form. Science 249:677.[ISI][Medline]
  28. Azuma, M., Cayabyab, M., Buck, D., Phillips, J. H. and Lanier, L. L. 1992. CD28 interaction with B7 costimulates primary allogeneic proliferative responses and cytotoxicity mediated by small, resting T lymphocytes. J. Exp. Med. 175:353.[Abstract]
  29. Nakanishi, S., Kakita, S., Takahashi, I., Kawahara, K., Tsukuda, E., Sano, T., Yamada, K., Yoshida, M., Kase, H. and Matsuda, Y. 1992. Wortmannin, a microbial product inhibitor of myosin light chain kinase. J. Biol. Chem. 267:2157.[Abstract/Free Full Text]
  30. Vlahos, C. J., Matter, W. F., Hui, K. Y. and Brown, R. F. 1994. A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4- morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J. Biol. Chem. 269:5241.[Abstract/Free Full Text]
  31. Lu, Y., Rodriguez, R., Bjorndahl, J., Phillips, C. A. and Trevillyan, J. M. 1996. CD28-dependent killing by human YT cells requires phosphatidylinositol 3-kinase activation. Eur. J. Immunol. 26:1278.[ISI][Medline]
  32. Teng, J. M. C., Liu, X., Wills, G. B. and Dupont, B. 1996. CD28-mediated cytotoxicity by the human leukemic NK cell line YT involves tyrosine phosphorylation, activation of phosphatidylinositol 3-kinase, and protein kinase C. J. Immunol. 156:3222.[Abstract]
  33. Chen, J. W., Murphy, T. L., Willingham, M. C., Pastan, I. and August, J. T. 1985. Identification of two lysosomal membrane glycoproteins. J. Cell Biol. 101:85.[Abstract]
  34. Cefai, D., Schneider, H., Matangkasombut, O., Kang, H., Brody, J. and Rudd, C. E. 1998. CD28 receptor endocytosis is targeted by mutations that disrupt phosphatidylinositol 3-kinase binding and costimulation. J. Immunol. 160:2223.[Abstract/Free Full Text]
  35. Meyers, R. and Cantley, L. C. 1997. Cloning and characterization of a wortmannin-sensitive human phosphatidylinositol 4-kinase. J. Biol. Chem. 272:4384.[Abstract/Free Full Text]
  36. Nakanishi, S., Catt, K. J. and Balla, T. 1995. A wortmannin-sensitive phosphatidylinositol 4-kinase that regulates hormone-sensitive pools of inositolphospholipids. Proc. Natl Acad. Sci. USA 92:5317.[Abstract]
  37. Cross, M. J., Stewart, A., Hodgkin, M. N., Kerr, D. J. and Wakelam, M. J. 1995. Wortmannin and its structural analogue demethoxyviridin inhibit stimulated phospholipase A2 activity in Swiss 3T3 cells. Wortmannin is not a specific inhibitor of phosphatidylinositol 3-kinase. J. Biol. Chem. 270:25352.[Abstract/Free Full Text]
  38. Bonser, R. W., Thompson, N. T., Randall, R. W., Tateson, J. E., Spacey, G. D., Hodson, H. F. and Garland, L. G. 1989. Demethoxyviridin and wortmannin block phospholipase C and D activation in the human neutrophil. Br. J. Pharmacol. 264:617.
  39. Jones, S. M. and Howell, K. E. 1997. Phosphatidylinositol 3-kinase is required for the formation of constitutive transport vesicles from the TGN. J. Cell Biol. 139:339.[Abstract/Free Full Text]
  40. Martys, J. L., Wjasow, C., Gangi, D. M., Kielian, M. C., McGraw, T. E. and Backer, J. M. 1996. Wortmannin-sensitive trafficking pathways in Chinese hamster ovary cells. Differential effects on endocytosis and lysosomal sorting. J. Biol. Chem. 271:10953.[Abstract/Free Full Text]
  41. Robinson, M. S. 1994. The role of clathrin, adaptors and dynamin in endocytosis. Curr. Opin. Cell Biol. 6:538.[ISI][Medline]
  42. Cowles, C. R., Odorizzi, G., Payne, G. S. and Emr, S. D. 1997. The AP-3 adaptor complex is essential for cargo-selective transport to the yeast vacuole. Cell 91:109.[ISI][Medline]
  43. Stepp, J. D., Huang, K. and Lemmon, S. K. 1997. The yeast adaptor protein complex, AP-3, is essential for the efficient delivery of alkaline phosphatase by the alternate pathway to the vacuole. J. Cell Biol. 139:1761.[Abstract/Free Full Text]
  44. Ward, S. G., Wilson, A., Turner, L., Westwick, J. and Sansom, D. M. 1995. Inhibition of CD28-mediated T cell costimulation by the phosphoinositide 3-kinase inhibitor wortmannin. Eur. J. Immunol. 25:526.[ISI][Medline]
  45. Crooks, M. E., Littman, D. R., Carter, R. H., Fearon, D. T., Weiss, A. and Stein, P. H. 1995. CD28-mediated costimulation in the absence of phosphatidylinositol 3- kinase association and activation. Mol. Cell. Biol. 15:6820.[Abstract]
  46. Lu, Y., Phillips, C. A. and Trevillyan, J. M. 1995. Phosphatidylinositol 3-kinase activity is not essential for CD28 costimulatory activity in Jurkat T cells: studies with a selective inhibitor, wortmannin. Eur. J. Immunol. 25:533.[ISI][Medline]
  47. Ni, H. T., Deeths, M. J. and Mescher, M. F. 1996. Phosphatidylinositol 3 kinase activity is not essential for B7-1- mediated costimulation of proliferation or development of cytotoxicity in murine T cells. J. Immunol. 157:2243.[Abstract]
  48. Truitt, K. E., Shi, J., Gibson, S., Segal, L. G., Mills, G. B. and Imboden, J. B. 1995. CD28 delivers costimulatory signals independently of its association with phosphatidylinositol 3-kinase. J. Immunol. 155:4702.[Abstract]
  49. Taub, D. D., Murphy, W. J., Asai, O., Fenton, R. G., Peltz, G., Key, M. L., Turcovski, C. S. and Longo, D. L. 1997. Induction of alloantigen-specific T cell tolerance through the treatment of human T lymphocytes with wortmannin. J. Immunol. 158:2745.[Abstract]
  50. Wilson, A., Sansom, D., Parry, R., Westwick, J. and Ward, S. 1995. The phosphoinositide 3-kinase inhibitor wortmannin inhibits CD28-mediated T cell co-stimulation. Biochem. Soc. Trans. 23:282.[ISI][Medline]