| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
,
Departments of
*
Microbiology and Immunology and
Medicine, and
Howard Hughes Medical Institute, University of California, San Francisco, CA 94143;
§
Department of Medicine, Committee on Immunology and the Gwenn Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; and
¶
Medical Research Council Cellular Immunology Unit, Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Ab. CD134-deficient
mice controlled infection with Leishmania major,
Nippostrongylus brasiliensis, and Theiler’s murine
encephalomyelitis virus, and they made overtly normal Ab responses to a
variety of antigens. Thus, CD134 is not essential for many T cell
responses in vivo, nor is it required for the provision of help to B
cells. Nonetheless, a subtle role in the regulation of T cell
reactivity is suggested by the effect of CD134 deficiency on in vitro T
cell responses. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
B
and AP-1 (7, 12). Mutant mice that lack expression of TNF
receptors, their ligands, or TRAFs show a range of defects in lymphoid
homeostasis and immune responses (3, 5, 6, 13, 14, 15, 16). The CD134 Ag was first identified as a 50-kDa glycoprotein target for the MRC OX40 Ab that was selectively expressed on activated rat CD4+ T cells (17). Mouse and human CD134 show a similar pattern of expression to the rat form, except they are also found on activated CD8+ T cells (18, 19). The ligand for CD134 (CD134L) is a type II trimeric transmembrane protein whose mRNA is markedly induced in human T cell leukemia virus 1-infected cells through the action of the viral tax transactivator (19, 20). Trimeric CD134L binds to cell surface CD134 with high affinity (Kd = 0.2–0.4 nM) and a slow off-rate (koff = 4 x 10-5 s-1) (8).
In several experimental settings, CD134 acts as a costimulator for T cells. For instance, anti-CD134 mAb augments the response of rat T cells activated with allogenic MHC (17) or anti-CD3 (21). Similarly, mouse and human T cells make more robust proliferative and cytokine responses when activated in the presence of transfected cells expressing CD134L (19, 20, 22). This costimulatory activity appears to be synergistic with costimulation through CD28 and is particularly potent in prolonging the responses of differentiated effector T cells (23). Interestingly, a similar type of costimulatory activity has also been described for CD137 (4-1BB), which is a related member of the TNF receptor family that is also induced on activated T lymphocytes (24, 25, 26). Costimulatory signals delivered through CD134 have been implicated in selectively promoting the differentiation of Th2 cells (27, 28, 29), but other studies suggest they may also regulate Th1 development (23, 30, 31). The cytoplasmic tail of CD134 can associate with TRAF2, TRAF3, and TRAF5 and may well mediate its costimulatory effect through these interactions (12, 32, 33). Finally, recent data indicate that like anti-CD137 (34), anti-CD134 signaling can block activation-induced T cell death in mice treated with superantigens (35).
The physiological significance of costimulatory signaling through CD134 has not yet been resolved. CD134 is expressed on inflammatory T cells in vivo, as in the CNS of rodents with experimental allergic encephalomyelitis (36), in the joints of humans with rheumatoid arthritis (37), in the peripheral blood of rats with acute graft-vs-host disease (38), or on tumor-infiltrating lymphocytes (39). CD134 is also expressed on T cells in the intestines of mice with inflammatory bowel disease wherein blockade of the CD134-CD134L interaction can lessen the severity of the disease (40). Cumulatively, these expression and functional data raise the possibility that CD134 signaling may help to prolong Ag-specific proliferative responses or otherwise influence the persistence, differentiation, or reactivation of effector/memory populations.
The mRNA for CD134L is expressed in activated T cells, B cells, and also in the mouse brain and kidney (19). In humans, CD134L mRNA is found in the heart, skeletal muscle, pancreas, testes, and ovary (19). Human dendritic cells express CD134L, and its ligation on these cells induces them to differentiate and secrete cytokines (41). CD134L is also expressed on various types of endothelium, including that from umbilical cord, foreskin, and the aorta (42, 43). Thus, the expression of CD134L would allow for a role as a costimulatory ligand for T cells, but it also raises the possibility that CD134 may regulate the extravasation of activated T cells under certain circumstances.
A possible function for CD134 in the regulation of T cell-dependent B cell responses has been proposed based on the finding that a rabbit anti-mouse CD134 antiserum can inhibit Ag-specific B cell responses in mice (44). Treated mice developed germinal centers in response to trinitrophenyl-keyhole limpet hemocyanin (TNP-KLH) immunization, but they were impaired in their secretion of TNP-specific IgG Abs and they also lacked TNP-reactive B cell foci in the spleen. Nevertheless, these mice made adequate secondary responses, suggesting that the CD134 signal might regulate differentiation into Ig-secreting plasma cells but not memory B cells. The induction of CD134L expression on activated B cells and the provision of a proliferative signal to B cells through it (45) would be consistent with this hypothesis. Costimulation through CD134 has also been implicated in the up-regulation of the chemokine receptor BLR-1 on T cells (29), which could be important for recruiting Ag-specific T cells into germinal centers and thereby influencing Ab responses. However, a unique role for CD134 in this context has not yet been established.
To study the function of CD134 in more detail, we have generated CD134-deficient mice by gene targeting in embryonic stem (ES) cells. The phenotype of these mice suggests that CD134 is not a primary regulator of many T cell responses in vivo, nor is it essential for B cell differentiation and Ab secretion. Nonetheless, in vitro assays of T cell activation suggest that signals from CD134 may help to promote T cell proliferation under certain circumstances.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The isolation of 129/Sv genomic phage 
clones that contained
the cd134 gene has been described previously
(46). These phage clones were further characterized by
subcloning, restriction enzyme, and Southern blot analyses. A targeting
construct was generated by inserting a 3.5-kb
KpnI-SacI fragment that contained exons 5, 6, and
7 into the pL2neo-2 vector (modified from
pL2neo, kind gift from Dr. Hua Gu, National
Institutes of Health, Bethesda, MD). The pL2neo
vector contains a neomycin resistance cassette
(neor) under the control of the MC1
(HSV-thymidine kinase) promoter. Neor and the
CD134 genomic sequence were then reexcised using Hind III
and inserted into pBluescript that contained a 4-kb
KpnI-HindIII upstream fragment of the
cd134 gene.
A total of 2 x 107 RF8 ES cells (47) were transfected with 20 µg of the linearized targeting vector by electroporation using a Bio-Rad Gene Pulser (Bio-Rad, Richmond, CA) set at 500 µF and 250 V. The cells were plated on primary embryonic feeder cells, and G418 selection (200 µg/ml) was applied 36 h after electroporation. Ten days later, individual G418-resistant clones were picked to 96-well plates containing feeder cells. The clones were expanded and then split in half so that a portion of each could be frozen in 96-well plates, and the remaining portion could be expanded for DNA extraction and Southern blot analysis. A probe on the 5' side of the targeted region was used on a BglII digest of the genomic DNA to identify targeted clones. The structure of the mutant allele was subsequently confirmed using a 3' probe on genomic DNA that had been digested with ApaI or BamHI. Approximately 1% of neor colonies were mutant at the cd134 locus. Chimeric mice were produced by injection of mutant ES cells into 3.5-day-old blastocysts according to standard procedures (48). These chimeras were then bred to C57BL/6 females, and germline transmission of the cd134 mutation was confirmed by Southern blot analysis of tail DNA. CD134+/- mice were interbred to produce the mice that were used in the experiments described below.
Flow cytometric analysis
All flow cytometry was performed using a Becton Dickinson FACScan flow cytometer (Becton Dickinson, Mountain View, CA) with CellQuest software Becton Dickinson). Fluorescent mAbs were purchased from PharMingen (San Diego, CA) and Caltag (South San Francisco, CA). The OX86 (18) and OX89 (D. Shipton, A. Al-Shamkhani, M. Puklavek, and A. N. Barclay, unpublished data) mAbs were used as supernatant or purified Ig from the respective hybridomas. OX86 and OX89 are specific for mouse CD134 and CD134L, respectively. Labeling and analysis of cells with 5- (and 6-)carboxyfluorescein diacetate succinimidyl (CFSE; diester Molecular Probes, Eugene, OR) was performed as described previously (49, 50).
In vitro T cell assays
Cell preparation and culture were performed in RPMI 1640
supplemented with 10% FCS, 2 mM L-glutamine, 50 µM 2-ME,
100 µM nonessential amino acids, 50 U/ml penicillin, and 50 µg/ml
streptomycin. Lymph node (LN) cells were isolated from
CD134-/- and control (littermate) mice using a
40 µM nylon cell strainer (Becton Dickinson). To purify
CD4+ T cells, LN cells were incubated at 20
x 106/ml in M5/114 or BP107 supernatant
(anti-I-A mAbs) and 0.5 µg/ml purified 2.43 (anti-CD8 mAb).
After two washes in medium, the LN cells were treated at 37°C with a
1/10 dilution of guinea pig complement (Accurate Chemicals, Westbury,
NY) at 20 x 106/ml for 30 min. After two
additional washes in medium, the cells were rocked at room temperature
for 30 min with sheep anti-mouse Ig and sheep anti-rat
Ig-coated magnetic beads according to the manufacturer’s instructions
(Dynal, Oslo, Norway). Bead-coated cells were then removed from the
culture using a magnet. The purity of the final preparation was
determined by flow cytometry and was typically >90%. For anti-CD3
proliferation assays, 5 x 104 purified
CD4+ LN T cells were incubated in microtiter
wells with 5 x 105 irradiated splenocytes
(2000 rad) from C57BL/6 x 129/Sv mice in the presence of
anti-CD3 mAb (purified 145-2C11) for 1, 2, 3, 4, or 5 days. The
plates were then incubated with 1 µCi/well
[3H]thymidine for 18–24 h. For mixed
lymphocyte responses, 0.5, 1, 2, or 4 x 105
purified CD4+ LN T cells were incubated with
5 x 105 allogenic stimulator cells from
BALB/c (H-2d) or CBA (H-2k)
mice in a volume of 200 µl in flat-bottom 96-well plates for 1, 2, 3,
4, or 5 days.
To confirm the lack of expression of CD134 in the CD134-deficient mice, LN cells from CD134-/- and control mice were stimulated with PMA (1 ng/ml) and ionomycin (500 ng/ml) for 3 days.
Immunizations, ELISAs, and ELISPOT assays
Mice were immunized i.p with T-dependent and T-independent Ags,
including 10 µg TNP-KLH, 10 µg TNP-Ficoll, and 5 µg nitrophenyl
(NP)-OVA (all given in alum). For ELISAs, plates were precoated with
0.5 µg/ml TNP-KLH, TNP-Ficoll, or NP (13)-BSA (Solid
Phase Sciences, San Rafael, CA) in PBS. The plates were blocked with
10% FCS in PBS containing 0.1% (v/v) Tween 20 and then incubated
overnight at room temperature with 2-fold serial dilutions of sera from
CD134-/- or control mice in PBS containing 2%
FCS. TNP plates were washed and then incubated with biotinylated goat
anti-murine IgM, IgG1, IgG2a, IgG2b, IgG3, IgA, or IgE
(PharMingen). This was followed by an additional wash before the
addition of 1 µg/ml HRP coupled to streptavidin (Jackson
ImmunoResearch, West Grove, PA). After 30 min at room temperature, the
plates were washed and then
2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) in a citric
acid/phosphate buffer and 0.003%
H2O2 was added as a
substrate. Detection was performed using a Molecular Dynamics plate
reader (Molecular Dynamics, Sunnyvale, CA) set at 1–2, with
1 = 405 nm and 2 = 490 nm. Results for the TNP-KLH
assays were quantitated by comparison to standard anti-TNP mAbs
(kindly provided by Dr. Robert Coffman, DNAX, Palo Alto, CA). NP ELISAs
were similar except alkaline phosphatase-coupled anti-mouse Ig Ab
(PharMingen) and Sigma 104 substrate (Sigma, St. Louis, MO) were
used.
To measure Abs produced against Theiler’s murine encephalomyelitis virus (TMEV), serial dilutions of sera were incubated for 45 min at 37°C in commercially available ELISA plates coated with TMEV or control Ags (Charles River Breeding Laboratories, Wilmington, MA). The plates were washed and then incubated with alkaline phosphatase coupled anti-IgG Ab (Jackson ImmunoResearch) for 45 min at 37°C. Sigma 104 was used as a substrate as above.
IgE responses were determined as described previously (51). Briefly, ELISA plates were coated with an anti-IgE Ab (B.1E.3) in PBS overnight at 4°C and then blocked with 10% FCS in PBS/Tween 20. After several washes in PBS/Tween 20, serial dilutions of the sera were added and the plates were incubated at room temperature. The plates were washed again and incubated with a secondary biotinylated anti-IgE Ab (EM-95). After another round of washes, the plates were incubated with alkaline phosphatase coupled to streptavidin (The Jackson Laboratory, Bar Harbor, ME). Sigma 104 substrate was added after a final wash.
For enzyme-linked immunospot (ELISPOT) assays, ELISA plates were coated with anti-IL-4 Ab (11B11) in PBS overnight at 4°C. The plates were then blocked with 10% FCS in PBS before incubation at 37°C overnight with cells (2-fold serial dilutions starting at 1 x 105). They were then washed again immediately before separate incubations with a secondary anti-Il-4 Ab (BUD6), followed by streptavidin-alkaline phosphatase. After a final round of washes, the plates were treated for 30 min in the dark with 1 mg/ml 5-bromo-4-chloro-3-indolylphosphate p-toluidine (Sigma) in 0.1 M alkaline buffer/0.6% agarose. IL-4-secreting cells were detected as blue spots on the bottom of the wells.
Infections and delayed-type hypersensitivity assays
Mice were inoculated with 104 PFU intracranially or 106 PFU i.p. of the DA strain of TMEV as described (52, 53). Sera were collected on day 7 after i.p. inoculation or on day 90 after intracranial inoculation. Total RNA was prepared from PBS-perfused brain and spinal cord using Tri-Reagent (Molecular Research Center, Cincinnati, OH). Five-fold serial dilutions starting with 10 µg RNA were filtered onto Hybond C-extra membranes (Amersham, Arlington Heights, IL) and hybridized with a virus-specific or GAPDH 32P-labeled probe. Positive controls for the detection of viral RNA included brain and spinal cord RNA from a C57BL/6 mouse 7 days after TMEV inoculation and viral cDNA.
Mice were inoculated in the hind footpads with 4 x 105 metacyclic promastigotes of Leishmania major. The progression of the disease was monitored weekly using a metric caliper to measure the hind footpad lesions. Parasite burden was determined as described previously (54).
Infective third stage larvae of Nippostrongylus brasiliensis were isolated from the feces of experimentally infected rats as described (51). The larvae were washed extensively with saline, counted, and injected s.c. at the base of the tail into CD134-deficient and control mice using 500 worms/mouse in 0.2 ml of PBS. After 10 days, the number of adult worms in the intestines was determined by direct visualization. The lungs of infected mice were completely blanched with ice-cold PBS, excised, minced into fine fragments, and dispersed into a single-cell suspension in PBS using a syringe plunger. Cells were passed through a nylon strainer and adjusted to 107 cells/ml in culture medium for use in cytokine ELISPOT assays.
For delayed-type hypersensitivity responses, mice were sensitized with 100 µl of 100 mg/ml oxazolone (4-ethoxymethylene-2-phenyloxazolone; Sigma) in acetone/olive oil (4:1) applied evenly on a shaved hind flank (55, 56). Five days later, 5 µl of the same oxazolone solution was applied to each side of the right ear. Acetone/olive oil without oxazolone was applied to the left ear in a similar fashion. The thickness of the central portion of each ear was then measured at various times using a Dial thickness gauge.
Adoptive transfer experiments with TCR transgenic T cells
These experiments were performed essentially as described (57). In brief, cells from the LN of female H-2dd DO11.10 TCR transgenic mice (CD134-/- or controls, on a B6/129/B10.D2 background) were injected i.v. into the tail veins of B10.D2 female mice. Donor populations were normalized by FACS so that each recipient received the same quantity of transgenic CD4+ T cells (6–7 x 107). In some experiments, the donor populations were labeled with CFSE as above. After 24 h, the recipient mice were then immunized s.c. with 300 µg of the chicken OVA peptide (residues 323–329) in CFA. FACS analysis with the KJ-126 Ab was then used to monitor the expansion of DO11.10 transgenic T cells in the brachial and axillary LN at several time points after immunization.
Activation-induced cell death
A total of 2 x 106 LN cells/ml were
stimulated with 5 µg/ml Con A for 48 h. The cells were then
washed extensively before incubation for 48 h with high (100 U) or
low doses (3 U) of IL-2 to predispose for apoptosis as described
previously (58). Cells were then stimulated with
plate-bound anti-CD3 Ab to induce apoptosis. Viable cells were
distinguished from dead cells by size (forward light scatter) and
propidium iodide incorporation.
CD134-deficient and control mice were injected i.p. with 100 µg of staphylococcal enterotoxin B (SEB; Sigma). FACS analysis was used to determine the proportions of LN T cells expressing Vß8.1 or 8.2 and Vß6 on day 0, 1, 2, 3, 4, and 7.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The gene encoding CD134 (46) was inactivated in
murine ES cells according to the strategy depicted in Fig. 1
A. Four exons of the gene
encoding the first 143 residues of the CD134 protein were deleted and
replaced by a neomycin resistance cassette. The expected structure of
the resultant mutant allele was verified by Southern blot using probes
from the 5' and 3' side of the targeted region (Fig. 1B) and
also using neomycin and CD134 cDNA probes (data not shown).
CD134-mutant mice were generated from the targeted ES cells by standard
blastocyst injection procedures (48, 59).
|
A). No other receptor for
CD134L could be detected on T cells in the absence of CD134 using
soluble CD134L-Fc. The expression of CD134L was significantly increased
on activated CD134-/- T cells and thymocytes
relative to wild-type cells as detected with either soluble CD134 or
the OX89 mAb (Fig. 2, A and B). It remains to be
established whether this increase in CD134L expression has a
transcriptional or posttranslational origin and whether it has any
impact on the phenotype of the mutant mice.
|
C; Table I and data not
shown). Thus, at first inspection, the absence of CD134 had no
detectable impact on lymphocyte development and homeostasis.
|
Several previous reports have implicated CD134 in costimulation
during T cell responses (17, 19, 20, 21, 22, 23, 35, 40). Therefore,
we performed a series of experiments to test for abnormalities in
proliferative responses made by CD134-deficient T cells. As shown in
Fig. 3A,
CD134-/- CD4+ T cells
proliferated weakly when stimulated with anti-CD3 mAb in vitro,
with the peak response made by CD134-/- T cells
typically five times lower than that of CD134-expressing control cells.
This defect in T cell proliferation could not be corrected either by
the addition of recombinant IL-2 to the cultures or by treatment with
anti-CD28 mAb (Fig. 3A). Decreased proliferation was
readily apparent when cells were activated with soluble but not
plate-bound anti-CD3 mAb (data not shown). CFSE-labeling
experiments showed that proliferating CD134-/-
cells progressed through successive cell divisions at a rate that was
largely indistinguishable from control cells (data not shown and Fig. 3D). Furthermore, while proliferating, the cells
up-regulated the expression of activation Ags in a normal fashion.
There was no apparent increase in the frequency of apoptotic cells
identified by staining with annexin V (data not shown), suggesting that
the defect in proliferation was not caused by enhanced apoptosis.
Curiously, other in vitro and in vivo assays of T cell proliferation,
such as mixed lymphocyte responses, peptide-specific responses of
transgenic T cells, or stimulation with SEB, did not reveal the same
sort of defect (Fig. 3, B–D, and data not shown). We also
found that CD134-/- T cells underwent
activation-induced cell death in vitro and in vivo with apparently
normal kinetics in response to anti-CD3 or SEB stimulation,
respectively (Fig. 3E and data not shown).
|
C).
Furthermore, CFSE-labeling experiments showed that the mutant T cells
also progressed through the cell cycle in vivo with apparently normal
kinetics (Fig. 3D).
As additional in vivo tests of the responses of
CD134-/- T cells, we challenged the mutant
animals with three kinds of infectious organisms. L. major
and N. brasiliensis were used to evaluate the capacity of
the mice to mount Th1- and Th2-dependent responses, respectively
(61, 62), whereas TMEV was employed as a neuroinflammatory
agent whose clearance from the CNS depends primarily on the action of
cytotoxic T cells (63). As shown in Fig. 4 and described in more detail below, in
all three cases, CD134-/- mice made equivalent
responses to those of control littermates.
|
A) and by a direct determination of the parasite burden in
the spleen and footpads (data not shown). Thus, CD134 is not essential
for T cells to adopt the Th1 fate and mediate effective resistance to
Leishmania.
The clearance of N. brasiliensis from the intestines of
infected mice is dependent on Th2 cells and is therefore a useful in
vivo assay for the function of these cells (62). Nine days
after infection, both CD134-/- and wild-type
animals had cleared the parasite from their intestinal tract and showed
evidence of a robust IL-4-dependent Th2 response in the form of
IL-4-secreting cells in their lungs (Fig. 4B) and elevated
levels of IgE in their sera (Fig. 6C). Thus, by this assay,
the absence of CD134 did not impair Th2 capability in vivo.
|
As a final specific test of the functional properties of
CD134-deficient T cells, we examined the capacity of
CD134-/- mice to mount a delayed-type
hypersensitivity response after topical application of oxazolone
(55, 56). As shown in Fig. 5, CD134-/- mice
showed normal ear swelling after s.c. injection of oxazolone. This
result indicated that the absence of CD134 did not obviously impair the
capacity of T cells to initiate an inflammatory response.
|
Antagonizing the CD134 ligand/receptor with a rabbit
anti-mouse CD134 antiserum was previously reported to interfere
with B cell differentiation and Ab secretion (44).
Therefore, we measured serum Ab titers after immunization with model
protein Ags and after infection with the three agents mentioned above.
Immunization with TNP-KLH (Fig. 6A) or NP-OVA (Fig. 6B) resulted in robust and diverse Ag-specific responses
that were no different from those made by control animals. The mice
also made enhanced responses after reimmunization (Fig. 6A),
indicating that immunological memory was not impaired by the absence of
CD134. CD134-/- mice made strong responses to
Theiler’s virus (Fig. 6D) and they showed elevated IgE
levels that were equivalent to those of control-infected mice after
inoculation with L. major and N. brasiliensis
(Fig. 6C). These last observations and the general finding
that isotype diversity was unaffected (Fig. 6A) indicate
that Th2 responses persist despite the absence of CD134. Finally,
CD134-/- mice made normal responses to the T
cell-independent Ag TNP-Ficoll (Fig. 6E). Cumulatively, the
results indicate that B cell responses are not obligatorily dependent
on the function of the CD134 receptor/ligand system.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Although we have uncovered a defect in T cell proliferation in vitro,
the underlying mechanism and physiological significance of this defect
remain to be established. Annexin V staining experiments indicate that
anti-CD3-activated CD134-/- T cells are not
abnormally prone to apoptosis (data not shown). CFSE-labeling
experiments also show that they divide at a similar rate to that of
wild-type T cells in vivo (Fig. 3D) or in vitro (data not
shown). Moreover, while dividing, the cells change their pattern of
expression of differentiation markers in an apparently normal fashion
(data not shown). Additional kinetic experiments also indicated that
the requirement for CD134 function was evident at early time points
after stimulation, consistent with the very rapid and therefore
atypical (23) up-regulation of CD134 under these in vitro
conditions (data not shown).
The in vitro assay employing soluble anti-CD3 mAb generated a
dependency on CD134 that was not apparent when T cells were activated
under other conditions (e.g., allo- or peptide-specific responses; Fig. 3, B and C). At this point, it is unclear why
there was a requirement for CD134 under one set of circumstances, but
not the other. It is possible, however, that the anti-CD3 assay was
deficient in supplying forms of costimulation that were more abundant
in the other assays. Costimulation that might substitute for CD134
signaling appeared to be distinct from that provided by CD28 because
anti-CD28 mAb could not restore the proliferative response.
Understanding the basis of the proliferative defect is likely to
provide important insight into the physiological conditions that
establish a need for the CD134 molecule. Such conditions will also
probably be revealed through the use of more sensitive in vivo assays
than the ones employed here.
The data presented here are consistent with previous work suggesting that CD134 provides a costimulatory function that can promote T cell proliferation (19, 20, 22). Whereas this costimulatory function is apparently not essential for T cell responses to several model challenges, it may act to sustain the duration of T cell immunity in vivo and perhaps enhance the formation or persistence of memory T cells. These possibilities are currently under analysis.
In vivo, CD134 expression is a characteristic of T cells involved in chronic inflammatory syndromes such as experimental allergic encephalomyelitis (31), rheumatoid arthritis (37) and inflammatory bowel disease (40). It seems feasible therefore that costimulatory signals from CD134 may help to sustain T cell reactivity under these types of chronic inflammatory circumstances. Indeed, recent studies employing soluble CD134 in vivo lend experimental support to this notion (30, 40). It remains unclear, however, whether the impact of CD134 signaling in such cases is most significant for the T cell while in the LN draining an inflamed site, or, alternatively, within the inflamed tissues themselves. In this regard, CD30 (also a member of the TNF receptor family) has recently been implicated in the regulation of T lymphocyte proliferation in situ in the pancreatic islets (66). Although the reported effect of CD30 in this case is to suppress T lymphocyte responses, it is possible that CD134 may have the opposite effect and instead drive the localized expansion of Ag-reactive T cells. A chronic inflammatory response may therefore depend on the sustained inhibition and potentiation of signals through CD30 and CD134, respectively.
The finding that B cell responses are apparently unaffected by the loss of CD134 is superficially at variance with predictions from a study employing a rabbit antiserum specific for mouse CD134 (44). In this study, the blockade of the CD134 receptor-ligand interaction interfered with the secretion of IgG, a result that was suggestive of an important role for CD134 in T:B cell collaboration and B cell differentiation in vivo. Interestingly, the blockade did not impair recall responses to the priming Ag, indicating that the differentiation of Ag-primed B cells into memory cells was unaffected. Why the absence of CD134 would have a different impact on B cell responses than a blocking Ab is not immediately clear, but it is possible that the anti-OX40 antiserum had unanticipated or indirect effects on the survival of T cells or on their capacity to interact with B cells. Given that CD134 is expressed only on activated peripheral T cells, it seems unlikely that its absence would have a significant effect on the composition of the naive T cell population. Thus, the capacity of CD134-/- T cells to provide adequate help to B cells is probably not due to the selection of an atypical precursor population that is abnormally CD134 independent in its function. Nonetheless, it is possible that the loss of CD134 may have a subtle effect on the capacity of T cells to promote certain B cell responses, and this effect may not be obvious in the settings that we have examined, perhaps because they involve powerful antigenic stimulation. We have observed germinal centers in the spleens of unimmunized mice and in the LN of immunized mice, but further work is required to determine whether these germinal centers are different from those present in normal mice, either in their rate of formation, their constituents, or their function. Other assays of T:B cell interaction, such as sensitive adoptive transfer systems, may be useful in determining whether the absence of CD134 has an effect on B cell function beyond that which might be due solely to diminished T cell expansion (67, 68).
The identity of cell surface molecules that might perform similar
functions to CD134 and thereby ameliorate the impact of its absence is
an issue of significance in terms of defining the physiological
function of CD134. Candidates in this respect would include the CD137
molecule as well as other members of the TNF receptor family. CD137 is
attractive because of its similar pattern of expression and because it
can activate NF-B through TRAF2, as can CD134 (24, 33, 69). Thus, it will be of interest to examine the phenotype of
mice that lack expression of both CD134 and CD137. These mice would
likely provide information concerning related functions of the two
molecules while also permitting more subtle tests to study their
individual functions.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
2 S.D.P. and C.P-R. contributed equally to this work.
3 Current address: Systemix Inc., Palo Alto, CA.
4 Current address: Ares-Serono International S.A., 14, Chemin des Aulx, 1228 Plan-les Ouates, Switzerland.
5 Address correspondence and reprint requests to Dr. Nigel Killeen, Department of Microbiology and Immunology, University of California, San Francisco, CA 94143-0414. E-mail address:
6 Abbreviations used in this paper: TRAF, TNF receptor-associated factor; TNP-KLH, trinitrophenyl-keyhole limpet hemocyanin; ELISPOT, enzyme-linked immunospot; NP, nitrophenyl; CFSE, carboxyfluorescein diacetate succinimidyl diester; LN, lymph node; TMEV, Theiler’s murine encephalomyelitis virus; SEB, staphylococcal enterotoxin B; ES, embryonic stem.
Received for publication July 20, 1999. Accepted for publication October 7, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol. Rev. 156:137.[Medline]
and tumor necrosis factor in organogenesis and spatial organization of lymphoid tissue. Eur. J. Immunol. 27:2600.[Medline]
B activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient mice. Immunity 7:715.[Medline]
and beta revealed in lymphotoxin ß-deficient mice. Immunity 6:491.[Medline]
B activation. J. Biol. Chem. 273:5808.B. Mol. Cell. Biol. 18:558.
or interleukin 4 during the resolution or progression of murine leishmaniasis: evidence for expansion of distinct helper T cell subsets. J. Exp. Med. 169:59.ß T lymphocytes for apoptosis. Nature 353:858.[Medline]
, and Stat6 are required for the expulsion of the gastrointestinal nematode parasite Nippostrongylus brasiliensis. Immunity 8:255.[Medline]
B. Biochem. Biophys. Res. Commun. 242:613.[Medline]
This article has been cited by other articles:
|
|
 |
|
  D. Hirschhorn-Cymerman, G. A. Rizzuto, T. Merghoub, A. D. Cohen, F. Avogadri, A. M. Lesokhin, A. D. Weinberg, J. D. Wolchok, and A. N. Houghton OX40 engagement and chemotherapy combination provides potent antitumor immunity with concomitant regulatory T cell apoptosis J. Exp. Med., May 11, 2009; 206(5): 1103 - 1116. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Klinger, J. K. Kim, S. A. Chmura, A. Barczak, D. J. Erle, and N. Killeen Thymic OX40 Expression Discriminates Cells Undergoing Strong Responses to Selection Ligands J. Immunol., April 15, 2009; 182(8): 4581 - 4589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Xiao, A. Kroemer, W. Gao, N. Ishii, G. Demirci, and X. C. Li OX40/OX40L Costimulation Affects Induction of Foxp3+ Regulatory T Cells in Part by Expanding Memory T Cells In Vivo J. Immunol., September 1, 2008; 181(5): 3193 - 3201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. J. Gough, C. E. Ruby, W. L. Redmond, B. Dhungel, A. Brown, and A. D. Weinberg OX40 Agonist Therapy Enhances CD8 Infiltration and Decreases Immune Suppression in the Tumor Cancer Res., July 1, 2008; 68(13): 5206 - 5215. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kroemer, X. Xiao, M. D. Vu, W. Gao, K. Minamimura, M. Chen, T. Maki, and X. C. Li OX40 Controls Functionally Different T Cell Subsets and Their Resistance to Depletion Therapy J. Immunol., October 15, 2007; 179(8): 5584 - 5591. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Vu, X. Xiao, W. Gao, N. Degauque, M. Chen, A. Kroemer, N. Killeen, N. Ishii, and X. Chang Li OX40 costimulation turns off Foxp3+ Tregs Blood, October 1, 2007; 110(7): 2501 - 2510. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q. Liu, Z. Liu, C. T. Rozo, H. A. Hamed, F. Alem, J. F. Urban Jr., and W. C. Gause The Role of B Cells in the Development of CD4 Effector T Cells during a Polarized Th2 Immune Response J. Immunol., September 15, 2007; 179(6): 3821 - 3830. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Humphreys, A. Loewendorf, C. de Trez, K. Schneider, C. A. Benedict, M. W. Munks, C. F. Ware, and M. Croft OX40 Costimulation Promotes Persistence of Cytomegalovirus-Specific CD8 T Cells: A CD4-Dependent Mechanism J. Immunol., August 15, 2007; 179(4): 2195 - 2202. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Soroosh, S. Ine, K. Sugamura, and N. Ishii OX40-OX40 Ligand Interaction through T Cell-T Cell Contact Contributes to CD4 T Cell Longevity J. Immunol., May 15, 2006; 176(10): 5975 - 5987. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. E. Foulds and H. Shen Clonal Competition Inhibits the Proliferation and Differentiation of Adoptively Transferred TCR Transgenic CD4 T Cells in Response to Infection. J. Immunol., March 1, 2006; 176(5): 3037 - 3043. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Fukushima, T. Yamaguchi, W. Ishida, K. Fukata, H. Yagita, and H. Ueno Roles of OX40 in the Development of Murine Experimental Allergic Conjunctivitis: Exacerbation and Attenuation by Stimulation and Blocking of OX40 Invest. Ophthalmol. Vis. Sci., February 1, 2006; 47(2): 657 - 663. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Serghides, J. Bukczynski, T. Wen, C. Wang, J.-P. Routy, M.-R. Boulassel, R.-P. Sekaly, M. Ostrowski, N. F. Bernard, and T. H. Watts Evaluation of OX40 Ligand as a Costimulator of Human Antiviral Memory CD8 T Cell Responses: Comparison with B7.1 and 4-1BBL J. Immunol., November 15, 2005; 175(10): 6368 - 6377. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. R Blazar and W. J Murphy Bone marrow transplantation and approaches to avoid graft-versus-host disease (GVHD) Phil Trans R Soc B, September 29, 2005; 360(1461): 1747 - 1767. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Song, X. Tang, K. M. Harms, and M. Croft OX40 and Bcl-xL Promote the Persistence of CD8 T Cells to Recall Tumor-Associated Antigen J. Immunol., September 15, 2005; 175(6): 3534 - 3541. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. M. C. Gaspal, M.-Y. Kim, F. M. McConnell, C. Raykundalia, V. Bekiaris, and P. J. L. Lane Mice Deficient in OX40 and CD30 Signals Lack Memory Antibody Responses because of Deficient CD4 T Cell Memory J. Immunol., April 1, 2005; 174(7): 3891 - 3896. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Valzasina, C. Guiducci, H. Dislich, N. Killeen, A. D. Weinberg, and M. P. Colombo Triggering of OX40 (CD134) on CD4+CD25+ T cells blocks their inhibitory activity: a novel regulatory role for OX40 and its comparison with GITR Blood, April 1, 2005; 105(7): 2845 - 2851. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M.-Y. Kim, V. Bekiaris, F. M. McConnell, F. M. C. Gaspal, C. Raykundalia, and P. J. L. Lane OX40 Signals during Priming on Dendritic Cells Inhibit CD4 T Cell Proliferation: IL-4 Switches off OX40 Signals Enabling Rapid Proliferation of Th2 Effectors J. Immunol., February 1, 2005; 174(3): 1433 - 1437. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Dawicki, E. M. Bertram, A. H. Sharpe, and T. H. Watts 4-1BB and OX40 Act Independently to Facilitate Robust CD8 and CD4 Recall Responses J. Immunol., November 15, 2004; 173(10): 5944 - 5951. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Florido, M. Borges, H. Yagita, and R. Appelberg Contribution of CD30/CD153 but not of CD27/CD70, CD134/OX40L, or CD137/4-1BBL to the optimal induction of protective immunity to Mycobacterium avium J. Leukoc. Biol., November 1, 2004; 76(5): 1039 - 1046. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zingoni, T. Sornasse, B. G. Cocks, Y. Tanaka, A. Santoni, and L. L. Lanier Cross-Talk between Activated Human NK Cells and CD4+ T Cells via OX40-OX40 Ligand Interactions J. Immunol., September 15, 2004; 173(6): 3716 - 3724. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Xiao, J. Hendriks, P. Langerak, H. Jacobs, and J. Borst CD27 Is Acquired by Primed B Cells at the Centroblast Stage and Promotes Germinal Center Formation J. Immunol., June 15, 2004; 172(12): 7432 - 7441. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Weinberg, D. E. Evans, C. Thalhofer, T. Shi, and R. A. Prell The generation of T cell memory: a review describing the molecular and cellular events following OX40 (CD134) engagement J. Leukoc. Biol., June 1, 2004; 75(6): 962 - 972. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. K. Lathrop, C. A. Huddleston, P. A. Dullforce, M. J. Montfort, A. D. Weinberg, and D. C. Parker A Signal through OX40 (CD134) Allows Anergic, Autoreactive T Cells to Acquire Effector Cell Functions J. Immunol., June 1, 2004; 172(11): 6735 - 6743. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Bansal-Pakala, B. S. Halteman, M. H.-Y. Cheng, and M. Croft Costimulation of CD8 T Cell Responses by OX40 J. Immunol., April 15, 2004; 172(8): 4821 - 4825. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Takeda, S. Ine, N. Killeen, L. C. Ndhlovu, K. Murata, S. Satomi, K. Sugamura, and N. Ishii Distinct Roles for the OX40-OX40 Ligand Interaction in Regulatory and Nonregulatory T Cells J. Immunol., March 15, 2004; 172(6): 3580 - 3589. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Bertram, W. Dawicki, B. Sedgmen, J. L. Bramson, D. H. Lynch, and T. H. Watts A Switch in Costimulation from CD28 to 4-1BB during Primary versus Secondary CD8 T Cell Response to Influenza In Vivo J. Immunol., January 15, 2004; 172(2): 981 - 988. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
N. Martin-Orozco, Z. Chen, L. Poirot, E. Hyatt, A. Chen, O. Kanagawa, A. Sharpe, D. Mathis, and C. Benoist Paradoxical Dampening of Anti-Islet Self-Reactivity but Promotion of Diabetes by OX40 Ligand J. Immunol., December 15, 2003; 171(12): 6954 - 6960. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. A. Prell, D. E. Evans, C. Thalhofer, T. Shi, C. Funatake, and A. D. Weinberg OX40-Mediated Memory T Cell Generation Is TNF Receptor-Associated Factor 2 Dependent J. Immunol., December 1, 2003; 171(11): 5997 - 6005. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Humphreys, G. Walzl, L. Edwards, A. Rae, S. Hill, and T. Hussell A Critical Role for OX40 in T Cell-mediated Immunopathology during Lung Viral Infection J. Exp. Med., October 20, 2003; 198(8): 1237 - 1242. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. R. Humphreys, L. Edwards, G. Walzl, A. J. Rae, G. Dougan, S. Hill, and T. Hussell OX40 Ligation on Activated T Cells Enhances the Control of Cryptococcus neoformans and Reduces Pulmonary Eosinophilia J. Immunol., June 15, 2003; 170(12): 6125 - 6132. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. R. Blazar, A. H. Sharpe, A. I. Chen, A. Panoskaltsis-Mortari, C. Lees, H. Akiba, H. Yagita, N. Killeen, and P. A. Taylor Ligation of OX40 (CD134) regulates graft-versus-host disease (GVHD) and graft rejection in allogeneic bone marrow transplant recipients Blood, May 1, 2003; 101(9): 3741 - 3748. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P.-J. Linton, B. Bautista, E. Biederman, E. S. Bradley, J. Harbertson, R. M. Kondrack, R. C. Padrick, and L. M. Bradley Costimulation via OX40L Expressed by B Cells Is Sufficient to Determine the Extent of Primary CD4 Cell Expansion and Th2 Cytokine Secretion In Vivo J. Exp. Med., April 7, 2003; 197(7): 875 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Totsuka, T. Kanai, K. Uraushihara, R. Iiyama, M. Yamazaki, H. Akiba, H. Yagita, K. Okumura, and M. Watanabe Therapeutic effect of anti-OX40L and anti-TNF-alpha MAbs in a murine model of chronic colitis Am J Physiol Gastrointest Liver Physiol, April 1, 2003; 284(4): G595 - G603. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Fillatreau and D. Gray T Cell Accumulation in B Cell Follicles Is Regulated by Dendritic Cells and Is Independent of B Cell Activation J. Exp. Med., January 20, 2003; 197(2): 195 - 206. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. J. Ekkens, Z. Liu, Q. Liu, J. Whitmire, S. Xiao, A. Foster, J. Pesce, J. VanNoy, A. H. Sharpe, J. F. Urban, et al. The Role of OX40 Ligand Interactions in the Development of the Th2 Response to the Gastrointestinal Nematode Parasite Heligmosomoides polygyrus J. Immunol., January 1, 2003; 170(1): 384 - 393. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Ohlrogge, F. Haag, J. Lohler, M. Seman, D. R. Littman, N. Killeen, and F. Koch-Nolte Generation and Characterization of Ecto-ADP-Ribosyltransferase ART2.1/ART2.2-Deficient Mice Mol. Cell. Biol., November 1, 2002; 22(21): 7535 - 7542. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Murata, M. Nose, L. C. Ndhlovu, T. Sato, K. Sugamura, and N. Ishii Constitutive OX40/OX40 Ligand Interaction Induces Autoimmune-Like Diseases J. Immunol., October 15, 2002; 169(8): 4628 - 4636. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Cannons, E. M. Bertram, and T. H. Watts Cutting Edge: Profound Defect in T Cell Responses in TNF Receptor-Associated Factor 2 Dominant Negative Mice J. Immunol., September 15, 2002; 169(6): 2828 - 2831. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. M. Bertram, P. Lau, and T. H. Watts Temporal Segregation of 4-1BB Versus CD28-Mediated Costimulation: 4-1BB Ligand Influences T Cell Numbers Late in the Primary Response and Regulates the Size of the T Cell Memory Response Following Influenza Infection J. Immunol., April 15, 2002; 168(8): 3777 - 3785. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Yamada, A. D. Salama, and M. H. Sayegh The Role of Novel T Cell Costimulatory Pathways in Autoimmunity and Transplantation J. Am. Soc. Nephrol., February 1, 2002; 13(2): 559 - 575. [Full Text] [PDF]
|
|
|
|
|
|
H.-C. Wang and J. R. Klein Multiple Levels of Activation of Murine CD8+ Intraepithelial Lymphocytes Defined by OX40 (CD134) Expression: Effects on Cell-Mediated Cytotoxicity, IFN-{gamma}, and IL-10 Regulation J. Immunol., December 15, 2001; 167(12): 6717 - 6723. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Ndhlovu, N. Ishii, K. Murata, T. Sato, and K. Sugamura Critical Involvement of OX40 Ligand Signals in the T Cell Priming Events During Experimental Autoimmune Encephalomyelitis J. Immunol., September 1, 2001; 167(5): 2991 - 2999. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. L. Cannons, P. Lau, B. Ghumman, M. A. DeBenedette, H. Yagita, K. Okumura, and T. H. Watts 4-1BB Ligand Induces Cell Division, Sustains Survival, and Enhances Effector Function of CD4 and CD8 T Cells with Similar Efficacy J. Immunol., August 1, 2001; 167(3): 1313 - 1324. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Baba, Y. Takahashi, J. Lichtenfeld, R. Tanaka, A. Yoshida, K. Sugamura, N. Yamamoto, and Y. Tanaka Functional CD4 T Cells after Intercellular Molecular Transfer of OX40 Ligand J. Immunol., July 15, 2001; 167(2): 875 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Nakae, M. Asano, R. Horai, N. Sakaguchi, and Y. Iwakura IL-1 Enhances T Cell-Dependent Antibody Production Through Induction of CD40 Ligand and OX40 on T Cells J. Immunol., July 1, 2001; 167(1): 90 - 97. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
V. Malmstrom, D. Shipton, B. Singh, A. Al-Shamkhani, M. J. Puklavec, A. N. Barclay, and F. Powrie CD134L Expression on Dendritic Cells in the Mesenteric Lymph Nodes Drives Colitis in T Cell-Restored SCID Mice J. Immunol., June 1, 2001; 166(11): 6972 - 6981. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. G.-H. Jember, R. Zuberi, F.-T. Liu, and M. Croft Development of Allergic Inflammation in a Murine Model of Asthma Is Dependent on the Costimulatory Receptor Ox40 J. Exp. Med., February 5, 2001; 193(3): 387 - 392. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Nohara, H. Akiba, A. Nakajima, A. Inoue, C.-S. Koh, H. Ohshima, H. Yagita, Y. Mizuno, and K. Okumura Amelioration of Experimental Autoimmune Encephalomyelitis with Anti-OX40 Ligand Monoclonal Antibody: A Critical Role for OX40 Ligand in Migration, But Not Development, of Pathogenic T Cells J. Immunol., February 1, 2001; 166(3): 2108 - 2115. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. M. M. den Haan and M. J. Bevan A novel helper role for CD4 T cells PNAS, November 21, 2000; 97(24): 12950 - 12952. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
