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*
Department of Infectious Disease and Immunology, Okinawa-Asia Research Center of Medical Science, Faculty of Medicine, University of the Ryukyus, Okinawa, Japan;
Department of Microbiology, School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; and
Department of Microbiology and Immunology, School of Medicine, Tohoku University, Miyagi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various proteins belonging to TNF superfamily and TNFR superfamily expressed on the immune cells have functions in cell growth and death (8). OX40 (CD134), one member of the TNFR superfamily, is expressed on activated CD4+ T cells (9, 10). The costimulation of CD4+ T cells via OX40 by OX40 ligand (OX40L)3 results in enhancement of T cell growth and cytokine production (10, 11, 12, 13, 14, 15). In addition to the costimulatory function, OX40 has been shown to be involved in T cell migration (15, 16). OX40L (gp34, CD134 ligand) has been shown to be expressed on restricted immune cell populations, including dendritic cells (DCs) and activated B cells, as well as endothelial cells (10, 12, 17, 18, 19, 20, 21). It has been shown that the stimulation via OX40L of DCs enhances their activation and maturation (20), and that that of B cells induces elevated response of humoral immunity (22, 23). Although the functional importance of OX40/OX40L interaction in the immune responses has been intensively investigated, the mechanism of the modification or the regulation of this reaction still remains unclear.
In this report, we describe an intercellular molecular transfer of functional OX40L to CD4+ from not only OX40L transfectants but also human T cell leukemia virus type I (HTLV-I)-infected cells, monocytes, and HUVECs. These findings provide us with a new idea to help clarify the regulation and modification mechanisms of OX40/OX40L interaction.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Sigma, St. Louis, MO), 100 U/ml penicillin, and 100 µg/ml streptomycin was used as a basic medium (referred to as RPMI medium). Human PBMC were separated from heparinized peripheral blood obtained from volunteer healthy donors by using lympholyte-H (Cedarlane Laboratories, Hornby, Ontario, Canada). For preparation of CD4+ T cell-enriched cells, PBMC were depleted of CD8+ and CD14+ cells with anti-CD8 and -CD14 Abs-conjugated magnetic beads (Dynal, Lake Success, NY). CD4+ T cells were activated by immobilized OKT-3 mAb or the magnetic beads (Dynal) coated with OKT-3 and anti-CD28 mAb for 3–7 days in RPMI medium containing 50 U/ml recombinant human IL-2 (Shionogi, Osaka, Japan). Alternatively, CD4+ T cells were activated in RPMI medium containing either 20 ng/ml recombinant human IL-12 (R&D Systems, Minneapolis, MN), or 20 ng/ml recombinant human IL-4 (R&D Systems). Establishing and maintaining the IL-2-stimulated NK cell lines were described elsewhere (24). CD14+ monocytes were separated from normal PBMC by a monocyte-negative isolation kit (Dynal). Human monocytic leukemia cell line THP-1 (American Type Culture Collection (ATCC), Manassas, VA), human T lymphoma cell lines Jurkat and HUT-78, HTLV-I-infected human T cell line MT-2, human epithelial cell line HeLa-S3, and monkey kidney cell line COS-1 were also maintained in RPMI medium. Other HTLV-I-infected human T cell lines, ILT-AKI, ILT-626, and ILT-NIS, were maintained in RPMI medium containing 20 U/ml recombinant IL-2. Establishment and maintenance of human OX40L transfectants of Jurkat cell line J34-10-9 (N. Ishii, K. Murata, Y. Tanaka, K. Sugamura, manuscript in preparation), mouse fibroblast cell line transfected with cDNA of OX40L (SVT2/OX40L) or mock vector (SVT2/control), and HIV-1 latent infected T cell line transfected with cDNA of human OX40 (ACH2/OX40) or vector alone (ACH2/moc), will be described elsewhere (41). HUVECs were purchased (Kurabo, Osaka, Japan) and were cultured in Humedia-EG2 medium (Kurabo).
Purchased mAbs were anti-CD3 (OKT3; ATCC), anti-CD4 (OKT4;
Beckman Coulter, Fullerton, CA), anti-CD14-FITC (Dako, Carpinteria,
CA), anti-CD28-FITC (Dako), anti-CD40-FITC (Ancell, Bayport,
MN), anti-CD69-FITC (Dako), anti-CD95-FITC (Dako), anti-HLA
class I (W6/32; ATCC),
anti-
2-microglobulin (BBM.1; ATCC),
anti-HLA-DR-FITC (Dako), anti-TNF-
(R&D Systems),
anti-TNF- (R&D Systems), anti-OX40 (Nichirei, Tokyo, Japan),
goat anti-mouse IgG-FITC (American Qualex, San Clemente, CA),
streptavidin-PE (Dako), streptavidin-PerCP (BD Biosciences, San Jose,
CA), and streptavidin-Cy3 (Amersham Pharmacia Biotech, Piscataway, NJ).
Mouse anti-human OX40L mAbs, 5A8, 8F4, TAG34 (25), a
new rat anti-OX40L IgG1 mAb,
W9-14 and a new mouse
anti-OX40 IgG1 mAb, B7B5 (41) were prepared in our laboratory. Rat
anti-HTLV-I envelope gp46 mAb, Rey-7, was used for negative control
staining (26). mAbs were purified from ascites fluids by
gel filtration with Superdex G-200 (Amersham Pharmacia Biotech) and
were labeled with N-hydroxysuccinimide-FITC (Sigma), normal
human serum-biotin (Sigma), or Cy5 Bis functional dye (Amersham
Pharmacia Biotech) following the manufacturer’s instructions.
Expression vector construction and transfection of COS-1 cells
For the construction of the expression vector of OX40L with FLAG peptide on the C terminus of OX40L, complementary DNA of OX40L lacking the stop codon on the 3' end were amplified from pSGP34 (17) with OX40L specific primer set (5'-GGCAAGCTTCCATTCTTCATCTTCCC-3', 5'-GGCGGATCCAAGGACACAGAATTCACC-3') (Espec Oligo Service, Tsukuba, Japan) containing HindIII or BamHI recognition sequence by PCR (DNA thermal cycler, Perkin-Elmer, Boston, MA). PCR product of OX40L-FLAG was cloned into the pCR2.1 vector (Invitrogen, Carlsbad, CA) by the TA cloning method and recloned into the transient mammalian expression vector pFLAG-CMV-5a (Sigma) after confirmation of their sequences by DNA sequencer 4200 (LI-COR, Lincoln, NE) using M13 reverse and forward primers (ALOKA, Tokyo, Japan). COS-1 cells were transfected with pFLAG-CMV-5a carrying OX40L cDNA (pFLAG-CMV-5a/OX40L) or vector alone using FuGENE (Boehringer-Mannheim, Mannheim, Germany) following the manufacturer’s instructions. Two days after transfection, the expression of OX40L-FLAG proteins on the COS-1 cells were confirmed by staining with anti-OX40L mAb and biotinylated anti-FLAG peptide mAb (M2; Sigma) followed by staining with streptavidin-PE.
OX40L transfer and flow cytometry analysis
OX40L-expressing transfectants, SVT2/OX40L, COS/OX40L-flag, J34-10-9 and negative control cells treated with or without fixation by 4% paraformaldehyde (PFA; Wako Biochemicals, Osaka, Japan) for 10 min at room temperature were cocultured with activated human CD4+ T cells, freshly isolated human PBMC, or other cell lines for 2 h at either 37°C or on ice. Alternatively, CD4+ T cells and SVT2 transfectants or J34-10-9 cells and SVT2 transfectants were cultured in separated chambers through a membrane with 0.4-µm pores (Transwell; Costar, Corning, NY). Transfer of OX40L protein onto the recipient cells was analyzed by multicolor staining. Anti-pan HLA class I mAb (W6/32) was used to distinguish recipient cells from the mouse SVT2 transfectants and other tumor cell lines, and anti-HLA class II mAb was used for distinguishing recipient cells from COS-1 transfectants. For staining of the cells, prewashed cell mixtures were incubated with 100 µg/ml human IgG in PBS containing 2% FCS and 0.1% NaN3 (referred to as FACS buffer) for 15 min at 4°C for blocking the FcR, and subsequently incubated with the appropriate concentration (5–10 µg/ml) of labeled mAbs for 30 min on ice. After washing with FACS buffer, cells were fixed with 1% PFA for 10 min and membrane staining was analyzed with FACSCalibur (BD Biosciences). In the analysis of transferred OX40L under the acidic condition, for distinguishing SVT2 transfectants from CD4+ T cells, the donor cells were prelabeled with a PKH67 fluorescent kit (Zynaxis, Malvern, PA). The PFA-fixed prelabeled SVT2 transfectants were removed by Ficoll density gradient medium (Cedarlane Laboratories) after a 2-h coculture with CD4+ T cells on ice. Separated recipient CD4+ T cells were incubated in 1.0 ml of 0.2 M glycine-HCl buffer (pH 2.8) for 10 min on ice and then stained with mAbs as described above (27).
Immunoprecipitation
The transferred OX40L on the recipient cells was analyzed by immunoprecipitation followed by Western blotting. After coculture of the CD4+ T recipient cells with PFA-fixed OX40L-expressing cells, the live recipient cells were separated by a gradient centrifugation followed by the positive separation of CD4+ T cells using immunobeads. Then, 5 x 105 of the sorted CD4+ T cells were surface biotinylated with Sulfo-N-hydroxysuccinimide-biotin (Pierce, Rockford, IL), and solubilized in 500 µl of low-salt lysis buffer (10 mM Tris-HCl, pH 8.0, 0.14 M NaCl, 3 mM MgCl2, 1 mM PMSF, 0.5% Nonidet P-40). The postnuclear lysates were precleaned by incubating with 125 µl protein G-Sepharose (Amersham Pharmacia Biotech) and then subjected to immunoprecipitation with 2 µg anti-OX40L mAb, 5A8, and 12.5 µl protein G-Sepharose. The Sepharose was washed four times and then eluted with the 12 µl of sample buffer (0.5 M Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, 0.1% bromophenol blue, and 4% 2-ME) for 5 min at 100°C. The immunoprecipitates were separated on a 10% SDS-polyacrylamide gel and transferred onto Clear Blot Membrane-p (ATTO, Tokyo, Japan) by the electric Western blotting system (ATTO). Biotinylated protein bands were visualized by streptavidin-HRP and ECL chemiluminescent substrate (Amersham Pharmacia Biotech) followed by analysis with Fluor-S MAX MultiImager (Bio-Rad, Hercules, CA).
Detection of mRNA of OX40L by RT-PCR
For analysis of mRNA of OX40L in the OX40L-transferred cells after coculture with OX40L-expressing cells, RT-PCR experiments were performed. Two million stimulated CD4+ T cells or THP-1 cells after culture with fixed SVT2/control or SVT2/OX40L for 2 h were separated by gradient centrifugation and Ab-coated magnetic beads. After washing these cells with PBS, total RNA was isolated by TRIzol reagent (Life Technologies), and first-strand DNA was prepared by using reverse transcriptase with oligo(dT) primers (Life Technologies). These first-strand DNA were amplified with a OX40L specific primer set or G3PDH specific primer set (5'-ACCACAGTCCATGCCATCAC-3', 5'-TCCACCACCCTGTTGCTGTA-3') with 30 cycles of PCR. The amplified products were determined on 1% agarose gel. The first-strand DNA from OX40L-expressing MT-2 cells was used as positive control.
HIV-1 induction assay
For studying the function of the transferred OX40L protein on
the CD4+ T cells, we used the ACH2/OX40 cells.
Cross-linking of OX40 on the ACH2/OX40 by stimulation with
OX40L-expressing cells have been confirmed to induce the ACH2/OX40
cells to produce HIV-1 virions in the culture supernatants (41).
In this study, ACH2/OX40 or ACH2/moc cells were cocultured with an
equal numbers of PFA-fixed OX40L-transferred CD4+
T cells for 2 days in the presence of 10 µg/ml of both
anti-TNF- and anti-TNF- mAbs (R&D Systems) to avoid the
effect of TNF. Then HIV-1 p24 released in the culture supernatants was
measured by an ELISA kit (Zeptometrix, Buffalo, NY).
Immunofluorescence confocal microscopy
Coculture of CD4+ T cells and SVT2 transfectants were set by the same methods for FACS analysis of OX40L transfer as described above. After washing and blocking with human IgG, cells were incubated with FITC-labeled W9-1 and biotinylated OKT4 for 30 min at 4°C. Subsequently, cells were incubated with streptavidin-Cy3 for 30 min at 4°C, followed by resuspension with FACS buffer with 1% PFA. A volume of 5 µl of cell suspension was sealed between a glass slide and coverslip, and examined by confocal laser microscopy (Fluo View BX-50; Olympus, Tokyo, Japan) with a x40 objective lens, using laser excitation at 488 and 543 nm. The widths of FITC and Cy3 emission channels were set such that bleed-through across channels was negligible.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previously, we found that coculture of normal activated
CD4+ T cells with OX40L+
HTLV-I-infected MT-2 cells induced OX40L expression on the
CD4+ T cells that was specifically blocked by
anti-OX40L mAb (our unpublished data). In these studies, we
used a SVT2 mouse fibroblast cell line and a Jurkat cell line
transfected with cDNA of human OX40L (designated as SVT2/OX40L and
J34-10-9, respectively). For negative controls, the same
fibroblast-transfected cell line with mock vector (SVT2/control) and a
parental Jurkat cell line were used. The expression of OX40L proteins
on these cells was confirmed by both flow cytometry and Western
blotting analyses using mAbs specific for OX40L (Fig. 1
A and data not shown). We first examined the modification of
the cell surface expression levels of various costimulatory and related
membrane proteins, including OX40L, OX40, CD25, CD40, CD69, CD95, and
CD28, on the activated CD4+ T cells after
coculture with the SVT2/OX40L. In the flow cytometry analysis, the
mouse SVT2 transfectants were easily gated out from the mixed
population, based on the HLA class I negative phenotype and the pattern
of forward and side scatter (data not shown). As shown in Fig. 1B, CD4+ T cells became OX40L positive
after coculture with SVT2/OX40L but not with SVT2/control cells. No
significant change in the expression levels of CD25, CD40, CD69, CD95,
and CD28 was observed on the surface of CD4+ T
cells after coculture with SVT2 transfectants. The level of OX40
expression on CD4+ T cells was slightly
down-regulated only after coculture with SVT2/OX40L.
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To determine the possibility that OX40L protein was transferred
from the SVT2/OX40L cells during the period of coculture, we first
surface-biotinylated OX40L+ J34-10-9 cells and
Jurkat cells, and then cocultured with the recipient
CD4+ T cells. After a 2-h coculture,
CD4+ T cells were stained with Cy5-conjugated
anti-CD4 mAb and PE-conjugated streptavidin. The cell mixtures were
simultaneously stained with anti-OX40L mAb (TAG34-FITC) to confirm
the expression of OX40L on CD4+ T cells. As shown
in Fig. 2A, 16% of CD4+ T cells cocultured with
J34-10-9 cells became OX40L positive. This OX40L acquisition on
CD4+ T cells was almost completely abrogated by
pretreatment of J34-10-9 cells with anti-OX40L mAb, W9-1. Although
low levels of biotinylated substances (10–11%) were detected on the
CD4+ T cell population cocultured with
biotinylated Jurkat cells, 38% of the CD4+ T
cells cocultured with biotinylated J34-10-9 cells expressed high levels
of biotin-related substances on the cell surface. Acquisition of the
biotinylated substances was significantly inhibited by the
anti-OX40L, suggesting that OX40L is the major biotinylated protein
transferred from the OX40L+ cells to
CD4+ T cells. To confirm the intercellular
molecular transfer of OX40L protein, we established COS-1 cells
transiently expressing OX40L-FLAG peptide fusion protein
(COS/OX40L-flag) and cocultured them with
CD4+ T cells. The expression of OX40L-flag fusion
protein was examined by flow cytometry using various anti-OX40L
mAbs and M2 anti-FLAG Ab (Fig. 2B). Immunoprecipitation
with these specific Abs was also performed (data not shown). These
results showed that CD4+ T cells acquired OX40L
from the donor COS-1 cell transfectant. Interestingly, the FLAG-labeled
OX40L transfer was also observed on the human OX40-negative monocytic
tumor cell line, THP-1 (Fig. 2C). These data indicate that
the newly appeared OX40L protein on OX40-positive
CD4+ T cells and OX40-negative THP-1 cells were
produced in the donor OX40L transfectants and then transferred to the
recipient cells.
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It has been reported that various human hemopoietic cell
populations including HTLV-I-infected T cells, activated B cells,
macrophages, and DCs, as well as endothelial cells, express OX40L
(10, 12, 17, 18, 19, 20, 21, 25). We investigated whether various
OX40L-expressing HTLV-I-infected T cell lines are capable of
transferring OX40L onto activated CD4+ T
cells. Fig. 3A shows that cells with high OX40L expression including MT-2,
ILT-NIS, and ILT-626 successfully induced OX40L transfer on the
CD4+ T cells, as in the case of SVT2/OX40L. Low
levels of molecular transfer of OX40L were seen from ILT-AKI
expressing low levels of OX40L. We also determined the capability of
transferring OX40L of normal cells including monocytes and HUVECs. To
enhance OX40L expression on monocytes, they were cocultured with
allogenic Jurkat cells for 24 h before examining the capability
of OX40L transfer. Coculture of CD4+ T cells with
these two OX40L+ cells resulted in OX40L
expression on the CD4+ T cells at low levels
(Fig. 3B).
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C). Comparison of efficiency of OX40L transfer and the
level of the OX40 expression among CD4+ T cells
activated with different cytokines showed that IL-12-activated
CD4+ T cells acquired slightly higher levels of
OX40L than those activated with IL-4 (Fig. 3D).
Interestingly, not only CD4+ T cells but also
resting CD8+ T cells, fresh NK cells, and
IL-2-stimulated NK cells from healthy donors became OX40L positive by
coculture with SVT2/OX40L (Fig. 3C). Although PHA-activated
CD14+ monocytes do not express detectable levels
of OX40L or OX40, OX40L was also detected after coculture with
SVT2/OX40L (Fig. 3C). Various cell lines without OX40
expression including THP-1, Molt-4, HUT-78, Raji, and HeLa-S3 cells
were capable of receiving OX40L to significant levels (Fig. 3E).
To determine whether mRNA of OX40L was synthesized in
CD4+ T cells and THP-1 cells during a 2-h
coculture with SVT2/OX40L, total RNA of these cells was examined by
RT-PCR. Whereas the OX40L mRNA was detected in positive control MT-2
cells, no OX40L mRNA was detected in either OX40L-positive normal
CD4+ T cells nor THP-1 cells (Fig. 3F), suggesting that the induced expression of OX40L
protein on the surface of the CD4+ T cells and
THP-1 cells is independent of the de novo synthesis of OX40L protein.
These observations indicate that intercellular molecular transfer of
OX40L occurs regardless of the existence of the receptor OX40 on the
cell surface even in an Ag-independent manner.
OX40L transfer occurs in various conditions
To determine the mechanism of OX40L transfer, the efficiency of
transfer under different cell culture conditions was compared. As shown
in Fig. 4A, the molecular transfer of OX40L from SVT2/OX40L to
CD4+ T cells did not occur when they were
cultured in separated chambers through a membrane with 0.4-µm pores.
Neither the culture supernatants from SVT2/OX40L cells that contained
soluble OX40L at a high level as determined by a sandwich ELISA nor
immobilized anti-OX40L Ab-captured OX40L from SVT2/OX40L cell
lysates on the culture plate induced OX40L transfer on
CD4+ T cells (data not shown). These data
indicate that cell-to-cell contact is necessary for OX40L transfer.
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B), showing that the OX40L transfer occurs without
being involved by the vesicle formed with detached membrane fragments.
Based on the reports describing the proteolytic cleavage processes
existing in other TNF family molecules (28, 29, 30, 31), we were
concerned with the possibility that cleaved OX40L by activated
metalloproteinase was transferred to CD4+ T
cells. However, under the coculture conditions in the presence of
various proteinase inhibitors, including aprotinin, pepstatin,
leupeptin, epoxy, rhamno, bestatin, and captopril, no inhibition
of OX40L transfer to CD4+ T cells or other cell
lines was observed (data not shown). Furthermore, the efficiency of
OX40L transfer was not affected by coculture on ice, avoiding the
activation of possible proteinase (Fig. 4C). Thus it is
unlikely that the mechanism of OX40L transfer was based on the cleavage
of OX40L from the cells.
To confirm that the intact molecule of OX40L moved to
CD4+ T cells without any cleavage, we compared
the molecular mass between transferred OX40L protein expressed
on CD4+ T cells and that of donor cells by
immunoprecipitation followed by Western blotting analysis.
CD4+ T cells cocultured with prefixed SVT2/OX40L
were separated by gradient centrifugation and were further purified by
magnetic bead-conjugated anti-CD4 mAb for depletion of
OX40L-expressing transfectants. These sorting procedures removed the
fixed cells >99.5% as determined by flow cytometry (data not shown).
As shown in Fig. 4D, a specific protein precipitated with
5A8 from OX40L-transferred CD4+ T cells was of
the same molecular mass, 34 kDa, identical with that from SVT2/OX40L.
Essentially the same results were obtained when we used COS/OX40L-flag
as the source of OX40L and immunoprecipitated with anti-FLAG Ab
from OX40L-transferred CD4+ T cells (data not
shown). Therefore, it is likely that a whole intact molecule of OX40L
on the OX40L-expressing cells moves to the recipient cells by
cell-to-cell contact. Acid treatment was used to distinguish
surface-bound OX40L from OX40L stabilized in the transmembrane of
recipient CD4+ T cells (Fig. 4E). No
significant change of OX40L staining was observed in
CD4+ T cells after treatment at pH 2.8 and pH
7.0. These results suggest that transferred OX40L on
CD4+ T cell is stabilized in the transmembrane of
the recipient cells.
To obtain more information about transferred OX40L on
CD4+ T cells, cells after coculture were examined
by confocal microscopy. Representative surface staining of individual
cells is shown in Fig. 4F. The pattern of ring staining of
OX40L on SVT2/OX40L (green) and that of CD4 on
CD4+ T cells (red) denotes the cell surface.
Images of cell surface staining were overlaid on the Nomarski images
(Fig. 4F, a–c). Whereas no positive staining of
OX40L was detected on CD4+ T cells cocultured
with SVT2/control cell (Fig. 4Fb), discrete punctate
staining for OX40L was shown on CD4+ T cells
cocultured with SVT2/OX40L (Fig. 4Fd). An essentially
identical discrete punctate pattern of cell surface staining for OX40L
was observed on CD4+ T cells cultured with
PFA-fixed SVT2/OX40L cells (data not shown). These results confirmed
the existence of OX40L or CD4+ T cells and
provided the pattern of distribution of transferred OX40L on
CD4+ T cells.
Function of transferred OX40L
Finally, we examined the kinetics of transferred OX40L on the cell
membrane of CD4+ T cells for understanding its
biological function. The transferred OX40L on the surface of
CD4+ T cells, after removal of cocultured
SVT2/OX40L cells, could be detected even 24 h after coculture
(Fig. 5A). The continued presence of OX40L on the cells led us to
expect a possible biological function of transferred OX40L. To
determine the stimulatory function of transferred OX40L, we used HIV-1
latently infected cells transfected with OX40 (ACH2/OX40), which
produce HIV-1 under stimulation via OX40 in vitro. Fig. 5B
shows that OX40L-transferred CD4+ T cells
specifically stimulated HIV-1 production in the ACH2/OX40 cells.
Coculture with the CD4+ T cells with
OX40-untransfected ACH2 cells did not stimulate HIV-1 production. The
HIV-1 production by the OX40L-transferred CD4+ T
cells was efficiently inhibited by anti-OX40L mAb, 5A8. These data
demonstrate that the transferred OX40L proteins are functional to
stimulate OX40-expressing cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Various types of cells expressing high levels of OX40L such as HTLV-I-infected human T cell lines and OX40L transfectants of mouse fibroblast or monkey kidney cell origins were capable of transferring OX40L onto the surface of various types of recipient cells including normal CD4+ T cells. Therefore, it is likely that intercellular transfer is a general phenomenon for OX40L molecules; this is independent of the other cellular factors of the OX40L donor cells. Although it has been shown that OX40 is the primary counterreceptor of OX40L, we found that OX40L was transferred not only to OX40-expressing cells but also to OX40-negative cells. These data suggest that there are OX40-dependent and -independent pathways in the intercellular transfer of OX40L. The former pathway is supported indirectly by our observations. Activated CD4+ T cells that expressed high levels of OX40 on the cell surface had more capacity for acquiring OX40L than resting OX40- CD4+ T cells. Although the slight increase of the acquisition of OX40L by IL-12-activated CD4+ T cells seemed to depend on the higher OX40 expression, it also implies a possible relationship between the OX40L acquisition and helper T cell differentiation status. That the level of OX40 on the activated CD4+ T cells was slightly down-modulated by OX40L transfer indicates either that the epitope of OX40 recognized by the anti-OX40 Ab used was masked by binding with the transferred OX40L molecule, or that the conformation of the OX40 molecule might be changed. However, we failed to coprecipitate OX40L protein by immunoprecipitation of OX40 from OX40L-transferred CD4+ T cell lysates with anti-OX40 mAb, B7B5 (data not shown). Additional experiments using a variety of anti-OX40 mAbs may clarify a possible OX40 involvement in the intercellular OX40L transfer to activated CD4+ T cells. The kinetics of transferred OX40L protein indicated that the transferred OX40L remained longer on the surface of CD4+ T cells than the other reported molecules. Because the high affinity and slow kinetics of OX40/OX40L interaction has been reported by using the magnetic resonance method (BIAcore biosensor) (38), we speculate that the OX40L transfer and continued presence of OX40L may partially depend on OX40. However, the present results showing an OX40-independent OX40L transfer also suggested an involvement of a yet unknown mechanism.
Our present data showed that cell-to-cell contact was essential for OX40L transfer. Because we could detect a full length of OX40L protein on the recipient CD4+ T cells, it is unlikely that the molecular transfer is mediated by an enzymatic cleavage mechanism. The OX40L transfer occurred from PFA-fixed OX40L+ cells, suggesting that focal membrane fusion and membrane vesicle formation are not applicable either. Furthermore, acid treatment did not reduce the level of OX40L expression on the surface of the recipient CD4+ T cells, suggesting that transferred OX40L molecules were anchored onto the cell membrane. In addition, confocal laser microscopy revealed the discrete punctate formation of transferred OX40L, which is similar to the morphological features of acquired MHC or B7-1 molecules (36, 37). Taken together, it is more likely that the carboxyl terminus of type II membrane protein OX40L can penetrate the recipient cell membrane in association with the unknown membrane molecule.
The OX40L transfer, which we have observed in an Ag-independent cell-to-cell interaction system using OX40L single-positive transfectants, may occur more frequently or efficiently in the Ag-specific T-APC interaction system accompanied by OX40/OX40L costimulation and in the T cell adhesion onto the endothelial cells. However, because the expression level of OX40L on normal cells was much lower than that on the transfectants, it has been difficult to detect transferred OX40L protein on CD4+ T cells from normal OX40L+ cells. Nevertheless, HUVECs and allostimulated monocytes were capable of transferring OX40L onto CD4+ T cells at low levels, suggesting that OX40L transfer may occur in inflammation conditions.
Although there were some reports showing the intercellular molecular transfer and internalization of membrane proteins in the T-APC interaction (7, 34, 35, 36), the functional roles of these phenomena are still not clarified. One possible function of the transferred protein is to provide a new phenotype to the recipient cells. By the process of the acquisition of peptide-MHC complex from APC by T cells, it was shown that the T cells became sensitive to peptide-specific lysis by neighboring T cells (36). Naive T cells after acquisition of CD80 from APCs were also reported to be capable of acting as APCs (7). The intercellular transfer of the chemokine receptor CCR5 by membrane-derived microparticles to CCR5-negative cells has been reported to render them susceptible to infection with macrophage-tropic CCR5 using HIV-1 (39). In this study, the ability of transferred OX40L to stimulate OX40+ cells, as revealed by an HIV-1 induction assay, indicates that transferred OX40L retains the active binding site to OX40 and keeps its proper protein conformation on the recipient cells. These observations strongly suggest that the OX40L-transferred recipient cells may acquire new ability to stimulate bystander OX40-positive cells and thus may potentially give rise to enhance an immune response via T cell-to-T cell costimulation accompanied with interactions of TCR/peptide/MHC complex or other costimulatory molecules (7).
Another possible biological function of OX40L transfer is to terminate the cell-to-cell interaction for allowance of cell dissociation (4, 6). Recently impaired down-regulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor in autosomal dominant periodic fever syndrome patients was reported to increase the inflammatory response (40). Because OX40L transfer may partially depend on OX40 on the recipient cells as mentioned above, it may be possible that transferred OX40L remains on the recipient cell surface and limits the further receptor-ligand interaction by blocking the binding site of OX40. For the verification of this hypothesis, analysis of the cells expressing the molecularly modified OX40L, which has an impaired capacity being transferred, may be suitable. These works and establishing a line of anti-OX40 or anti-OX40L mAbs with different blocking activities are now underway.
In conclusion, the findings of intercellular molecular transfer of OX40L protein provides us a new mechanism of modification of cell-to-cell interaction in a wide variety of immune responses.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eishi Baba at the current address: Department of Cancer Therapy and Research, Graduate School of Medical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail address: e-baba{at}tumor.med.kyushu-u.ac.jp, or Dr. Yuetsu Tanaka, Department of Infectious Disease and Immunology, Okinawa-Asia Research Center of Medical Science, Faculty of Medicine, University of Ryukus, 207 Uehara, Nishihara, Okinawa 903-0215, Japan. E-mail address: yuetsu{at}ma.kcom.ne.jp
3 Abbreviations used in this paper: OX40L, OX40 ligand; HTLV-I, human T cell leukemia virus type I; PFA, paraformaldehyde; DC, dendritic cell.
4 J. Lichetenfeld, Y. Takahashi, A. Yoshida, E. Baba, R. Tanaka, N. Yamamoto, and Y. Tanaka. Identification and measurement of OX40 ligand in various human cell lines using a library of specific monoclonal antibodies. Submitted for publication.
Received for publication November 27, 2000. Accepted for publication May 9, 2001.
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. Nature 385:733.[Medline]
from cells. Nature 385:729.[Medline]
and Notch. Cell 90:589.[Medline]
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