* Department of Hematology/Oncology and
Department of Tumor Biology, Graduate School of Medicine, Kyoto University, Japan
Correspondence: Toshiyuki Hori, MD, Department of Hematology/Oncology, Graduate School of Medicine, Kyoto University, 54 Shogoin-kawaracho, Sakyo, Kyoto, 606-8507, Japan. E-mail: thori{at}kuhp.kyoto-u.ac.jp
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chain and
anti-IL-2R ß chain mAbs abolished the costimulatory effects of HUVEC.
Thus, the augmentation of T-cell proliferation appears to be
attributable to increased IL-2 production. These results suggest that
gp34 expressed on HUVEC plays a role in potentiation of T-cell immune
response by providing OX40+ T cells with costimulatory
signals.
Key Words: endothelial biology • T lymphocytes • costimulation • TNF receptor family
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Tissue infiltration, as well as physiological homing of circulating leukocytes, involves a series of events initiated by interaction with vascular endothelial cells (EC). The evidence to date indicates that leukocytes adhere to EC sequentially through many adhesion molecules, such as selectins and integrins, and eventually transmigrate to the extravascular spaces in various organs [14 , 15 ]. In our previous study, we have demonstrated that gp34 is expressed on human vascular EC without stimulation in vitro and that the OX40/gp34 system mediates the adhesion of activated or HTLV-I-transformed T cells directly to vascular EC in vitro [16 ]. Although the distribution of gp34 expression in vivo has yet to be determined, it is conceivable that the OX40/gp34 system plays a certain role in the migration and recruitment of OX40+ T cells. In fact, we showed that peripheral blood mononuclear cells (PBMC), as well as tissue-infiltrating leukemic cells of most adult T-cell leukemia (ATL) patients, expressed OX40 constitutively and were able to adhere to human umbilical vein endothelial cells (HUVEC) via the OX40/gp34 system in vitro [17 ], suggesting that this system may be involved in tumor infiltration of various organs, such as lung, skin, and intestine.
Most of the adhesion molecules have been able to transmit intracellular signals [18 19 20 21 22 23 ], resulting in activation of cellular function. In the case of the OX40/gp34 system, its costimulatory function had been known before we showed that cell adhesion could also be mediated by this system. Accordingly, it is natural to speculate that EC provide T cells with costimulatory signals via the OX40/gp34 system. Previous studies have indicated that EC can augment T-cell response to antigens or mitogens in vitro [24 , 25 ]. Although some adhesion molecules, such as lymphocyte function-associated antigen (LFA)-3, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1, have been shown to play roles in such augmentation, the precise mechanism responsible for the costimulatory function of EC has not been elucidated fully, however.
So far, the involvement of the OX40/gp34 system with the costimulatory function of has not been explored. Therefore, in this study, we examined whether the OX40/gp34 system is involved in costimulation of T cells by EC. To address this question, we stimulated peripheral blood CD4+ T cells with anti-CD3-coated beads, phytohemagglutinin (PHA), or concanavalin A (Con A) in the presence of irradiated HUVEC, and we evaluated the effects of anti-OX40 or anti-gp34 monoclonal antibody (mAb) on T-cell proliferation. Here, we demonstrate that the OX40/gp34 system constitutes one of the distinct pathways through which HUVEC provide T cells with costimulatory signals. Data on IL-2 production during this proliferation and comparisons with other adhesion molecule systems are also presented.
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CD4+ T cells were purified from fresh PBMC by negative magnetic bead immunoselection, as described elsewhere [26 ]. Briefly, PBMC were incubated with CD8 (OKT8), CD11b (OKM1), CD14 (TÜK14), CD16 (3G8), and CD20 (1F5) at saturating concentrations, and immunomagnetic beads coated with anti-mouse immunoglobulin G (IgG; Dynabeads M-450, Dynal AS, Oslo, Norway) were added to the incubates to isolate the CD4+ T cells. The purity of the isolated CD4+ population assessed by FACScan analysis was over 94%, and the isolated CD4+ T cells were used for the proliferation assay and CTLL-2 bioassay.
HUVEC were isolated by collagenase digestion using the procedure described previously [27 ]. HUVEC monolayers were maintained in HuMedia-EG2 (Kurabo, Osaka, Japan), and cells at the second through fifth passages were used in the subsequent assays. A portion of HUVEC was fixed with 1% paraformaldehyde for 10 min at 25°C and washed five times with sterile phosphate-buffer saline (PBS).
CTLL-2, a murine IL-2-dependent cell line, was cultured in RPMI 1640 medium supplemented with 10% heat-inactivated FCS, 30 µg/ml tobramycin, 5 nM 2-mercaptoethanol (2-ME), and 0.5 nM recombinant human IL-2 (kindly provided by Shionogi Pharmaceutical).
mAbs
An anti-OX40 mAb, 131 (IgG1), and anti-gp34 mAb, ik1 (IgG1),
were generated in our laboratory as described previously
[16
, 28
]. Anti-Tac (anti-IL-2R ) and 2RB
(anti-IL-2R ß) were described elsewhere [29
,
30
]. The hybridomas of 1F5 (anti-CD20), OKM-1
(anti-CD11b), and OKT8 (anti-CD8), and a myeloma cell line, P3X63Ag8 (a
mouse mAb, control IgG1), were obtained from American Type Culture
Collection (Rockville, MD). 3G8 (anti-CD16) was a kind gift from Dr.
J. C. Unkeless (Mount Sinai Medical Center, New York). These mAbs
were purified from ascitic fluids using a mouse IgG purification kit
(Amersham Life Science, Arlington Heights, IL). AICD58 (anti-CD58,
LFA-3), 1G11 (anti-CD106, VCAM-1), 84H10 (anti-CD54, the extracellular
part of ICAM-1), and MAB104 (anti-CD80, B7-1) were purchased from
Immunotech S.A. (Marseille, France). IT2.2 (anti-CD86, B7-2) was
purchased from PharMingen (San Diego, CA). TÜR14
(anti-CD14) was purchased from Dako (Glostrup, Denmark).
The fluorescein isothiocyanate (FITC)-conjugated mAbs; Leu-2a (anti-CD8), Leu-3a (anti-CD4), Leu-4 (anti-CD3), Leu-11a (anti-CD16), Leu-16 (anti-CD20), Leu-M3 (anti-CD14), HLA-DR, and the phycoerythrin (PE)-conjugated anti-CD25 mAb were purchased from Beckton Dickinson (San Jose, CA). FITC-conjugated anti-OX40 mAb (FITC-315) was prepared in our laboratory as described previously [17 ].
Immunofluorescence staining and flow cytometric analysis
Cells were stained by direct or indirect immunofluorescence
using a FACScan (Becton Dickinson) as described previously
[31
]. Data were analyzed using CELLQuest software
(Becton Dickinson).
Proliferation assay
The proliferation assay was performed in 200 µl reaction
mixtures in 96-well, flat-bottomed microplates. Purified
CD4+ T cells (1x105 cells/well) were
stimulated with 5000/well anti-CD3-coated beads [Dynabeads M-450 (Pan
T), Dynal AS], 0.5 µg/ml PHA (Difco Labs, Detroit, MI), or 0.1
µg/ml Con A (Sigma Chemical Co., St. Louis, MO) and cultured in the
presence of 20 Gy irradiated or paraformaldehyde-fixed HUVEC. Cultures
were incubated at 37°C in a humidified atmosphere containing 5%
CO2 for 5 days. Tritium-labeled thymidine
([3H]TdR) (0.5 µCi/well; Du Pont/NEN Research Products,
Boston, MA) was added to each well 16 h before the harvest of the
culture, and cell proliferation was measured by the incorporation of
[3H]TdR. The radioactivity of the [3H]TdR
incorporation into the cells was measured by a microplate scintillation
counter (Packard Instrument Co., Downers Grove, IL).
Measurement of IL-2 production
CD4+ T cells were incubated under the conditions
described above for the proliferation assay. The culture supernatants
were collected after incubation for 48 h. The amount of IL-2 was
determined by bioassay using a murine IL-2-dependent cell line, CTLL-2,
as follows. Five thousand CTLL-2 cells were cultured with 50%
supernatants for 20 h, 0.5 µCi/well [3H]TdR was
added to each culture, and proliferation was measured after incubation
for a further 8 h, as described previously [32
].
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Figure 1. Expression of gp34, LFA-3, ICAM-1, and VCAM-1 on cultured HUVEC before
and after irradiation or fixation with paraformaldehyde. Intact
cultured HUVEC, irradiated HUVEC, and paraformaldehyde-fixed HUVEC were
stained with anti-gp34 (ik1), anti-LFA-3 (AICD58), anti-ICAM-1 (84H10),
anti-VCAM-1 (1G11), anti-CD80 (MAB104), or CD86 (IT2.2) mAbs by
indirect immunofluorescence and were subjected to flow cytometric
analysis. Dotted lines indicate staining with control mouse IgG1
antibody.
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Figure 2. Dose-dependent effects of anti-OX40 or anti-gp34 mAb on
CD4+ T-cell proliferation cultured with HUVEC.
CD4+ T cells were stimulated with: (A) 5000/well
anti-CD3-coated beads; (B) 0.5 µg/ml PHA; and (C) 0.1 µg/ml Con A.
Anti-OX40 mAb (131), anti-gp34 mAb (ik1), or control IgG at various
concentrations was added to CD4+ T cells in 96-well plates
in the presence or absence of irradiated HUVEC. [3H]TdR
incorporation of triplicated wells was measured on day 5. The results
are presented as the mean cpm ± SE.
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Figure 3. CD4+ T-cell proliferation cultured with
paraformaldehyde-fixed HUVEC. CD4+ T cells were cultured in
the presence or absence of paraformaldehyde-fixed HUVEC (10,000
cells/well) and were stimulated with 5000/well anti-CD3-coated beads in
the presence of anti-OX40 mAb (131), anti-gp34 mAb (ik1), or control
IgG (50 µg/ml) in 96-well plates. [3H]TdR incorporation
of triplicated wells was measured on day 5. The results are presented
as the mean cpm ± SE.
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Figure 4. Induction of IL-2R, HLA-DR, and OX40 on CD4+ T cells
cultured with HUEVC. CD4+ T cells were cultured with
5000/well anti-CD3-coated beads in the presence or absence of
irradiated HUVEC. We also added 50 µg/ml anti-gp34 mAb (ik1) in this
culture system. CD4+ T cells were harvested after 5 days
and examined for their expression of IL2R (CD25), HLA-DR, and OX40 by
direct immunofluorescence and flow cytometric analysis. Dotted lines
indicate staining with control mouse antibody.
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chain,
anti-CD25) and/or 2RB (anti-IL-2R ß chain) added to this culture
system inhibited HUVEC-induced CD4+ T-cell proliferation
strongly (Fig. 6
). We obtained similar results when CD4+ T cells were
stimulated with PHA or Con A (data not shown). Clearly, these results
show that HUVEC promoted T-cell proliferation via the OX40/gp34 system
by increasing IL-2 production.
 View larger version (40K): [in a new window] |
Figure 5. IL-2 production by CD4+ T cells stimulated by
anti-CD3-coated beads and HUVEC. CD4+ T cells were plated
in 96-well plates in the presence or absence of irradiated HUVEC and
stimulated with 5000/well anti-CD3-coated beads in the presence of
anti-OX40 mAb (131), anti-gp34 mAb (ik1), or control IgG (50 µg/ml).
For analysis of IL-2 production, supernatants were taken after 48 h and tested by CTLL-2 bioassay. CTLL-2 (5000/well) was cultured for
28 h with each supernatant, and [3H]TdR was added
during the last 8 h of the culture. All the experiments were
performed in triplicate. The results are presented as the mean cpm ± SE.
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 View larger version (41K): [in a new window] |
Figure 6. Effects of anti-IL-2R mAbs on the proliferation elicited by HUVEC.
CD4+ T cells were cultured with 5000/well anti-CD3-coated
beads in the presence or absence of irradiated HUVEC. Anti-Tac
(anti-IL-2R chain) and/or 2RB (anti-IL-2R ß chain) at 50 µg/ml
were added in this culture system. [3H]TdR incorporation
of triplicated wells was measured on day 5. The results are presented
as the mean cpm ± SE.
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Figure 7. Effects of anti-LFA-3 mAb, anti-ICAM-1, anti-VCAM-1, and anti-gp34 mAbs
on CD4+ T-cell proliferation cultured with HUVEC.
CD4+ T cells were cultured with 20 µg/ml of anti-gp34 mAb
(ik1), anti-LFA-3 mAb (AICD58), anti-ICAM-1 mAb (84H10), anti-VCAM-1
mAb (1G11), and control IgG in the presence or absence of irradiated
HUVEC. CD4+ T cells were stimulated with: (A) 5000/well
anti-CD3-coated beads; (B) 0.5 µg/ml PHA; and (C) 0.1 µg/ml Con A.
[3H]TdR incorporation of triplicated wells was measured
on day 5. The results are presented as the mean cpm ±
SE.
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Figure 8. Effects of combinations of anti-LFA-3 mAb, anti-ICAM-1, and anti-gp34
mAbs on CD4+ T-cell proliferation cultured with HUVEC.
CD4+ T cells were stimulated with: (A) 5000/well
anti-CD3-coated beads; (B) 0.5 µg/ml PHA; and (C) 0.1 µg/ml Con A
in the presence of irradiated HUVEC. Anti-gp34 mAb (ik1), anti-LFA-3
mAb (AICD58), anti-ICAM-1 mAb (84H10), and control IgG (20 µg/ml)
were added in this culture system at various combinations.
[3H]TdR incorporation of triplicated wells was measured
on day 5. The results are presented as the mean cpm ±
SE.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The mechanism of the costimulation by HUVEC is not a result of secondary effects of newly induced cell-surface molecules or HUVEC-derived cytokines, because paraformaldehyde-fixed HUVEC exerted similar costimulatory activity. We do not exclude the possibility that the OX40/gp34 system mediated only adhesion between T cells and that this caused the subsequent interaction between other preexisting costimulatory molecules or other adhesion molecules generating costimulatory signals. However, because OX40 is as a member of the TNF/NGF receptor family and can transmit costimulatory signals by itself on the ligand binding, it is more likely that gp34 expressed on HUVEC activated directly OX40+ T cells.
Augmented proliferation of T cells in the presence of HUVEC seems to be
ascribed to increased IL-2 production. It has been shown that the
ligand binding of OX40 together with T-cell antigen receptor (TCR)
triggering leads to enhancement of IL-2 production
[8
9
10
]. Because T-cell costimulation is often
determined as a measure of the augmentation of IL-2 production
[38
], our results are mostly compatible with those
studied previously. The intracellular signaling pathway of OX40 that
leads to IL-2 production has not been clarified thoroughly. We showed
that OX40 stimulation causes nuclear factor (NF)-
B activation
through TNF-R-associated factors (TRAF)2 and TRAF5 [39
].
Although nuclear factor of activated T cells (NF-ATs) are thought to be
mainly responsible for IL-2 induction [40
], recent
evidence indicated that c-Rel family-transcription factors including
NF-B also play important roles [41
, 42
].
Therefore, further investigations to determine whether OX40-induced
IL-2 production is mediated by NF-B or other transcription factors
should be carried out.
Comparing the CD2/LFA3, LFA-1/ICAM-1, and VLA-4/VCAM-1 systems indicated that the OX40/gp34 system constitutes one of the distinct pathways of costimulation by HUVEC. Previously, Savage et al. [23 ] pointed out that EC used at least two ligands; one is LFA-3 and the other has not yet been identified but does not appear to be ICAM-1, VCAM-1, CD44, or B7/BB1. We think that the other ligand on EC is likely to be gp34, because we found that only anti-gp34 mAb was an effective inhibitor of T-cell proliferation in response to anti-CD3-coated beads, PHA, or Con A. Moreover, the inhibitory effect of anti-gp34 mAbs was prominent when added together with other mAbs. Especially when T cells were stimulated with anti-CD3-coated beads or PHA, anti-LFA-3 and anti-gp34 mAbs together abolished such proliferation. The reason why anti-VCAM-1 mAb did not inhibit the augmentation of T-cell proliferation may be that HUVEC we used express little VCAM-1. Furthermore, in the case of these adhesion molecules, the same argument as mentioned above should be taken into consideration: Cell adhesion can trigger secondary interactions of other functional molecules. Simple inhibition assays using blocking mAbs may not be sufficient to define the roles of the individual molecules exactly.
Members of the NGF/TNF receptor family and their ligands are known to have various biological functions, such as cell growth, activation, differentiation, and survival [5 6 7 ]. To clarify the physiological significance of these molecules, it is important to know how they are expressed and distributed in vivo. We and others [43 , 44 ] have studied OX40 expression in lymphatic tissues in vivo under nonmalignant and malignant circumstances. Specifically, strong OX40 expression was detected on fresh leukemic cells from most ATL patients and on infiltrating lymphocytes from patients with autoimmune diseases [16 ]. However, the major problem with ascertaining the physiological relevance of the members of this family is that the expression of the ligands in vivo is elusive. For example, little is known about the expressions of Fas and CD27 ligands. Although we have detected gp34 expression in blood vessels in some skin and muscle biopsy specimens from patients with inflammatory skin diseases [44 ], its expression in the normal state has not been examined in detail. We need to elucidate the distribution of gp34 expression in vivo and how this expression is regulated, to understand better the roles of the OX40/gp34 system in T cell-EC interaction.
Received September 30, 1999; revised February 8, 2000; accepted February 10, 2000.
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chain-IL-2 complex in the formation of the ternary complex of IL-2 and high-affinity IL-2 receptor Int. Immunol. 2,521-530-dependent pathway J. Clin. Invest. 96,2839-2844B activation J. Biol. Chem. 273,5808-5814B J. Immunol. 147,416-420[Abstract]B/Rel transcription factors J. Exp. Med. 187,143-146This article has been cited by other articles:
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