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of General Virology (2001), 82, 831–835. Printed in Great Britain
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SHORT COMMUNICATION
Vascular cell adhesion molecule-1 induced by human T-cell
leukaemia virus type 1 Tax protein in T-cells stimulates
proliferation of human T-lymphocytes
He! le' ne Valentin,1, 2 Samir Hamaia,1 Ste! phane Ko$ nig1 and Louis Gazzolo1
1
Virologie et Pathoge! ne' se Virale (UMR5537), CNRS-UCB Lyon 1, Faculte! de Me! decine Laennec, Rue Guillaume Paradin,
69372 Lyon Cedex 08, France
2
Immunobiologie Fondamentale et Clinique, INSERM U503, 21 Avenue Tony Garnier, 69365 Lyon Cedex 07, France
Human T-cell leukaemia/lymphotropic virus type 1
(HTLV-1), aetiologically linked to lymphoproliferative as well as inflammatory diseases,
infects and activates CD4M helper T-cells and thus
alters immunoregulatory pathways. The viral regulatory Tax protein has been shown previously to
induce the expression of vascular cell adhesion
molecule-1 (VCAM-1) by T-cells. To determine the
functional role of this adhesion molecule, Jurkat Tcells stably expressing either Tax or both Tax and
Rex (another viral regulatory protein) were used in
binding and coculture assays performed with either
control Jurkat cells or primary human T-lymphocytes. Evidence was provided that VCAM-1 acting
in synergy with leucocyte function-associated
antigen-3 promotes T-cell–T-cell interactions and
increases T-cell proliferation. Interestingly, Rex was
found to modulate these events. These data establish that VCAM-1 induced by Tax on T-cells thus
contributes to the immunopathological process
triggered by HTLV-1 infection.
Among individuals infected with human T-cell leukaemia\
lymphotropic virus type 1 (HTLV-1), a small percentage
develop a lymphoproliferative disease, adult T-cell leukaemia
(ATL) or a chronic inflammatory disease, such as tropical
spastic parapesis\HTLV-1-associated myelopathy (TSP\
HAM) (reviewed by Uchiyama, 1997). HTLV-1 is considered
to be a retrovirus that alters immunoregulatory pathways. In
vivo and in vitro studies have demonstrated that HTLV-1
infects CD4+ helper T-cells, which are then characterized by an
increased expression of activation markers and spontaneous
proliferation. Both processes are dependent on the activity of
Author for correspondence : He! le' ne Valentin at INSERM.
Fax j33 4 37 28 23 41. e-mail valentin!cervi-lyon.inserm.fr
0001-7543 # 2001 SGM
the viral regulatory protein Tax, which is involved in the
transcriptional activation of the provirus and expression of
many cellular genes implicated in T-cell activation and
proliferation. More specifically, Tax has been shown to
intervene in T-cell–T-cell interactions by enhancing the
expression of an adhesion molecule, leukocyte functionassociated antigen-3 (LFA-3 or CD58), which is the ligand for
CD2 (Tanaka et al., 1995). The interaction of LFA-3 expressed
by HTLV-1-infected T-cells with CD2 on resting T-cells
defines an important costimulatory pathway in triggering the
proliferation of resting T-cells (Wucherpfennig et al., 1992). In
addition, other adhesion molecule interactions, such as intercellular adhesion molecule-1 (ICAM-1 or CD54) with LFA-1
(CD11a\CD18), have been suggested to participate, first, in
HTLV-1-mediated activation of uninfected T-cells, facilitating
lymphoproliferation, and second, in increased adhesion to
endothelial cells, resulting in tissue or organ infiltration
(Ichinose et al., 1992, 1994).
We have reported previously the expression of vascular
cell adhesion molecule-1 (VCAM-1 or CD106) by Jurkat cells
stably expressing the tax gene and shown that Tax was transactivating the VCAM-1 gene via two NF-κB sites present in
the gene promoter (Valentin et al., 1997). Moreover, this
VCAM-1 up-regulation has been confirmed in freshly isolated
T-cells from TSP\HAM patients (Valentin et al., 1997).
VCAM-1 may serve as either an accessory molecule or a
potential coreceptor for HTLV-1-induced cell fusion (Daenke et
al., 1999 ; Hildreth et al., 1997). VCAM-1 is normally expressed
by stimulated endothelial cells, therefore favouring the adhesion of these cells to T-lymphocytes via VLA-4, which is the
VCAM-1 ligand that is constitutively expressed by T-cells.
Thus, this adhesion pathway may be important in mediating
emigration of leukaemic T-cells into the organs of ATL
patients and entry of activated T-cells into the central nervous
system of individuals with TSP\HAM (Ishikawa et al.,
1993 ; Uchiyama, 1997). Furthermore, a recent report has
underlined the role of VCAM-1 in the induction of matrix
metalloproteinase-2, which is an important mediator for the
extravagation of T-cells and the degradation of the subendo-
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H. Valentin and others
Fig. 1. Comparative analysis by flow cytometry of cell surface (A) LFA-3
(CD58), (B) VCAM-1 (CD106) and (C) VLA-4 (CD29/CD49d)
expression by parental Jurkat cells, by control neomycin-resistant Jurkat
cells (C9), and by Jurkat cells stably expressing either Tax (50) or both
Tax and Rex (C11 and E12). Bars represent the mean of fluorescence
intensity. Data represent the meanpSD from 3 to 10 experiments. The
asterisks (C) indicate a statistically significant difference compared with
control, as calculated by Student’s t-test (P 0n05).
thelial basement membrane (Kambara et al., 1999). Alternatively, we have proposed that VCAM-1 expressed by HTLV1-infected T-cells might favour the interaction of these cells
with each other and with uninfected T-cells expressing VLA-4.
IDC
Indeed such interactions may lead to cell fusion and\or
transmission of HTLV-1.
In the present study, we have investigated the functional
consequences of Tax-induced VCAM-1 expression by T-cells
in the activation and proliferation of normal resting T-cells. To
that aim, we have used, in addition to parental Jurkat and Taxnegative neomycin-resistant Jurkat cells, three clones of Jurkat
cells stably expressing either Tax (clone 50) or Tax and Rex,
another viral regulatory protein, but acting at a posttranscriptional level (clones C11 and E12) (Lemasson et al.,
1997). We first performed a flow cytometry analysis to
compare the expression of VCAM-1 to that of LFA-3 by the
different Jurkat cell lines. We confirmed that VCAM-1 was
significantly expressed by Tax-expressing Jurkat cells (C11,
E12 and 50) compared with the Tax-negative C9 and parental
T-cells (Fig. 1 B) (Valentin et al., 1997). Furthermore, the
expression of LFA-3 was found to be up-regulated in the three
Tax-positive clones when compared to the Tax-negative cells
(Fig. 1 A). We then analysed the profile of VLA-4 (CD29\
CD49d) expression. In contrast to CD29 (β1 integrin) expression, CD49d (α4 integrin) down-regulation was statistically correlated, by the Student t-test, to Tax expression in
T-cells (Fig. 1 C). These data propose that Tax expression
might induce a switch of VLA-4 expression to its VCAM-1
ligand on T-cells. As the level of CD49d expression has been
reported to vary among ATL and HTLV-1-infected cases (AlFahim et al., 1999 ; Dhawan et al., 1993 ; Ishikawa et al.,
1993 ; Uchiyama et al., 1996 ; Wake et al., 1995), it cannot be
excluded that the expression of this integrin may be dependent
on the level of Tax or of any viral or cellular protein.
Next, we investigated the functional consequences of
VCAM-1 and\or LFA-3 expressed by Jurkat cells stably
expressing Tax. We first evaluated the ability of Jurkat Taxpositive cells to adhere to Tax-negative T-cells by rosette
assay analysis. Briefly, 1i10& Tax-negative T-cells were
labelled with Hoechst H33342 fluorochrome (Sigma), as
described previously (Weston & Parish, 1990), and then mixed
with either Tax-negative or Tax-expressing T-cells for 15 min
at 37 mC. No rosette formation was observed between parental
Jurkat cells, between parental Jurkat and Tax-negative
neomycin-resistant Jurkat (C9) cells or between C9 cells. On
the contrary, from 15 to 48 % of Tax-negative T-cells were
found to bind to C11, E12 and 50 Tax-expressing T-cells
(Table 1 and data not shown). These results underline the fact
that Tax is mediating T-cell–T-cell interactions. To determine
the involvement of adhesion molecules, Tax-expressing cells
were first incubated with specific MAbs (10 µg\ml for 40 min
at 4 mC) and then with labelled Tax-negative T-cells. Contrary
to an incubation with an irrelevant MAb (to CD46) which was
unable to interfere with rosette formation, inhibitions of 38 and
48 % were observed when Tax-expressing T-cells were preincubated with blocking MAbs to LFA-3 or VCAM-1,
respectively (Table 1). This inhibition increased up to 75 %
when Tax-expressing cells were treated with both MAbs.
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Tax-induced VCAM-1 and T-cell proliferation
Table 1. Effect of blocking VCAM-1 and LFA-3 MAbs on
binding to parental Jurkat cells
Control parental Jurkat cells were labelled with Hoechst H33342
fluorochrome and incubated with Jurkat E12 cells stably expressing
Tax and Rex, at a ratio of 10 : 1, respectively, for 30 min at 4 mC. E12
cells were pre-incubated with medium (none), an irrelevant MAb to
CD46 (MCI20.6) or with blocking MAbs to LFA-3 (AICD58) or
VCAM-1 (1G11) at 10 µg\ml. Cells were then observed with a
fluorescence microscope (Nikon-Diaphot ELW D 0.3). A rosette was
scored when at least three labelled cells were binding one unlabelled
cell. Data represent the mean of three separate experiments (SD
10 %). Results are expressed as percentage rosette formation (labelled
parental Jurkat cells bound to E12 cells) or percentage inhibition of
rosette formation observed with MAb-treated E12 cells to that
observed with untreated E12 cells.
Pre-treatment
of E12 cells
None
Anti-CD46
Anti-LFA-3
Anti-VCAM-1
Anti-LFA-3 j anti-VCAM-1
Rosette
formation
(%)
Inhibition
of rosette
formation (%)
36n5
34n6
22n5
19n0
9n0
–
5n2
38n3
47n9
75n3
These data clearly provide evidence that VCAM-1 induced in
Tax-positive T-cells is involved in T-cell–T-cell interactions
and that both VCAM-1 and LFA-3 are acting synergistically in
this adhesion process.
These data suggest that these adhesion molecules may
increase T-cell proliferation by enhancing binding of Taxexpressing T-cells to normal T-cells. To verify this hypothesis,
human T-lymphocytes were purified and cultured, as described
elsewhere (Huet et al., 1986). Briefly, mononuclear cells,
obtained by Ficoll–hypaque centrifugation of peripheral blood
from normal healthy donors (E! tablissement de Transfusion
Sanguine de Lyon), were incubated for 1 h at 37 mC in plastic
culture dishes. Nonadherent cells were then depleted of
phagocytic cells by carbonyl iron ingestion and passage over
a magnet. Accessory cells were lysed after incubation with leucine methyl ester. After eliminating residual HLA-DR cells,
this preparation was analysed by flow cytometry and found to
contain more than 95 % CD2+CD3+ cells (data not shown).
Under these conditions, these T-lymphocytes were unable to
proliferate when stimulated via the CD3\TCR pathway unless
they were incubated with accessory cells. We therefore
investigated whether the Tax-expressing Jurkat cells were
endowed with such a property. To investigate this, Taxnegative or -positive cells were fixed with 1 % paraformaldehyde. No significant [$H]thymidine incorporation by these
cells could be detected (less than 300 c.p.m.). Then, 2i10%
fixed cells were incubated with 5i10% T-lymphocytes in the
Fig. 2. Comparative analysis of the ability of parental Jurkat cells, control
neomycin-resistant Jurkat cells (C9) and Jurkat cells stably expressing
either Tax (50) or both Tax and Rex (C11 and E12) to induce
proliferation of normal human T-lymphocytes. (A) Purified T-lymphocytes
incubated in 96-well plates coated with anti-CD3 MAb for 1 day and then
cocultivated for 3 days at 37 mC with paraformaldehyde-fixed Jurkat cells.
Cells were then incubated with 1 µCi [3H]thymidine for 16 h. Data are
expressed as [3H]thymidine (c.p.m.) incorporated by T-lymphocytes.
(B) Inhibition of T-lymphocyte proliferation as stimulated in (A) and
cocultivated with Jurkat cells (stably expressing Tax or Tax and Rex) pretreated for 1 h with anti-CD46, anti-VCAM-1 (1G11) or anti-LFA-3
(AICD58) MAb. Data are shown as percentage proliferation of Tlymphocytes cocultivated with untreated Jurkat cells. Data represent the
meanpSD of three separate experiments.
presence of 5 µg\ml CD3 MAb pre-adsorbed onto a 96-well
plate. The proliferation of these T-lymphocytes was assessed
following the addition of [$H]thymidine. As shown in Fig.
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H. Valentin and others
2 (A), Tax-negative Jurkat cells were unable to induce DNA
synthesis whereas Tax-positive cells induced a significant level
of [$H]thymidine incorporation. These observations indicate
that a costimulatory signal is delivered by Tax-positive Jurkat
cells. To determine whether VCAM-1 induced by Tax is
involved in the proliferation of CD3-stimulated Tlymphocytes, we performed blocking experiments in which
Tax-expressing Jurkat cells were pre-treated with 10 µg\ml
VCAM-1-blocking MAb for 30 min at 4 mC, fixed and then
cocultivated with CD3-stimulated T-lymphocytes. A 15 to
20 % inhibition of the proliferation of CD3-stimulated Tlymphocytes cocultivated with C11 and E12 cells (which
express both Tax and Rex) was observed, whereas a 90 %
inhibition was reached in the presence of cells from clone 50
(which expresses only Tax) (Fig. 2 B). Likewise, [H$]thymidine
incorporation by CD3-stimulated T-lymphocytes was reduced
by about 70 % only with fixed cells of clone 50 which had been
pre-treated with the LFA-3-blocking MAb (Fig. 2 B). The
discrepancy in the inhibition of T-cell proliferation between
Jurkat cells expressing Tax and Rex and those expressing only
Tax proposes that the stimulatory pathway induced by
VCAM-1 and LFA-3 is the only one operating in cells
expressing Tax alone. Consequently, other molecules in cells
expressing both Tax and Rex might be required to fully
activate resting T-cells (reviewed by Ho$ llsberg, 1999). Collectively, these data indicate that Rex may be involved in the
expression of these molecules and\or in the down-regulation
of the stimulatory potential linked to VCAM-1 and LFA-3.
This former hypothesis is supported by previous observations
indicating that Rex has the capability to increase cellular gene
expression. Indeed, it has been reported that Rex may
contribute to human interleukin-2 gene expression regulation
and in stabilizing the interleukin-2 receptor α-chain mRNA in
T-cells (Kanamori et al., 1990 ; McGuire et al., 1993). More
interestingly, a recent study has underlined that the concomitant expression of Tax and Rex in Jurkat cells resulted in
the overexpression of a protein tyrosine kinase, FynB, probably
through a splicing mechanism controlled by Rex (Weil et al.,
1999).
In conclusion, VCAM-1 induced by HTLV-1 Tax protein in
infected T-cells is able to promote T-cell–T-cell adhesion, thus
contributing to the proliferation of uninfected T-lymphocytes.
Such events may have particular relevance to an understanding
of the immunopathogenesis of HTLV-1 and of the spontaneous
proliferation of T-cells of HTLV-1-infected individuals, which
contributes to an amplification of immune system activation in
an antigen-independent manner, thus favouring HTLV-1
dissemination and maintenance of the disease.
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Received 13 November 2000 ; Accepted 26 December 2000
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