By
From the * Laboratoire d'Immunologie, Institut de Recherches Cliniques de Montréal, H2W 1R7
Montréal, Canada; Laboratoire d'Immunochimie Analytique, Institut Pasteur, 75015 Paris, France;
and § Department of Microbiology and Immunology, Universite de Montréal, H3C 3J7 Montréal,
Canada and Department of Microbiology and Immunology, and Department of Experimental
Medicine, McGill University, H3A 2B4 Montréal, Canada
Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class
II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer
usually leads to the activation and deletion of T cells expressing responsive V
s. However,
there is no direct molecular evidence for such a transfer. We have developed an in vitro system
which confirms this property of vSAGs. vSAG7 was transfected into a class II
murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V
s led to high levels of IL-2 production which was specifically inhibited by
vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II
cells in
mice led to expansion of V
6+ CD4+ cells. We show that this transfer activity is paracrine but
does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable
membranes. Transfer can occur both from class II
and class II+ cells, indicating that MHC
class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with
well defined binding sites showed that the transferred vSAG7 fragment binds to the
1 domain
of HLA-DR.
Superantigens (SAGs)1 are a family of proteins which can
induce the stimulation, followed in vivo by the deletion of
a subset of T cells sharing particular TCR V Endogenous superantigens are encoded in the mammalian genome by retroviruses (MMTVs) that have integrated
in the germline host DNA. Most of these integrated proviruses have lost their ability to form virions, but continue to
express viral proteins. More than 30 sites of integration
(Mtv) for distinct MMTV on different chromosomes are
known, with most of the common murine strains possessing two to eight of these loci [11, 12]. The 3 The primary sequence of different vSAGs is highly conserved except in the COOH terminus region (18, 19),
which imparts V Different studies have demonstrated that vSAG presentation is not strictly restricted to particular alleles of class II.
However, some murine alleles of MHC class II do present
vSAGs better than others, and the hierarchy was established
as: I-E >I-A >I-Aq (24). As demonstrated by Labrecque
et al. (28), human DR alleles can also present vSAGs to human PBMCs. Moreover, human class II alleles and isotypes
show a hierarchy in vSAG presentation (29).
Several reports have suggested that vSAGs can be intercellularly transferred from class II These in vivo studies have provided strong arguments
for the transfer of vSAGs from cells which do not express
class II (or which express class II isotypes that fail to present
vSAG7) to class II+ cells proficient in vSAG presentation.
However, it has never been possible to exclude that undetected expression or passive acquisition of MHC class II by
T cells rather than vSAG7 transfer is responsible for the observed V Mice.
5-wk-old CBA/CaJ female mice were purchased from
Jackson Laboratories (Bar Harbor, ME).
Culture Media.
PBMCs were cultured in RPMI 1640 supplemented with 5% FCS, 10 µM segments. These
SAGs can be either integrated in the host genome (endogenous superantigens) or can be produced by an increasing
array of pathogens (exogenous superantigens). These include microorganisms such as bacteria (staphylococci [1],
streptococci [2, 3]), or viruses (MMTVs [4, 5], rabies virus
[6]). The fact that they are conserved in such a wide array
of pathogens indicates that they might play a pivotal role in
the pathogenic process. Indeed, in the case of mouse mammary tumor virus (MMTV) encoded superantigens, it has
been clearly demonstrated that viral infection is directly associated with the expansion of cells carrying the V
elements responding to the vSAGs (7). Moreover, the presence of V
6+ cells which are responsive to the rabies virus
SAG is required to induce paralysis of rabies-infected mice
(6, 10).
LTR of Mtvs
contains an open reading frame encoding the viral superantigen (vSAG) (13), which has been characterized as a
type II transmembrane protein (16) with five glycosylation
sites and three furin-like cleavage sites (17).
specificity (20, 21). When vSAG7, the
3
LTR product of Mtv7 is presented at the cell surface by
MHC class II molecules, T cells expressing TCR V
6, 7, 8.1, or 9 chains are triggered to proliferate in vitro or are
deleted in vivo (4, 5, 22, 23).
cells that express vSAGs
to class II+ cells (30): first, it was shown that the response of a T cell clone to minor lymphocyte stimulatory (Mls) required the presence of B cells and of splenic
adherent cells (SAC) (30), suggesting that SAC cells
present an Mls fragment transferred from B cells. However, these experiments failed to demonstrate whether
spleen cells provide a costimulatory signal or if they stimulate
the T cell clone after the transfer of vSAG7 from B cells. It
was then shown that F1
parent chimeras between mtv7+
I-E
mice from the H-2q haplotype (which do not delete
V
6 cells) and mtv7
I-Ed mice leads to the elimination of
V
6+ cells (31). Lastly, injection of mtv7
mice with
CD8+ cells from mtv7+ mice leads to the activation and
expansion of the mtv7 responsive V
6+ cells; it is noteworthy that the magnitude of the activation of V
6+ cells
was comparable after the injection of CD8+ cells which are
class II
and B cells which express high levels of class II,
suggesting the transfer of vSAG from CD8+ cells to class
II+ cells (32, 33).
-specific expansions. In this report we have developed an in vitro system to confirm at the molecular level
if vSAG7 transfer occurs and to get insights at the mechanisms leading to the intercellular transfer of vSAGs.
-mercaptoethanol, and 20 µg/ml
gentamycin (all GIBCO BRL, Gaithersburg, MD). Fibroblasts
were cultured in DMEM supplemented with 10% calf serum,
10
2 M Hepes (all GIBCO BRL), and 20 µg/ml gentamycin.
-mercaptoethanol, 20 µg/ml gentamycin, 4 mM Dextrose (Sigma Chemical Co., St. Louis, MO), essential and nonessential amino acids (GIBCO BRL), 1 mM Sodium
pyruvate (GIBCO BRL), and 10 mM Sodium Bicarbonate (Sigma).
Cell Lines.
We used DAP DR1 cells (DAP cells transfected
with MHC class II DR1 and
chains) (34), DAP vSAG7 cells
(DAP cells transfected with vSAG7 gene) and 3B2, called DAP DR1
vSAG7 cells thereafter (DAP cells transfected both with DR1 and
vSAG7 genes [28]). A panel of T cell hybridomas, Kmls 13.11, Kmls 12.6 (kindly provided by Drs. J. Kappler and P. Marrack,
National Jewish Hospital and Howard Hughes Institute, Denver,
CO), RG17 (kindly provided by Dr. B. Huber, Tufts University,
Boston, MA), and KR3 (kindly provided by Dr. O. Kanagawa,
Washington University, St. Louis, MO) was used in these experiments. Kmls 13.11, Kmls 12.6, and RG17 express V
6. KR3 expresses V
8.1.
Monoclonal Antibodies.
The mAb specific for the murine V3
(KJ25) was purified and biotinylated using standard procedures
(35). The mAb directed against V
6 (RR4-7) was used as a supernatant. The CD4 specific mAb (GK1-5) was purified and
FITC-conjugated using standard procedures (35). FITC mAb directed against human V
12 and V
13 were purchased from Immunotech (Marseille, France). OT145, a mAb directed against V
6.7, is a kind gift or Dr. D. Posnett (The New York Hospital, Cornell Medical Center, New York). PE-conjugated anti-CD4
(leu-3a) mAb was purchased from Becton Dickinson (Mountain
View, CA).
Transfections.
DAP cells were transfected using the Calcium
Phosphate precipitation technique (37) with the vSAG7 gene
cloned in the expression vector pH-Apr1-neo (38). G418 resistant clones were grown and screened for the expression of
vSAG7 by Northern blot analysis.
Northern Blot Analysis. RNA was isolated using RNAzol B (Cinna/Biotecx Laboratories, Friendswood, TX). 20 µg of RNA were fractionated on 1.2% formaldehyde-agarose gels (39), transferred onto nylon membranes (Amersham Corporation, Oakville, Ontario, Canada) and hybridized at 42°C in 50% formamide with vSAG7 (EcoRI-BglII fragment of 0.9 kb) (29) or actin (PstI fragment of 1.1 kb) (40) probes labeled using a random priming kit (Pharmacia, Uppsala, Sweden). Blots were washed at 65°C in 5× SSC, 1× SSC and finally in 0.1× SSC solutions, and exposed on Kodak XAR-5 film for 24 h.
Functional Assays. 60 × 104 DAP DR1 cells were cocultured with 60 × 104 T cell hybridomas and various concentrations of DAP vSAG7 for 24 h in 250 µl of DMEM supplemented with 10% calf serum. Supernatants were harvested, and IL-2 production was measured using the CTLL hexoaminidase assay (41).
Transfer experiments were carried out using transwells (Nunc, Naperville, IL) with 0.2-µm pores. 60 × 103 DAP DR1 and 60 × 104 hybridoma cells were added in the lower compartment, while various amounts of DAP vSAG7 cells were added in the upper compartment.PBMC Stimulation.
Blood was obtained from different
healthy donors, and PBMCs were purified using ficoll-hypaque
gradients (Pharmacia) as previously described (28). One million
PBMCs in 1 ml of RPMI 10% FCS were incubated for 7 d in 24well plates (Falcon, Becton Dickinson, Plymouth, UK) in the
presence of either DAP vSAG7 cells treated with mitomycin C
(100 µg/ml, 1 h at 37°C) or for 4 d in the presence of the bacterial superantigen Staphylococcal enterotoxin B (SEB) (Toxin
technologies, Sarasota, FL). Viral or bacterial superantigens were
either put in the same chamber as PBMCs, or were separated from PBMCs by a transwell (Costar, Cambridge, MA) with 0.4µm pores. Cells were then harvested and tested by flow cytometry for V expression.
Cytofluorimetry.
For human TcR V repertoire studies, 2-5 × 105 cells were stained with anti-V
antibodies at previously defined optimal titers for 20 min at 4°C in the dark and washed in
PBS containing 2% FCS. When necessary cells were then incubated with FITC-conjugated goat anti-mouse mAb, and 10%
normal mouse serum (Jackson Laboratories) in PBS. After washing, cells were finally incubated with PE- or PerCP-conjugated antiCD4 for 20 min in the dark. For murine TcR V
repertoire
studies, cells were first incubated with biotinylated anti-V
mAbs.
In a second step, they were incubated with FITC-conjugated antiCD4, and finally, with PE-conjugated streptavidin. For RR4-7, two additional steps (incubation with biotinylated goat anti-rat and then with normal rat serum 10% in PBS) were made before
adding FITC-conjugated anti-CD4. Acquisition and analysis of
cells were carried out using a FACScan® and the Lysis II software
(Becton Dickinson, Mountain View, CA). For each analysis, a minimum of 1 × 104 live cells gated by forward and side scatter were
analyzed.
The vSAG7 gene was transfected into the MHC class II
DAP3 murine fibroblastic line. Seven clones of G418-resistant cells were analyzed by Northern Blot. Equal loading of
all wells was confirmed by comparable levels of actin mRNA
in all clones tested (Fig. 1 a). When compared to other
clones, clones 17 and 18 showed significantly higher levels
of vSAG7 mRNA (Fig. 1 b). Clone 2 also expressed vSAG7 mRNA, although at lower levels. To verify the capacity of MHC class II
vSAG7+ cells to transfer vSAGs,
we developed a transfer assay in which equal numbers (6 × 104) of DAP DR1 cells and the V
6+ vSAG7-responsive
Kmls 13.11 hybridomas were cocultured for 16 to 20 h together with various amounts of the DAP vSAG7 cells. T cell
stimulation was assessed by measuring IL-2 present in the supernatants. The three vSAG7+ clones (clones 2, 17, and
18) were able to induce a dose dependent T cell activation
in several independent experiments. A representative experiment is shown in Fig. 1 c where increases in the production of IL-2 ranged from 10- to 20-fold as compared to
cocultures of DAP vSAG7 cells and untransfected DAP cells.
The latter indicated the absolute prerequisite for MHC
class II molecules in order to obtain vSAG7 presentation.
Levels of IL-2 production were directly correlated with the
number of cells expressing vSAG7 or with the levels of
vSAG7 expressed by the class II
fibroblasts. Indeed, little
or no stimulation was observed with less than 2 × 103 DAP
vSAG7 cells, whereas maximal response was obtained with
5 × 104 DAP vSAG7 cells per well.
Controls were performed to eliminate the possibility that this activity could be caused by the fusion of DAP DR1 with DAP vSAG7 cells. A flow cytometric assay was developed in which two different fibroblastic lines expressing distinct surface markers were cocultured for the duration of the above described functional assay (16 to 20 h). Results indicate that the percentage of fused cells was below 1% and fusion could thus not account for the superantigenic activity (data not shown). Our experiments thus indicate that a fragment of the vSAG7 protein carrying superantigenic activity is transferred intercellularly. Additional experiments were performed to compare the superantigenic activity in the transfer assay and in the direct presentation assay. Results illustrated in Fig. 1 (d and e) show that DAP vSAG7 and DAP DR1 vSAG7 express comparable levels of vSAG7 mRNA. The levels of stimulation induced by vSAG7 in transfer assays or in direct presentation assays are similar (Fig. 1, d-f ), indicating that transfer is an efficient process.
A similar strategy was used to monitor the transfer capacity of the exogenous vSAG GR. Results indicate that transfer of vSAG GR can occur from DAP DR1 vSAG GR to
CH12 cells and lead to the stimulation of KOX15, a
V15-expressing hybridoma (data not shown).
To further demonstrate the requirement for vSAG7
in order to obtain T cell stimulation we used the vSAG7specific mAb 6E1 to inhibit stimulation in the transfer assay. Results illustrated in Fig. 2 a clearly show that this
mAb inhibits vSAG7 activity in a dose-dependent manner,
as previously demonstrated for presentation of endogenously
expressed vSAG7. As a control of specificity, we show that
mAb 6E1 fails to inhibit SEB presentation (data not shown).
Inhibition experiments carried out with the MHC class II
DR-specific mAb XD5.117 further confirmed the requirement for DR expression in order to stimulate T cells in the
transfer assay (Fig. 2 b). Indeed, the XD5.117 mAb inhibited vSAG7 stimulation in a dose-dependent manner, further confirming the similarity between the transfer of vSAG7
and the presentation of endogenous vSAGs. Altogether, these
experiments confirmed that vSAG7 can be expressed at the
surface of MHC class II cells and is directly transferred to
MHC class II molecules expressed at the surface of DAP DR1
cells.
vSAG7 not
only reacts with V6-expressing T cells, but also V
8.1expressing T cells. A panel of V
6- (Kmls 13.11, Kmls
12.6 and RG17)- or V
8.1 (KR3)-expressing hybridomas
was used to demonstrate that transfer of vSAG7 was capable of stimulating T cells expressing different vSAG7responding TCRs. All these hybridomas were stimulated at
comparable levels by vSAG7 both in the transfer assay and
in direct presentation by DAP DR1 vSAG7 cells (Fig. 3 a).
In order to provide evidence that transfer of vSAG7 is not dependent on particular properties of DAP DR1 cells, we used CH12 cells, a murine B cell hybridoma expressing both I-Ek and I-Ak. CH12 cells did not stimulate Kmls 13.11 cells when cocultured with untransfected DAP cells, as indicated in Fig. 3 b. However, when CH12 cells were cocultured with DAP vSAG7 cells, efficient stimulation of Kmls 13.11 hybridoma occurred. Levels of T cell stimulation were directly correlated to the number of DAP vSAG7 cells added to the coculture and ranged in the same order of magnitude than those obtained using DAP DR1 cells (Fig. 3 b).
In Vivo Transfer of vSAG7.To further demonstrate that
the transfer activity bears physiological relevance, experiments were set up to verify if transfer of vSAG7 from DAP
vSAG7 occurs in vivo. For this purpose, mice were injected with 3 × 106 DAP vSAG7 in the hind footpad. Five
days following injection, draining lymph node cells were
analyzed by flow cytometry for TcR V6 expression. A
representative experiment is shown in Fig. 4. In this experiment, the percentage of V
6-expressing cells among the
CD4+ cells increases from 11% (mice injected with DAP
DR1 cells) in control mice to 25% in mice injected with
DAP vSAG7 cells. This enhancement is comparable to the
one observed after injection of DAP DR1 vSAG7 cells. As
previously shown following injection of B cells or CD8+
cells expressing vSAG7, we did not observe any expansion
of V
6+ CD8+ cells.
These results demonstrate that the fragment carrying the
superantigenic activity is thus transferred from class II DAP
vSAG7 to MHC class II+ cells in lymph nodes.
Labrecque et al. (28) have shown that
DAP DR1 vSAG7 cells are able to induce the proliferation
of human PBMCs. We show here that class II DAP vSAG7
cells are also able to stimulate PBMCs in a V
-restricted manner. DAP vSAG7 (2 × 105) were cocultured with
PBMCs (106) for 10 d. PBMCs were then harvested and
tested by cytofluorimetry for V
expression among CD4+
blasts. A representative experiment is shown in Fig. 5 and
demonstrates a three- to fourfold increase in the percentage
of V
12+ CD4+ blasts, as compared to PBMCs cocultured
with untransfected DAP cells. The percentage of V
6.7
cells, which are not responsive to vSAG7, remained unchanged.
Characterization of the Intercellular Transfer of vSAG7.
Whereas the above results undoubtedly showed that transfer of vSAGs is possible, they did not address the requirement for cellular interactions in this process. We thus performed transfer assays in which DAP vSAG7 cells were
separated from DAP DR1 cells and Kmls 13.11 hybridomas by a semi-permeable membrane. No stimulation could
be observed under these conditions (Fig. 6 a) even with concentrations of DAP vSAG7 cells as high as 2 × 106 per
ml. Conversely, SEB, even at low concentrations (1.5 ng/ml), was able to cross this membrane and stimulate the V6 hybridoma. These results raised the hypothesis that transfer is
mediated by an insoluble or unstable fragment. However,
we were not able to exclude the possibility that a small proportion of vSAG7 molecules are able to cross the membrane.
For this purpose, we tried to identify a more sensitive readout and compared stimulation of human PBMCs to stimulation of T cell hybridomas (Korman, A., personal communication). Titration experiments involving coculture of PBMCs with decreasing numbers of DAP vSAG7 cells indicated that stimulation of PBMCs required as few as 250 DAP vSAG7 cells (Fig. 6 b), while the stimulation of T cell hybridomas was repeatedly shown to require at least 2 × 103 DAP vSAG7 cells (Fig. 1 c). The use of human PBMCs thus provides a readout which is 10-fold more sensitive than that obtained using T cell hybridomas.
In vitro assays were then established in which DAP
vSAG7 cells were separated from PBMCs by a 0.4-µm semipermeable membrane. A threefold enrichment in CD4+
blasts expressing V12 was observed by flow cytometry
(Fig. 6 b). However, this enrichment was only detected
when high amounts (>3 × 104) of DAP vSAG7 cells were
used. This result clearly indicates that the fragment of
vSAG7 carrying the superantigenic activity is able to cross
the semi-permeable membrane. Our results also show that
at least 100-fold more DAP vSAG7 cells are required when
cells are separated by a transwell as compared to direct coculture of DAP vSAG7 cells and PBMCs, in order to obtain the same levels of V
12 expansion. It is thus possible
to estimate that only ~1% of the soluble vSAG7 molecules
are able to efficiently cross membranes. In comparison, SEB
was shown to freely cross the membrane, as the SEB-induced
stimulation of PBMCs is not altered by this compartmentalization, even at the lowest concentrations of bacterial
toxin (data not shown).
Similar experiments were performed using DAP DR1
vSAG7 cells in the upper compartment. Our results (Fig. 7)
show that the transferred fragment of vSAG7 crosses the
membrane and stimulates efficiently V12+ PBMCs. These
results were obtained using two different DAP DR1 vSAG7
clones, 3B2 and 3A5. The results obtained using DAP
vSAG7 or DAP DR1 vSAG7 (Figs. 6 b and 7) were very
similar: in both cases a small and comparable proportion of
vSAG molecules were able to cross the membrane and
stimulate PBMCs. These results indicate that the MHC
class II molecules expressed by DAP DR1 cells are not interfering with the transfer of vSAG7 molecules.
vSAG7 Interacts with the HLA-DR
Bacterial toxins bind to well identified sites on MHC class II. To provide a molecular characterization of the class II site which is
involved in the interaction with vSAG7, we have performed inhibition experiments of both vSAG7 in direct
and transfer presentation assays using bacterial toxins. We
show in Fig. 8 that the DAP DR1 vSAG7-induced stimulation of Kmls 13.11 is not inhibited by addition of SEA,
even at the highest concentration tested (50 µg/ml). On the
other hand, intercellular transfer of vSAG7 is significantly
affected by the simultaneous incubation of cells with high
concentrations of SEA (Fig. 8). This inhibition of vSAG7dependent T cell stimulation was directly correlated with
the amount of SEA. Addition of increasing concentrations
of SEA led to up to 95% inhibition of T cell stimulation.
These experiments allowed us to suggest that the molecular
interactions of the whole vSAG7 or of the transferred fragment with MHC class II are different.
SEA binds to the and
chains of MHC class II molecules (41a). We were thus interested to determine if the
binding sites for the bacterial toxin were also involved in
the binding of vSAG7. To address this question, we used a
mutant of SEA, SEA F47, which has lost its ability to bind
MHC class II through the
chain but is still able to bind
with high affinity to the
chain of MHC class II (41a, 42).
We show here (Fig. 9) that SEA F47 partially blocks the
activity of vSAG7 in the transfer assay, as compared to the
20-fold inhibition induced by SEA wt. Indeed, the addition of high concentrations of SEA F47 decreases T cell stimulation by less than a twofold (~40% of inhibition).
We concluded from these studies that vSAG7 interacts
mainly with MHC class II through its
chain. This result
was further confirmed by the fact that high concentrations
of TSST-1, a toxin known to bind exclusively to the
chain of MHC class II (43, 44), also exerted a partial
(~60%) but reproducible inhibition of vSAG7 presentation
in the transfer system (Fig. 9).
In this report we provide in vitro and in vivo evidence
indicating that a soluble form of vSAG7 can activate primary T cells of human or murine origin. This transfer is absolutely dependent on the presence of class II+ cells, and is
specific for the vSAG, as shown by inhibition experiments. Our demonstration that this transfer can occur across a semipermeable membrane clearly rules out the possibility that
fusion between class II+ and class II cells is responsible for
this effect.
Moreover, the use of the semi-permeable membrane
provided conclusive evidence that cell-to-cell contact is
not required for intercellular transfer of vSAG7. In these
conditions, however, transfer is not very efficient and involves only a small proportion (~1%) of vSAG7 molecules.
Indeed, titration curves of vSAG7 in the presence or in the
absence of a semi-permeable membrane shows that comparable V specific expansion requires 100-fold more vSAG7 cells in the presence of a semi-permeable membrane. The
fact that vSAG7 molecules can only cross membranes with
low efficiency suggests that vSAG7 is weakly soluble, and
that most of the transferable molecules remain uncovalently
bound at the cellular membrane to its NH2-terminal part
(45). These features leads us to conclude that vSAG7 transfer does not require strict cell-to-cell contact but rather cellular proximity. It can thus be considered as a paracrine
phenomenon. These results also provide convincing evidences that vSAG7 molecules can reach the cell surface in the absence of MHC class II molecules and are biologically active.
Titration experiments involving DAP DR1 vSAG7+
cells in the presence or in the absence of a semi-permeable
membrane indicate that DR molecules do not sequester
vSAGs. Indeed, the V skewing obtained using DAP DR1
vSAG7 and DAP vSAG7 shows that vSAG7 can be transferred efficiently from both cells. This suggests that vSAG
molecules are not sequestered by MHC class II at the surface of DAP DR1 vSAG7 cells. Alternatively, vSAGs could
be loosely associated to class II enabling the dissociation of
the transferred fragment and subsequent binding to another
class II+ cell. It is possible that the transferred fragment has
never been associated with class II molecules. The presence
of three cleavage sites for furin-like proteases could lead to
the dissociation of a COOH-terminal fragment, a situation
which is highly analogous to the one observed with the
HIV gp120, which readily dissociates from the membrane
gp41, and remains anchored at the cell surface (46).
Class II CD8+ T cells can delete vSAG-responsive T cells
more efficiently than class II+ B cells (32), although these
cells express comparable levels of vSAG7 mRNA (47).
This paradox could have been attributed to the sequestration of vSAGs by MHC class II+ molecules on the surface
of B cells. Our demonstration that both DR1+ and DR1
vSAG7+ cells are equally efficient in inducing the deletion
of V
6-expressing T cells indicates that the reported difference between CD8+ and B cells is not due to their differential expression of class II and subsequent sequestration of
vSAG7 by MHC class II, but rather to other cellular characteristics such as adhesion molecules, ability of being activated in different conditions or half life after injection.
We have then shown that SEA can compete with the transferred vSAG while it has no effect on the presentation of vSAG7 endogenously expressed by DAP DR1+ cells. Two models can be proposed to explain this discrepancy. First, it is possible that the transferred fragment of vSAG7 displays a lower affinity for class II MHC molecules than the full-length vSAG7 molecule. These differences could be attributed to additional contact points between the fulllength and the transferred fragment. Alternatively, when endogenously expressed, vSAG7 could induce a conformational modification of the vSAG7-class II complex generating a tighter association between the two molecules.
Although considerable progress has been made in the
understanding of TCR:vSAG interaction, little is available
concerning the sites involved in the binding of vSAGs to
MHC class II. However, different results indicate that the
chain of MHC class II is involved in the binding of vSAG7
(29, 48). Whereas SEA molecules efficiently abrogate vSAG7
transfer to DR1 molecules, a mutated SEA which fails to
interact with the
chain is much less efficient in exerting
this inhibitory effect. This result suggests that vSAG7 interacts with the DR
chain. Partial inhibition could be due
to the residual binding of SEA F47 to the DR
chain. Indeed our own results have suggested that SEA also binds to
the second loop of the DR
chain which includes residue
DR
39 (41a). Binding of vSAG7 to the DR
chain was
further confirmed using TSST-1, which predominantly
binds to the DR
chain (43, 44) and can also compete
(60% of inhibition) with the transferred fragment of
vSAG7. These results do not exclude however that vSAGs could also interact with the
chain of DR, as suggested by
the hierarchy in vSAG presentation by DR alleles, which
differ only through their
chain. (29)
vSAGs contain furin-like cleavage sites and are naturally cleaved. However, the cleaved fragment remains bound to the rest of the protein through non covalent bonds (45). These observations raise the hypothesis that the soluble molecule involved in the transfer phenomenon is a fragment of vSAG7 obtained by cleavage of vSAG7 at one of the three furin-like cleavage sites. The precise nature of this transferred fragment remains to be determined. In this context it is important to note that these cleavage sites have been conserved in most of the known sequences of exogenous and endogenous MMTVs. Such a degree of conservation would favor an important role for these cleavage sites. It is well established that the virus is strictly dependent for its replication on its capacity to stimulate a large pool of T cells (7, 9). Transfer of the vSAG fragment from infected cells to other MHC class II+ cells would favor T cell activation, upregulate viral production, and hence lead to viral dissemination in the host.
On the other hand, transfer of vSAGs could also provide
an advantage to the host; Moore et al. (49) have demonstrated that the expression of some endogenous vSAGs in
thymic class II+ cells is low or null, and that the bulk of vSAGs
is expressed in lymphocytes of the T lineage at different
stages of maturation. The efficient paracrine transfer of vSAGs
from MHC class II vSAG+ thymocytes (47) to class II+
vSAG
dendritic cells or macrophages would provide a
mechanism leading to the deletion of vSAG-responsive CD4+
or CD8+ cells. Transfer of vSAGs could then provide an
alternative way of thymic deletion of thymocytes expressing responsive V
s, this deletion being critical in order to
protect mice from exogenous infection by MMTVs sharing
the same V
specificity (7) in the first weeks after birth.
It is also quite likely that this transfer could play a role in peripheral deletion. CD8+ T cells express high levels of vSAGs (32, 47) which are upregulated upon T cell activation (15). This could lead to an increased transfer of vSAGs to adjacent class II+ cells and hence to the deletion of activated CD8+ T cells. Different reports demonstrate that vSAGs can also be expressed in cells of non-immune origin, such as lung cells, brain cells (47) or intestinal epithelial cells (50). A role of vSAG reservoir could be attributed to these cells, which could hence be involved in the deletion of activated T cells infiltrating these organs.
Finally, studies concerning binding of vSAGs to MHC class II, its intracellular transport, and cofactors involved in transfer, that remain unaddressed, should be made possible using the above described transfer assays.
Address correspondence to Rafick-Pierre Sekaly, Laboratory of Immunology, Institut de Recherches Cliniques de Montréal, 110, avenue des Pins Quest, H2W 1R7 Montréal, Canada.
Received for publication 21 October 1996
This work was supported by grants RG-544/95 from Human Frontier Science Project, MT-10055 from Medical Research Council of Canada, and 007273 from National Cancer Institute of Canada, attributed to R.P. Sékaly. R.P. Sékaly holds an MRC scientist award. F. Denis and J. Thibodeau are supported by fellowships from National Health Research and Development Program. M. Delcourt has a French government Assistant Moniteur Normalien fellowship.We would like to thank Pascal Lavoie and Dr. Michel Braun for critical review of the manuscript, and H. McGrath for excellent technical support.
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