1 Institute for Virology and Immunobiology, University of Würzburg, Versbacher Str. 7, D-97078 Würzburg, Germany
2 Children's Hospital, University of Würzburg, Josef-Schneider-Str. 2, D-97078 Würzburg, Germany
Correspondence
Sibylle Schneider-Schaulies
s-s-s{at}vim.uni-wuerzburg.de
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ABSTRACT |
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INTRODUCTION |
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As revealed by previous studies, the expression of the MV glycoprotein (gp) complex [consisting of the MV haemagglutinin (H) and the proteolytically activated fusion (F) protein] on a minority of cells [referred to as presenter cells' (PCs)] or UV-inactivated virus particles was necessary and sufficient to induce unresponsiveness to polyclonal and anti-CD3/CD28-stimulated proliferation of an excess amount of human and rodent primary lymphocytes [referred to as responder cells' (RCs)] (Schlender et al., 1996). In this system, any cell type expressing the MV proteins exerted this inhibitory activity. This also applied for expression of these proteins by human DCs, which was associated with loss of their T cell allostimulatory activity and acquisition of their inhibitory phenotype in the presence of mitogen (Dubois et al., 2001
; Klagge et al., 2000
).
Negative signalling by the effector complex did not prime for or induce T cell apoptosis, but rather caused a strongly retarded passage of the G1/S phase restriction point after mitogenic stimulation (Niewiesk et al., 1999; Schnorr et al., 1997a
). On a molecular level, deregulations of cyclin-associated kinases and p27Kip1 were seen (Engelking et al., 1999
). In spite of their nonproliferating state, mitogen-stimulated T cells produced normal levels of cytokines, including IL-2, and upregulated early activation markers, also including the IL-2R
-subunit (CD25). The latter findings suggested defects in IL-2R signalling and indeed, the IL-2-dependent activation of the phosphatidyl-inositol-3-kinase (PI3K)/Akt kinase pathway was disrupted (Avota et al., 2001
). Although this finding explained why MV gp-contacted T cells did not resume proliferation even upon the addition of exogenous IL-2, the molecular basis for the resistance of the unresponsive state to other exogenous stimuli, such as PMA/ionomycin and mitogens, is as yet unknown. Interestingly, cellular transformation in lymphocytic and monocytic human cell lines does not confer resistance to MV F/H-mediated negative signalling (Schlender et al., 1996
).
In humans, /
T cell receptor (TCR) T cells comprise on average 510 % of the total peripheral blood T cell population. In contrast to
/
TCR T cells, they lack CD4 and CD8 expression and recognize antigens independently of conventional MHC molecules (Kaufmann, 1996
). Early expansion and activation of
/
TCR T cells has been noted in a variety of infections; their role in compartmentalizing or controlling virus infections is, however, poorly understood (Hayday, 2000
). Although they can be activated by antigens expressed by herpes simplex virus-infected cells (Sciammas et al., 1994
), common compounds released upon cellular stress or transformation and phosphoantigens trigger
/
TCR T cell expansion, cytokine production and cytolytic activity (De Libero, 1997
). In vitro,
/
TCR T cells can be expanded from PBMCs by stimulation with phosphorylated nucleotide-containing microbacterial compounds or isoprenoid pyrophosphates such as isopentenyl pyrophosphate (IPP) in the presence of IL-2 (Wesch et al., 1997
).
Using this experimental system, we found that IPP/IL-2-stimulated expansion of human /
TCR T cells was inhibited efficiently by the MV F/H complex expressed on UV-inactivated MV or persistently MV-infected T or monocytic cell lines. In contrast, expansion of these cells was unaffected by MV-infected B cell lines or DCs, suggesting that these cells can provide additional signals that neutralize the inhibitory effect. This was, however, dependent on the presence of monocytes that required conditioning by B cells or DCs to provide a neutralizing, membrane contact-dependent signal to
/
TCR T cells. Overall, these findings show for the first time that interference with MV gp-mediated negative signalling to T cells is possible.
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METHODS |
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Preparation of cells and infection.
Human PBMCs were isolated by FicollPaque density gradient centrifugation (Amersham Pharmacia) of buffy coats obtained from the department of transfusion medicine, University of Würzburg, Germany. T cells were prepared by rosetting with AET-treated sheep red blood cells (90 % CD3+) (Virion). The T cell-depleted fraction was further depleted of B and NK cells using
-CD19- and
-CD56-coated magnetic beads (Miltenyi Biotech). The plastic-adherent fraction of the monocyte-enriched cell population (
90 % CD14+) was used directly in reconstitution assays (see below) or to generate immature DCs in vitro by culture in the presence of 2000 U rhuGM-CSF ml-1 (Novartis) and 3000 U rhuIL-4 ml-1 (Strathmann Biotech) for 6 days with fresh cytokines added on day 3. When indicated, 100 ng lipopolysaccharide (LPS) ml-1 (Sigma) was added on day 6 for 48 h. Monocyte-derived DCs were infected on day 7 with MV (m.o.i. of 0·1) or mock-infected for 2 h, subsequently washed and cultured in cytokine-containing medium.
Monoclonal antibodies (mAbs) and FACS analysis.
MV-specific and MxA-specific mAbs were generated in our laboratory. PBMCs and purified T cell populations were characterized using CD3-, CD19-, CD14-, CD16, CD56- and HLA-DR-specific antibodies, DCs were phenotypically analysed using HLA-DR-, CD83- and CD86-specific antibodies either directly FITC- (Dianova) or PE-conjugated or by labelled goat -mouse IgG antibodies (all antibodies were obtained from Immunotech).
/
TCR T cells were identified by CD3/pan-
/
TCR double staining (Immunotech). Directly conjugated irrelevant IgG1 and IgG2b mAbs served as isotype controls. Levels of cell staining were measured on a FACScan (Becton-Dickinson) using Lysis II and analysed with CELLQUEST software.
In vitro proliferation assays.
PBMCs (5x104 cells per well) or, when indicated, purified T cells (2·5x104 cells per well) were cultured in the presence of UV-inactivated MV (amounts added corresponding to that determined for live virus) or mock preparations (1·5 J cm-2), UV-inactivated cells lines (0·8 J cm-2) or allogeneic LPS-matured, mock- or MV-infected DCs each at the ratios indicated. Cocultures (set up in triplicates) were stimulated with 2·5 µg phytohaemagglutinin (PHA) ml-1 (Sigma) for 3 days in 96-well round-bottomed plates and pulsed with 0·5 µCi [3H]thymidine (Amersham) for the last 18 h, as described previously (Schlender et al., 1996). Mixed leucocyte reactions were set up using serial 1 : 3 dilutions of DCs cultured with 5x104 allogeneic CD3+ T cells in triplicates, in the presence of 100 µg z-fFG (Sigma), for 5 days in 96-well round-bottomed plates followed by a 0·5 µCi [3H]thymidine pulse. Cultures were harvested and incorporation rates were measured using a
-plate scintillation counter. Expansion of
/
TCR T cells was determined by double staining for CD3/pan-
/
-TCR and defining the percentage of
/
TCR+ T cells within the total CD3+ population after a 7 day stimulation with IL-2 (50 U ml-1) of PBMCs or purified T cells in the presence or absence of 0·25 µg IPP ml-1 (Sigma). When indicated, purified T cells were reconstituted with B cells or monocytes at ratios initially contained in the PBMC population prior to addition of UV-inactivated MV, UV-inactivated cell lines or DCs (PCs). Cocultures were also set up as transwell assays using tissue culture inserts (Anapore membrane, 0·2 µm pore size) (Nunc). Relative expansion indices (REI) were calculated from IPP/IL-2-stimulated cultures in the presence of UV-inactivated MV or MV-infected cells as compared to cultures containing mock preparations or uninfected cells. When indicated, PCs were centrifuged and replenished with supernatant of BJAB or BJAB pED cells prior to addition to PBMCs. All experiments were performed with PBMCs isolated from at least three different donors and values indicated represent the means of at least three independent experiments set up in triplicates. Means of triplicates for individual experiments were determined and these were used to calculate the final means and SD indicated. Statistical analysis was performed using Student's t-tests and significance levels were determined based on the respective controls, as indicated.
Cytokine assays.
Supernatants of BJAB, BJAB pED, Molt-4, Molt-4 pED, U-937 and U-937 pED cells (1x106 each) were harvested, transferred to BJAB cells for 48 h and induction of MxA protein was determined in lysates of these cells, and for comparison, BJAB cells treated with defined amounts of rhuIFN- (Strathmann Biotech). IFN-
production was analysed in supernatants of IPP/IL-2-stimulated PBMCs (5x104 per well) cocultured with BJAB, BJAB pED, U-937 or U-937 pED cells at the time intervals indicated by ELISA following the manufacturer's protocol (R&D Systems).
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RESULTS |
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DISCUSSION |
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While the ability of the PC populations to inhibit PHA-stimulated T cell proliferation correlated directly with the levels of their respective MV gp surface expression, persistently infected B cell lines failed to inhibit IPP/IL-2-driven /
TCR T cell expansion from PBMCs, even though they expressed high levels of these proteins (Table 1
and Fig. 2
). Thus, insufficient expression of the inhibitory complex does not clearly account for this effect. This is documented further by our finding that in purified T cell cultures, in the absence of accessory cells, these cell lines and lytically infected DCs efficiently impaired stimulated
/
TCR T cell expansion (Fig. 4b
). Thus, neutralization of negative signalling is not a property of these particular PC populations but is mediated by monocytes conditioned by these cells (Fig. 4c
). It is unclear how persistently infected B cells or lytically infected DCs condition monocytes in our system. Apparently, however, soluble mediators released from these cultures may play a role in this process (Fig. 5b
). As documented by a recent study, distinct CpG ODN sequences elicit strong V
9/V
2 T cell responses and increased their expansion from PBMC populations and this correlated with their ability to induce type I IFN (Rothenfusser et al., 2001
). This cytokine, however, is likely not involved in differential conditioning of monocytes in our system. Although type I IFN is amply produced from MV-infected DCs (Klagge et al., 2000
) and BJAB pED cells, in inhibitory systems it can be present (U-937 pED) or absent (Molt-4 pED and UV-inactivated MV). Thus, there is no correlation between production of this cytokine and the ability of PCs (or UV-inactivated MV) to differentially regulate IPP/IL-2-driven
/
TCR T cell expansion. Unlike for the expansion of
/
TCR T cells, differential effects on
/
TCR T cell expansion depending on the cell type expressing the MV gp complex were never observed nor has differential induction of cytokines been seen (Klagge et al., 2000
; Schlender et al., 1996
; Schnorr et al., 1997a
). Thus, it is unlikely that the efficiency of IPP/IL-2-driven expansion of
/
TCR T cells is differentially modulated by
/
TCR T cells present in our RC populations.
Our finding that reconstitution with monocytes is crucial for /
TCR T cells in spite of the inhibitory signal is not unexpected, since these cells are known to be able to assist in
/
TCR T cell expansion. Thus, pamidronate-driven activation of primary
/
TCR T cells, as indicated by proliferation and cytokine release, was strictly dependent on the presence of monocytes, indicating that antigen presentation by monocytes is required at least for certain ligands (Hayday, 2000
; Miyagawa et al., 2001
). There is evidence that certain antigens are presented to
/
TCR T cells by members of the CD1 complex, which are expressed on the surface of cells involved in antigen presentation (Ulrichs & Porcelli, 2000
). Although these molecules can be upregulated on human primary monocytes under inflammatory conditions (Giuliani et al., 2001
; Kasinrerk et al., 1993
), antigen presentation by this system is unlikely to account for the neutralizing role of monocytes in our system. This is because CD1 molecules are expressed on DCs and a subset of B cells, and both cell types do not overcome negative signalling to
/
TCR T cells in the absence of conditioned monocytes (Figs 4b and 5a
). It is presently unclear why persistently infected U-937 pED cells, which are of the monocyte lineage, fail to compensate the negative signal per se. This is possibly related to their highly immature monocyte-like phenotype and they are known to be refractory to most maturation signals.
Obviously, cellcell contacts between conditioned monocytes and /
TCR T cells are required (Fig. 5
). These could involve LFA-3CD2 interactions, which were found to be important, but not absolutely required, for proliferation of V
9/V
2 T cells (Wang & Malkovsky, 2000
). This study, however, also supported the crucial importance of IL-2, since
/
TCR T cell expansion, initially prevented in the presence of LFA-3 or CD2-specific antibodies, was fully restored in the presence of this cytokine. It is also possible that MICA or MICB (MHC class I-related chains A and B)NKG2D interactions are involved. MICA and MICB are polymorphic peptide-binding chains that are expressed ubiquitously and can stimulate, via NKG2D, an activating receptor complex also expressed on
/
TCR T cells. Engagement of NKG2D costimulates cytokine production and proliferation of V
9/V
2 cells. NKG2D forms homodimers that are associated with DAP10, an adaptor protein that signals in a manner similar to CD28 by recruitment of PI3K (Bahram et al., 1994
). This appears particularly interesting in view of our findings, that IL-2-triggered activation of the Akt kinase, which is dependent on PI3K activation, is blocked by the MV effector complex (Avota et al., 2001
). Thus, it is tempting to speculate that recruitment of PI3K and subsequently Akt kinase might be restored by activation of the NKG2D/DAP10 system. Remarkably, MICA expression can be stimulated in primary monocytes, although it is unknown as yet whether this can also be mediated by type I IFN. MICA is, however, not expressed on Molt-4 and U-937 cells (Zwirner et al., 1999
) and this may provide an alternative explanation of why U-937 pED cells per se cannot rescue IPP/IL-2-driven
/
TCR T cell expansion.
While some studies suggest a role of /
TCR T cells in virus infections (reviewed by Kaufmann, 1996
), fluctuations of
/
TCR T cell pools, their activation and their importance in measles pathogenesis have not been addressed as yet and cannot be predicted based on our in vitro studies. However, these findings represent a first example that interference with MV gp-induced T cell arrest is possible. This is again dependent on the interaction of surface molecules present on
/
T cells and conditioned monocytes. Identification of the molecules involved and the signalling pathways activated will provide important insights into how negative regulation of T cell expansion can be efficiently counteracted.
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ACKNOWLEDGEMENTS |
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Received 5 December 2002;
accepted 22 January 2003.
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