Expansion of human {gamma}/{delta} T cells in vitro is differentially regulated by the measles virus glycoproteins

Karen Bieback1, Claudia Breer1, Ralph Nanan2, Volker ter Meulen1 and Sibylle Schneider-Schaulies1

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


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Impaired proliferative response of lymphocytes after mitogenic stimulation ex vivo is a key feature of the generalized immunosuppression induced by measles virus (MV). Compelling evidence suggests that negative signalling by the MV glycoprotein (gp) complex and the surface of uninfected lymphocytes is essential for this effect. So far, the inhibitory activity of this complex applied to all lymphocyte subpopulations irrespective of the mode of stimulation and could not be overcome by external stimulation. This study shows that the isopentenyl pyrophosphate (IPP)/IL-2-stimulated expansion of human {gamma}/{delta} T cell receptor (TCR) T cells from peripheral blood mononuclear cells (PBMCs) is inhibited efficiently when the MV gp complex is expressed on the surface of persistently MV-infected T or monocytic cells. In contrast, persistently infected B cells or infected human dendritic cells (DCs) do not interfere with expansion of {gamma}/{delta} TCR T cells from PBMCs. These particular two cell populations, however, efficiently inhibit IPP/IL-2-stimulated expansion of {gamma}/{delta} TCR T cells from purified T cells and this is reverted by resubstitution with monocytes. As revealed by filter experiments, cocultivation with B cells and DCs empower monocytes, at least partially by soluble mediators, to provide membrane contact-dependent costimulatory signals that neutralize the inhibitory effect of the MV gp complex. Thus, {gamma}/{delta} TCR T cells are sensitive to MV gp-mediated inhibition; however, this is overcome efficiently by signals delivered from monocytes conditioned by B cells and DCs.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Immunosuppression induced by measles virus (MV) is the major cause of the high morbidity and mortality rates associated with acute measles. Marked lymphopenia, cytokine imbalance and strongly impaired proliferative responses of peripheral blood mononuclear cells (PBMCs) ex vivo to mitogenic stimulation are typically observed (Borrow & Oldstone, 1995; Schneider-Schaulies et al., 2001). Although cells of the haematopoetic lineage, such as lymphocytes and, particularly late in infection, monocytes, are target cells during natural infection (Esolen et al., 1993; Schneider-Schaulies et al., 1991), the overall frequency of infected PBMCs is usually low. Therefore, indirect mechanisms compatible with virus-induced interference with function and viability of professional antigen-presenting cells [mainly monocytes and dendritic cells (DCs)], imbalanced cytokine production by few infected cells, and direct signalling to T cells leading to apoptosis or proliferative arrest have been proposed to account for this effect (Addae et al., 1995; Karp et al., 1996; Schneider-Schaulies et al., 2001; Servet-Delprat et al., 2000; Vidalain et al., 2000).

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 {alpha}-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, {gamma}/{delta} T cell receptor (TCR) T cells comprise on average 5–10 % of the total peripheral blood T cell population. In contrast to {alpha}/{beta} TCR T cells, they lack CD4 and CD8 expression and recognize antigens independently of conventional MHC molecules (Kaufmann, 1996). Early expansion and activation of {gamma}/{delta} 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 {gamma}/{delta} TCR T cell expansion, cytokine production and cytolytic activity (De Libero, 1997). In vitro, {gamma}/{delta} 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 {gamma}/{delta} 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 {gamma}/{delta} TCR T cells. Overall, these findings show for the first time that interference with MV gp-mediated negative signalling to T cells is possible.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Cell lines and virus.
Uninfected or persistently MV Edmonston strain (ED)-infected lymphoblastoid B (BJAB and BJAB pED, 8866 and 8866 pED, Wil-2 and Wil-2 pED), T (Molt-4 and Molt-4 pED) and monocytic (U-937 and U-937 pED) cell lines were maintained in RPMI 1640 supplemented with 10 % foetal calf serum (FCS). MV vaccine strain ED-B was propagated on Vero cells in minimal essential medium containing 5 % FCS. Virus stocks (or mock supernatants) were harvested from Vero cells and clarified supernatants were obtained after stock virus centrifugation.

Preparation of cells and infection.
Human PBMCs were isolated by Ficoll–Paque 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 {alpha}-CD19- and {alpha}-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 {alpha}-mouse IgG antibodies (all antibodies were obtained from Immunotech). {gamma}/{delta} TCR T cells were identified by CD3/pan-{gamma}/{delta} 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 {beta}-plate scintillation counter. Expansion of {gamma}/{delta} TCR T cells was determined by double staining for CD3/pan-{gamma}/{delta}-TCR and defining the percentage of {gamma}/{delta} 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-{beta} (Strathmann Biotech). IFN-{gamma} 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).


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
IPP/IL-2-stimulated expansion of human {gamma}/{delta} TCR T cells is differentially regulated by MV
Human PBMC preparations were split in two and stimulated under conditions favouring expansion of mainly {alpha}/{beta} TCR T cells (PHA) or {gamma}/{delta} TCR T cells (IPP/IL-2, or for the control, IL-2 alone). Addition of IL-2 in the latter setting is necessary as {gamma}/{delta} TCR T cells require, but are unable to produce, this cytokine upon stimulation. Under IPP/IL-2 conditions, true expansion of {gamma}/{delta} but not {alpha}/{beta} TCR T cells occurred (as determined by incorporation of calibration beads and double staining for CD3/{gamma}/{delta} TCR+ cells) (Fig. 1a). Corroborating our previous studies (Schlender et al., 1996), PHA-stimulated proliferation of PBMCs was inhibited dose-dependently in the presence of UV-inactivated MV (Fig. 1b). This was also observed for the IPP/IL-2-driven expansion of {gamma}/{delta} TCR T cells, indicating that these were at least equally sensitive to MV gp-induced negative signalling (Fig. 1b). As seen previously, PHA-stimulated lymphoproliferation was impaired dose-dependently in the presence of UV-inactivated persistently MV-infected Molt-4 T cells (Molt-4 pED) or U-937 monocytic cells (U-937 pED), but not in uninfected Molt-4 or U-937 cells (PCs) (Fig. 2a, left panel). These PCs also inhibited the IPP/IL-2-stimulated expansion of {gamma}/{delta} TCR T cells and did so, compared to PHA-stimulation, even more efficiently with Molt-4 pED cells but was not strictly dose-dependent with U-937 pED cells (Fig. 2a, right panel). Surprisingly, persistently infected B cell lines, such as BJAB pED, 8866 pED and Wil-2 pED, only inhibited PHA-stimulated expansion (Fig. 2b, left panel), but not that of IPP/IL-2-stimulated {gamma}/{delta} TCR T cells and were even slightly stimulatory (Fig. 2b, right panel). The differential regulation of PHA- and IPP/IL-2-stimulated proliferation by PCs did not correlate with lower expression levels of the MV gp on these cells (Table 1). These findings suggest that {gamma}/{delta} TCR T cells are sensitive to MV-mediated inhibition; however, this signal can be neutralized by factors released from, or present on, persistently MV-infected B cells. Although IPP/IL-2-driven expansion of {gamma}/{delta} TCR T cells was differentially regulated, the stimulated release of IFN-{gamma} from these cells was comparable in the presence of all MV-infected PCs irrespective of their inhibitory activity (Table 2; data not shown for Molt-4 pED). This is in agreement with findings in mitogen-stimulated cultures where MV-infected PCs efficiently interfered with proliferation but not with cytokine release (Schnorr et al., 1997a).



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Fig. 1. UV-inactivated MV inhibits both PHA-driven lymphocyte and IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion. (a) Expansion of {gamma}/{delta} TCR T cells from PBMCs after 7 days of culture in the presence of IL-2 alone (upper panel) or IL-2/IPP (middle panel), as determined by CD3/pan-{gamma}/{delta} TCR double staining. Quantification of {gamma}/{delta} TCR, CD3 and {alpha}/{beta} TCR-positive cells 3 days after PHA stimulation (white bars) or 7 days of IL-2 stimulation in the absence (black bars) or presence of IPP (grey bars) (bottom panel). (b) UV-inactivated MV was added to PBMCs at the amounts indicated (corresponding to those determined for live virus) and inhibition of proliferation was determined 3 days after stimulation with PHA (white bars) or {gamma}/{delta} TCR T cell expansion 7 days after stimulation with IPP/IL-2 (black bars) (percentage {gamma}/{delta} TCR+ of total CD3+ T cells). PHA-driven proliferation was determined by incorporation of [3H]thymidine, IPP/IL-2-driven T cell expansion by CD3/pan-{gamma}/{delta} TCR double staining. Inhibition was determined compared to control values obtained with mock-treated cells. Data shown were obtained in four independent experiments (SD are indicated), with P values <0·01 with respect to the mock control. Statistically significant differences are indicated by asterisks.

 


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Fig. 2. Persistently MV-infected B cell lines do not inhibit IPP/IL-2-driven expansion of {gamma}/{delta} TCR T cells. PBMCs were stimulated for 48 h with PHA (a, b, left panels) or for 7 days with IPP/IL-2 (a, b, right panels) in the presence of decreasing amounts (PCs/RCs) of (a) UV-inactivated U-937 pED (white bars) or Molt-4 pED (black bars) cells, or (b) BJAB pED (black bars), 8866 pED (grey bars) or Wil-2 pED (open bars) cells. PHA-driven proliferation was determined by incorporation of [3H]thymidine (a, b, left panels), IPP/IL-2-driven T cell expansion by CD3/pan-{gamma}/{delta} TCR double staining (a, b, right panels). Inhibition was determined based on the values obtained in cultures where the respective uninfected cell population had been added (c.p.m. values of the controls were 56 602±3 556). Data shown were obtained in four independent experiments (SD are indicated), with P values <0·01 with respect to the respective uninfected control cells. Statistically significant differences are indicated by asterisks.

 

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Table 1. Surface expression levels of MV gp on persistently infected cell lines

Expression levels of MV F and H proteins (indicated as mean fluorescent intensities) were determined on persistently MV ED-infected cell lines by mAbs (each cell line was 100 % positive for these antigens).

 

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Table 2. IFN-{gamma} production from IPP/IL-2-stimulated PBMCs in the presence of PCs

PBMCs were stimulated with IPP/IL-2 in the absence or presence of PCs (PC : RC ratio, 1 : 50), supernatants were harvested after the time intervals indicated and analysed for IFN-{gamma} production by ELISA. Values indicated represent the mean of two independent experiments, each performed in triplicates. SD are indicated.

 
MV-infected DCs promote expansion of {gamma}/{delta} TCR T cells from PBMCs
To evaluate whether neutralization of the inhibitory signal is confined to persistently infected cells, we first added lytically MV-infected B cell lines to IPP/IL-2-stimulated PBMCs. Using these conditions, we always observed inhibition of IPP/IL-2-stimulated expansion of {gamma}/{delta} TCR T cells. This correlated with MV infection of these cells as we were unable to completely inhibit virus production from these PCs (data not shown). Therefore, we used primary infected human DCs as PCs, which produce only low amounts of infectious virus, and infection of RCs can be efficiently prevented by addition of a fusion inhibitory peptide. As reported earlier (Klagge et al., 2000; Schnorr et al., 1997b), MV infection caused maturation of immature DCs, as assessed by the induction of CD83, CD86 and upregulation of HLA-DR (Table 3), and MV-infected DCs did not efficiently stimulate allogenic T cell proliferation (Fig. 3a). In contrast, the very same DC samples, regardless of their maturation state or MV infection, did not interfere with IPP/IL-2-dependent expansion of {gamma}/{delta} TCR T cells from PBMCs (Fig. 3b), indicating that neutralization of MV F/H-mediated negative signalling is also exerted by DCs and is not confined to persistently infected B cell lines.


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Table 3. Phenotypic characterization of DCs used for the coculture with PBMCs or purified T cells

DCs were generated from monocytes and treated with LPS, mock preparations or infected with MV ED for 48 h. Expression levels of cellular and viral antigens are given as mean fluorescence intensities (HLA-DR, CD83, CD86) (100 % cells positive for these antigens) or % positive cells (for MV H).

 


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Fig. 3. MV-infected DCs fail to stimulate allogeneic T cell proliferation but do not interfere with IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion from PBMCs. DCs generated from monocytes were treated with mock supernatant (a, b, grey symbols and bars), LPS (black symbols and bars) or MV-infected (white symbols and bars) for 48 h and cocultured with allogeneic T cells alone (a) or in the presence of IPP/IL-2 (b) at decreasing amounts as indicated. Allogeneic T cell proliferation was determined by [3H]thymidine after 5 days (a), expansion of {gamma}/{delta} TCR T cells at day 7 after IPP/IL-2 stimulation. Stimulation indices (SI) (a) and REI (b) were determined based on T cell proliferation in the absence of DCs (a) or percentage of {gamma}/{delta} TCR T cells in the absence of IPP (b). Data shown were obtained in four independent experiments (SD are indicated), with P values <0·05 with respect to the mock control (c.p.m. values for the controls were 11 465±1 921). Statistically significant differences are indicated by asterisks.

 
Neutralization of negative signalling by persistently infected B cell lines and DCs requires the presence of monocytes
While IPP/IL-2-stimulated {gamma}/{delta} TCR T cell expansion from PBMCs was inhibited by U-937 pED, but not 8866 pED cells (and BJAB pED and Wil-2 pED cells, data not shown) or lytically infected DCs (Figs 2, 3 and Fig. 4a), all PC populations were inhibitory when purified T cells rather than PBMCs were used as RCs (Fig. 4b). This indicated that the neutralizing effect provided by persistently MV-infected B cell lines and infected DCs required a third cell population initially present in the PBMC preparation. Thus, purified B cells or monocytes were resubstituted to the RC populations (consisting of purified T cells) at ratios present initially in the PBMC samples (Table 4) prior to stimulation and addition of PCs. While expansion of {gamma}/{delta} TCR T cells by 8866 pED was still inhibited upon B cell reconstitution, resubstitution of the monocyte fraction enabled expansion of {gamma}/{delta} TCR T cells to levels similar to those seen with uninfected 8866 controls (Fig. 4c). Similarly, addition of monocytes efficiently reverted {gamma}/{delta} TCR T cell inhibition by MV-infected DCs (Fig. 4c). Thus, cross talk of PCs with monocytes as accessory cells seems to be required to neutralize MV F/H-mediated inhibition of IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion.



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Fig. 4. MV-induced inhibition of IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion from purified T cells can be rescued in the presence of monocytes. REI of {gamma}/{delta} TCR T cells from IPP/IL-2-stimulated PBMCs (a) or purified T cells (b) cocultured in the presence of uninfected or persistently MV-infected U-937 or 8866 cells or mock- or MV-infected DCs (PC : RC ratio, 1 : 50) were determined after 7 days by CD3/{gamma}/{delta}-TCR double staining. (c) Cocultures were set up as in (b) with each of the autologous purified B cell (black bars) or monocyte (white bars) fraction added back to the purified T cell populations at the ratios initially present in the PBMC preparations (Table 4). Data shown were obtained in independent experiments with three individual donors (SD are indicated), with P values <0·05 with respect to the uninfected control cell line. Statistically significant differences are indicated by asterisks.

 

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Table 4. Subset composition of PBMC preparations of three individual donors

Prior to separation into subset populations, PBMCs from three individual donors were analysed for their subset composition using a cocktail of CD3-, CD56-, CD19-, CD14 and HLA-DR-specific antibodies.

 
Resistance to inhibition requires membrane contact with monocytes rather than soluble mediators
It is only in the presence of monocytes that {gamma}/{delta} TCR T cells cocultivated with MV-infected DCs expanded in response to IPP/IL-2 stimulation (Figs 4c and 5a, lane 2). We now aimed to define whether soluble mediators or surface contact-mediated signals accounted for monocyte-mediated neutralization of MV F/H negative signalling. When separated from T cells by a membrane filter (pore size, 0·2 µM), MV-infected DCs did not interfere with IPP/IL-2-driven expansion of {gamma}/{delta} TCR T cells, corroborating our previous observations in PHA-stimulated cultures that T cell inhibition is surface contact-dependent (Fig. 5a, lanes 3, 4 and 6). When physical contact of the DC/T cell coculture and monocytes was abolished, these cells were unable to neutralize inhibition of {gamma}/{delta} TCR T cell expansion induced by MV-infected DCs (Fig. 5a, lane 5); this suggests that soluble mediators released from monocytes were most likely not involved. In support of this hypothesis, transfer of supernatants from MV-infected DC cultures, or conditioned media from LPS-matured DCs or lipopeptide-matured monocytes, did not rescue IPP/IL-2-driven expansion of {gamma}/{delta} T cells from purified T cell populations in the presence of MV-infected DCs (data not shown). Thus, signals provided by surface molecules on monocytes or monocytes preconditioned by persistently infected B cells or MV-infected DCs are instrumental in neutralizing MV F/H-mediated inhibition of stimulated {gamma}/{delta} T cell expansion.



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Fig. 5. Physical interaction between monocytes and DCs is required to rescue IPP/IL-2-driven expansion of {gamma}/{delta} TCR T cells from purified T cells, while persistently infected BJAB pED cells condition monocytes partially by soluble mediators. (a) DCs were LPS-matured (grey bars), mock- (white bars) or MV-infected (black bars) and added to IPP/IL-2-stimulated purified T cells alone (lane 1) (PC : RC ratio, 1 : 50) or in the presence of monocytes (lane 2) (at the ratio initially present in the PBMC sample). Alternatively, T cells were separated from DCs (lane 3), from a mixture of DCs and monocytes (lane 4), or from T cell/DC cocultures were separated from monocytes (lane 5) or T cell/monocyte cocultures from DCs (lane 6) by transwell filters. (b) PBMCs were cocultured with BJAB or BJAB pED cells (lane 1), Molt-4 or Molt-4 pED cells (lane 2) or Molt-4 pED cells resuspended in supernatant (SN) of BJAB cells or BJAB pED cells (lane 3) at a PC : RC ratio of 1 : 10. Relative {gamma}/{delta} TCR expansion levels were determined after 7 days by CD3/{gamma}/{delta} TCR double stainings. Data shown were obtained in independent experiments with three individual donors (SD are indicated), with P values <0·05 with respect to the mock (a) or BJAB cell (b) control. Statistically significant differences are indicated by asterisks.

 
Although monocytes are present in all PBMC preparations, they are only empowered to assist in IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion when MV-infected DCs or persistently infected B cell lines are used as PCs, indicating that these particular PCs may condition monocytes. Since this could involve soluble mediators, Molt-4 pED cells, which inhibit IPP/IL-2-driven {gamma}/{delta} TCR T cell expansion from PBMC cultures (Fig. 2a and Fig. 5b, lane 2), were replenished with supernatant of BJAB pED cells. Under these conditions, levels of inhibition induced by these cells dropped by on average 50 %, indicating that soluble mediators were at least partially involved (Fig. 5b, lane 3). These do not, however, include type I IFN, since there is no correlation of the levels of this cytokine present in the supernatants. Thus, high amounts of these cytokines are released from MV-infected DCs (Klagge et al., 2000) and BJAB pED cells, while production of type I IFN from inhibitory cell lines is equally high (U-937 pED) or undetectable (Molt-4 pED) (as tested by MxA induction in BJAB cells, data not shown).


   DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Lack of proliferative responses of especially T cells to polyclonal and antigen-specific stimulation is a hallmark of the general immunosuppression induced by MV (Schneider-Schaulies et al., 2001). To study the underlying mechanisms, in vitro systems based on mitogen- or anti-CD3-stimulated primary lymphocytes or lymphocytic cell lines have been widely used. Results obtained can be summarized in that inhibition of lymphocyte proliferation by MV can result from direct infection (Naniche et al., 1999; Yanagi et al., 1992) or by indirect mechanisms such as yet undefined soluble mediators released from infected cells (Fujinami et al., 1998) or surface contact-mediated signalling, which required interaction of the MV gp complex on few infected cells and an as yet unknown receptor on uninfected cells (Schlender et al., 1996). In the latter system, negative signalling by the MV effector F/H complex was shown to disrupt intracellular signalling pathways associated with the IL-2R heterotrimer (Avota et al., 2001), and this could explain why, in spite of normal expression levels of the IL-2R chains, mitogen-stimulated primary T cells cannot expand upon IL-2 addition after contact with the MV effector complex. The signal imposed on primary T cells did not induce cellular apoptosis but rather retarded S phase entry (Avota et al., 2001; Schnorr et al., 1997a). As yet, proliferative arrest after MV contact was observed for all stimuli applied to lymphocytes; even cellular transformation, in the case of cell lines of haematopoetic origin, did not prevent sensitivity to this negative signal (Schlender et al., 1996). {gamma}/{delta} TCR T cells expanded from PBMC preparations by IPP/IL-2 also generally do not represent an exception to that rule, since the MV F/H complex, when applied as UV-inactivated MV or on the surface of persistently infected Molt-4 or U-937 cells, causes their proliferative arrest (Fig. 1b and 2). Thus, although there is evidence that signalling properties of the {gamma}/{delta} TCR complex are quite different, and even superior to those of the {alpha}/{beta} TCR (Hayday, 2000; Hayes & Love, 2002), IPP/IL-2 stimulation does not rescue expansion of these cells. It is likely that, for MV-mediated inhibition of IPP/IL-2-stimulated {gamma}/{delta} TCR T cells, similar mechanisms are operative as for those induced by mitogens or anti-CD3/CD28 in PBMCs or purified T cell cultures. Thus, proliferation, but not cytokine release (Table 2), is affected, and TCR stimulation in the presence of IL-2 (as contained in all experiments) does not overcome negative signalling by the effector complex. It is thus very likely that, similar to the situation seen in mitogen-stimulated T cell and IL-2-dependent Kit-225 cells, IL-2-dependent activation of the Akt kinase pathway is interrupted in MV-contacted {gamma}/{delta} TCR T cells as well (Avota et al., 2001). This could not, however, be addressed experimentally in this system, since the frequency of {gamma}/{delta} T cells in PBMCs, particularly at the time when the signal is given (at the onset of the culture), is too low. Permanent human T cell lines of the {gamma}/{delta} TCR subtype analysed by us (which, for adult donors, consists mainly of the V{gamma}9/V{delta}2 subset) were not available to us, and our efforts to establish those by transformation by herpes virus saimiri (HVS) failed. {gamma}/{delta} TCR T cell lines obtained after HVS transformation are as yet of the {delta}1 subset (Fickenscher et al., 1997), and these were, when tested in our assay, all inhibited irrespective of the PCs used (data not shown). It is thus possible that the ability of certain PC types to neutralize the inhibitory effect is confined to the V{gamma}9/V{delta}2 TCR subset.

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 {gamma}/{delta} 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 {gamma}/{delta} 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{gamma}9/V{delta}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 {gamma}/{delta} TCR T cell expansion. Unlike for the expansion of {gamma}/{delta} TCR T cells, differential effects on {alpha}/{beta} 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 {gamma}/{delta} TCR T cells is differentially modulated by {alpha}/{beta} TCR T cells present in our RC populations.

Our finding that reconstitution with monocytes is crucial for {gamma}/{delta} TCR T cells in spite of the inhibitory signal is not unexpected, since these cells are known to be able to assist in {gamma}/{delta} TCR T cell expansion. Thus, pamidronate-driven activation of primary {gamma}/{delta} 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 {gamma}/{delta} 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 {gamma}/{delta} 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, cell–cell contacts between conditioned monocytes and {gamma}/{delta} TCR T cells are required (Fig. 5). These could involve LFA-3–CD2 interactions, which were found to be important, but not absolutely required, for proliferation of V{gamma}9/V{delta}2 T cells (Wang & Malkovsky, 2000). This study, however, also supported the crucial importance of IL-2, since {gamma}/{delta} 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 {gamma}/{delta} TCR T cells. Engagement of NKG2D costimulates cytokine production and proliferation of V{gamma}9/V{delta}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 {gamma}/{delta} TCR T cell expansion.

While some studies suggest a role of {gamma}/{delta} TCR T cells in virus infections (reviewed by Kaufmann, 1996), fluctuations of {gamma}/{delta} 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 {gamma}/{delta} 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.


   ACKNOWLEDGEMENTS
 
We thank Wilhelm Martin and Thomas Herrmann for helpful discussion; Marion Seufert, Claudia Rüth and Maren Klett for expert technical assistance; and the Deutsche Forschungsgemeinschaft and the Bundesministerium für Bildung und Forschung (IZKF 01 KS 9603) for financial support.


   REFERENCES
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Received 5 December 2002; accepted 22 January 2003.



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