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Immunology. Sep 1999; 98(1): 80–89.
PMCID: PMC2326898

Interleukin-10-induced CD8 cell proliferation

Abstract

Interleukin (IL)-10, a product of T helper 2 (Th2) lymphocytes, has been shown to be an important regulator of lymphoid and myeloid cells, inhibiting mitogen, peptide and alloantigen-induced T-cell proliferation and IL-2 production. The microenvironment at the time of cell activation, notably the presence or absence of cytokines such as IL-10, interferon-γ (IFN-γ) and IL-2, is believed to determine the lineage and magnitude of cell-mediated responses. In this study, we show that recombinant human IL-10 (rhIL-10) exerts a dose-dependent inhibitory effect on human peripheral blood mononuclear cells stimulated in vitro, when these cells have not previously been exposed to rhIL-10. Furthermore, incubation of these cells with high doses of rhIL-10, either before or at the time of activation, results in inhibition which is followed several days later by the emergence of a population of CD8 positive cells. This rhIL-10-responsive CD8, positive cell population still emerges even when the cells are washed following incubation with rhIL-10 prior to cell activation. Using purified CD8 populations this was shown to be a direct action of rhIL-10 on CD8 cells and not via CD4 positive cells and monocytes. This finding was only observed when cells were activated with a cross-linking anti-CD3 antibody and not when activated with phorbol-12-mystrate-13-acetate (PMA) and calcium ionophore (CaIon), suggesting that the effect is mediated through cell-surface receptors. Analysis of CD8 positive clones reveal production of Tc2 patterns of cytokines and reduced cell cytotoxicity to allogeneic, natural killer and lymphokine activated cell targets.

INTRODUCTION

Interleukin-10 (IL-10) has been shown to be an important regulator of lymphoid and myeloid cells.1 Since first being described as a product of murine T helper 2 (Th2) clones and monocytes which inhibited the proliferation of Thl cells, IL-10 has come to be recognised as a central immunoregulatory cytokine.2

The inhibitory effect of IL-10 on T-cell activation and proliferation has been shown to occur directly, through the interruption of IL-2 production,3,4 and indirectly, through the blockade of the action of interferon-γ (IFN-γ)5,6 leading to suppression of macrophage activation.7 However, the precise molecular and cellular pathways of this inhibition remain undefined. Liu and coworkers recently suggested that competition might occur between IL-10 and IFN-γ to bind common cell-surface receptors and that the balance of these two cytokines is an important step during cell proliferation.8 Interestingly, murine IL-10 (mlL-10) has also been shown to mediate a wide variety of biological functions stimulating the growth of mast cells,9 mature and immature thymocytes,10 Ly1-positive B cells,11 but most notably, the differentiation of murine cytotoxic T cells.12 In addition, mIL-10 has been reported to prolong the viability of cultured B lymphocytes in the absence of any evidence for cell activation13 and to increase the cell-surface expression of class II major histocompatibility complex (MHC) antigens without inducing cells to enter cell cycle.14

The microenvironment at the time of cell activation and the contribution of cytokines such as IL-10 and IFN-γ will be important factors influencing eventual disease outcome. Using murine models clear distinctions in the pattern of cytokines produced by CD4 cells can be associated with pathology;1 however, in humans this distinction is not as clear, although several diseases (allergy,15,16 hypereosinophilia17 and mycobacterial infections1820) have been attributed to dominant Th2-type responses. The signalling events which cause the emergence of a dominant Th2 cell proliferation are unclear and appear to require the presence of additional cytokines, including IL-4 and IL-5.

Previous studies using a murine model have shown that IL-10 inhibits mitogen-induced T-cell proliferation21 and using human cells Taga and Tosato demonstrated reduced IL-2 production.4 Whether IL-10 is only necessary at the time of activation or results from the combined effects of previous exposure remains the subject of investigation.

CD8 T cells expressing the CD57 (leu7) glycoprotein determinant are a small subset in healthy individuals that are associated with natural killer cell activity.22 D’Angeac and coworkers demonstrated that isolated CD8 cells negative for CD57 when cultured for 12 days with the cytokines interleukin-2 (IL-2) and interleukin-4 (IL-4) or with cells from a secondary mixed lymphocyte reaction (MLR) can be induced to express the CD57 antigen.23 Interestingly, CD8/57 positive cells have also been shown to predominate in clinical conditions in which low levels of IL-2 reside, such as following bone marrow transplantation and infection with the human immunodeficiency virus. Recent reports demonstrate the synergistic effects of IL-2 and IL-10 on natural killer (NK) cell activity. It is unclear if IL-2 and IL-10 act on all CD8 cells in the same way or whether they preferentially act on particular subsets such as CD8/CD57 coexpressing cells.

It has become evident in recent years that ligation of the T-cell receptor (TCR) can have a variety of outcomes. Non-responsiveness or partial activation can result from the lack of CD80/CD86 mediated costimulation.24 Chai and coworkers demonstrate that CD80 and CD86 are expressed on CD8 cells and that the ratio of these two molecules may be important regulators of cell activation.25 It is unclear as to the effect that IL-10 may have on CD8 costimulation and the role this may play in T:T antigen presentation.

In this study, we show that recombinant human IL-10 (rhIL-10) has a direct dose-dependent inhibitory effect on peripheral blood mononuclear cells stimulated in vitro when these cells have not previously been exposed to rhIL-10. We have extended previous studies by showing not only a primary inhibition with high doses of rhIL-10 (added either before or at the time of activation), but also a polyclonal expansion of CD8 cells several days later. Furthermore, using a combination of neutralizing antibodies and purified cell fractions we have shown that this expansion is independent of monocyte signalling and IL-4. CD8 clones responsive to high doses of IL-10 have reduced cell cytotoxicty, produce Tc2 cytokines and do not express CD57.

The study highlights important considerations for the therapeutic use of IL-10 as a biological modifier and any role it may play at sites of tissue pathology.

MATERIALS AND METHODS

Preparation of peripheral blood mononuclear (PBMN) cells

Peripheral blood from normal donors was collected into endotoxin-free lithium heparin (100 U/tube, Becton Dickinson, Luton, UK) and the mononuclear cells separated on Ficoll–Hypaque (Pharmacia LKB, Milton Keynes, UK), according to the manufacturer’s instructions. Cells recovered from the mononuclear layer were washed twice in Hank’s buffered salt solution (HBSS, pH 7·6, Flow ICN) and resuspended at a concentration of 1×106/ml in sterile filtered tissue culture medium consisting of RPMI-1640 (Sigma, Poole, UK) supplemented with 2 mm glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 mm HEPES buffer (pH 7·5) and 10% (v/v) heat inactivated fetal calf serum (Sigma).

Isolation of CD8 positive lymphocytes

CD8 cells were isolated using a MiniMACS separation system (Miltenyi Biotec). Briefly, separated PBMN cells were washed in unsupplemented RPMI-1640 and resuspended in wash buffer, consisting of 0·15 m phosphate-buffered saline (pH 7·5) with 0·5% (w/v) bovine serum albumin (Sigma) and 2 mm ethylenediamine tetra-acetic acid (EDTA; pH 7·2, Sigma). Twenty microlitres of CD8 Multisort Microbeads were added per 107 cells, mixed and incubated for 15 min at 4°. The cells were then washed, resuspended and passed through a MiniMACS column placed next to a magnet. The column was removed from the magnet and 1 ml of wash buffer rinsed through to harvest CD8 cells. These were then incubated with 20 μl of release reagent for 10 min at 4°. The cells were washed twice in wash buffer and resuspended at 1×106/ml in TCM. The purity was determined using flow cytometry and assays only established using CD8 populations of greater than 99% purity.

Maintenance of CD8 cells

At day 7–8 (proliferative phase) CD8 cells were harvested and seeded in individual wells at between 100 and 0·3 cells/well. Each culture contained 1×105 autologous irradiated PBMN cells (feeders). Cells were fed every 4 days with fresh media containing 100 U/ml of rhIL-10. At day 20 wells seeded with single cells that had grown were tested for cytokine production following stimulation with anti-CD3 as described below.

Pre-activation of cells with rhIL-10

Either PBMN or enriched CD8 positive cells were seeded at 1×106/ml in 5 ml cultures. To each cell suspension 0, 10, 25, 50 100 and 200 U/ml of rhIL-10 (Genzyme, Oxford, UK) was added. The absolute concentration of rhIL-10 was 20 ng/100 U established using the WHO International Standard 92/516. The primary cultures were incubated for 2 hr in 5% CO2 at 37° using polypropylene tubes to restrict cell adhesion. Following incubation, the cell suspension was mixed and divided into two halves. Two hundred microlitres of each primary culture was added to triplicate wells of a 96-well microtitre plate precoated with an anti-CD3. The second half of the primary culture was washed twice to remove exogenous IL-10 and then resuspended at 1×106/ml in TCM. Two hundred microlitres of this cell suspension was added to triplicate wells of a microtitre plate precoated with anti-CD3.

Stimulation of PBMN cells

Anti-CD3. A cross-linking anti-CD3 antibody independent of exogenous IL-2 was obtained from Immunotech (Coulter, Luton, UK). The optimal concentration for cell activation was established from the stimulation of PBMN cells from normal donors. A stock solution of 0·5 μg/ml of antibody was prepared in sterile RPMI-1640. Fifty microlitres of this stock solution was then added to each well of a microtitre plate and incubated for 2 hr, after which residual fluid was removed and 200 μl of each of the incubated cell suspensions added. Control cultures for each time-point consisted of cells which had been cultured in the absence of IL-10, and later stimulated with anti-CD3 or left unstimulated.

Phorbol-12-myristate-13-acetate (PMA)/calcium ionophore (CaIon). Optimal concentrations of PMA and the CaIon A1627 were established using PBMN cells from normal donors (data not shown).

Proliferation assay

Cell proliferation of each of the triplicate cultures was monitored by incorporation of tritiated thymidine. Cultures were pulsed with tritiated thymidine (0·5 μCi/well, Amersham, High Wycombe, UK) for 6 hr, harvested onto glass fibre filtermats (Wallac LKB, Milton Keynes, UK) and counted by liquid scintillation using a 1205 Betaplate (Wallac LKB). Results were recorded as counts per minute (c.p.m.).

Effect of anti-IL-4 neutralizing antibody on CD8 cell proliferation

A duplicate culture was prepared for each concentration of rhIL-10 in order to investigate the effects of endogenous IL-4. To each culture 2 μg/106 cells of a neutralizing monoclonal antibody to IL-4 (R & D Systems, Oxford, UK) was added immediately before cells were plated added to microtitre plates. This dose was established using the manufacturer’s instructions and confirmed by monitoring effect on the cell proliferation of T cells during a mixed lymphocyte reaction.

Immunophenotyping of cell cultures

A cocktail of directly labelled CD8 (Coulter Clone Cytostat, Luton, UK) and either CD3, TCR α/β, TCR γ/δ, CD11, CD18, CD25, CD95 (Serotech, Oxford, UK), CD8 α/β chain receptor, CD57 (Immunotech, Luton, UK) and CD80, CD86 (Pharmingen, Luton, UK) monoclonal antibodies were used to quantify cell populations. At each time point, 200 μl of the cell culture was removed and incubated either with 10 μl of the monoclonal antibody cocktail or an isotype-matched control for 10 min at room temperature. The cells were then fixed using Coulter Q-Prep as per manufacturer’s instructions and fluorescent intensity analysed by fluorescence-activated cell sorting (Coulter FACScan).

TCR variable beta gene (TCR-Vβ) family usage

Total RNA was isolated from control and IL-10 treated cells. Cells were lysed using TriReagent (Sigma) and total RNA extracted from the lysate according to the manufacturer’s instructions. RNA was reversed transcribed using a reverse transcription system (Promega, Southampton, UK). Polymerase chain reaction (PCR) was used to amplify specific segments of the first 20 TCR-Vβ families defined. Oligonucleotides corresponding to common region sequences of the 20 TCR-Vβ family members26 were used to amplify fragments of cDNA. Each 100 μl PCR reaction contained 2 μl cDNA, PCR buffer (10 mm Tris–HCl, pH 9·0, 50 mm KCl, 0·1% Triton-X-100, 0·01% (w/v) gelatin), 2·0 mm magnesium chloride, 200 mm dATP (adenosine triphosphate), dCTP (cytosine triphosphate), dGTP (guanosine triphosphate), dTTP (thymidine triphosphate), 25 mm C-β primer, 25 mm of the appropriate Vβ primer, 3·125 mm 5′ C-α primer, 3·125 mm 3′ C-α primer and 0·2 U SuperTaq (HT Biotechnology Ltd, Cambridge, UK). Amplification efficiency was monitored using primers to a constant region of the alpha TCR chain. PCR conditions were identical for each reaction and consisted of an initial step at 94° for 3 min, followed by 35 cycles of 94° for 1 min, 52° for 2 min, 72° for 3 min and a final extension of 72° for 10 min. In order to reduce the risk of contamination, PCR reactions were prepared in a laminar flow hood using aerosol-resistant filter pipette tips. The post-PCR products were then subjected to gel electrophoresis through 2% agarose gels in 1×Tris–acetate–EDTA (TAE) containing ethidium bromide. The products were visualized by exposure to UV light.

Cytotoxicity assays

After 7–9 days in culture CD8 responsive cells were assayed for cytotoxicity against K562 and Daudi and autologous cells. Target cells were labeled in suspension with 51chromium (51Cr) in 100 μl (100 μCi/106 cells) for 1 hr and washed three times prior to use. Three thousand targets were aliquoted into wells of a 96-well round-bottom microtitre plate; six wells were incubated with aqueous 1% Triton-X-100 (v/v) (BDH Chemicals, Dagenham, UK) for maximum release and six wells with medium alone for spontaneous release in each plate. The cultured CD8 cells were dispersed in their individual wells and equal volumes aliquoted into the target cells used. Medium was added to final volume of 200 μl in each well. The plates were incubated for 6 hr at 37°, 100 μl was harvested from each well and the released 51C measured.

Cytokine assays

A standard sandwich enzyme-linked immunosorbent assay (ELISA) technique was used. Briefly, 15 ng/ml of a primary goat polyclonal antibody (IL-2; AF202, IL-4; AF204, granulocyte–macrophage colony-stimulating factor (GM-CSF); AF215, IFN-γ; AF285 and tumour necrosis factor-α (TNF-α); AF610, R & D Systems) diluted 1:100 in coating buffer (Whitley Scientific, Preston, UK) were added to each well of a Nunc Immuno II plate (Gibco-BRL, Paisley, UK) and incubated overnight at 4°. The following morning the plates were washed and blocked for 3 hr at 37° with 1% (v/v) Tween-20 PBS, pH 7·4, containing 4% (v/v) human serum albumin (HSA; Sigma). Plates were washed and 100 μl of the second mouse antibody (IL-2; MAB602, IL-4; MAB604, GM-CSF; MAB615, IFN-γ; MAB285 and TNF-α; MAB210, R & D Systems) were added, respectively, and the plates incubated for a further 4 hr at 4°. Plates were washed and 100 μl of an alkaline phosphatase-conjugated rabbit antimouse antibody (Sigma) added and the plates incubated for 3 hr at room temperature. At this point, 100 μl of the substrate buffer (Whitley Scientific) containing 1·5 mg/ml p-nitrophenyl disodium phosphate was added. Absorbances were read at 405 nm when the top when the top standard read ≈1·6 optical density (OD) units. The assay was standardised against International Standards obtained from the National Institute of Biological Standards and Controls (Potters Bar, UK). The lower limit of detection was 10 pg/ml and the within batch coefficient of variation was IL-2, 10·9%; IL-4, 8·7%; GM-CSF, 4·2%; IFN-γ, 9·2% and TNF-α, 6·8%.

RESULTS

Dose-dependent effect of IL-10 on cell proliferation

A time course of cell proliferation was established using PBMN cells isolated from healthy individuals and stimulated with either immobilized anti-CD3 antibody or a combination of PMA and CaIon (Fig. 1a). The effect of IL-10 on cell activation was investigated at the time of maximum proliferation (96 hr). Addition of either 0, 1, 10, 25, 50, 100 and 200 U/ml rhIL-10 to individual cell cultures revealed a dose-dependent inhibition of cell proliferation (Fig. 1b) following stimulation with anti-CD3 but not PMA and CaIon. Maximal inhibition was observed with 25 U/ml of rhIL-10 at 96 hr. Interestingly, a reduced inhibitory effect on cell proliferation was shown with higher concentrations (50, 100 and 200 U/ml) of rhIL-10. No difference in the degree of proliferation was observed between the 100 and 200 U/ml and for this reason all subsequent investigations were performed using 100 U/ml concentration. To determine whether the reduced cell inhibitory effect resulted from increased cell viability or proliferation, cultures were maintained for 10 days following activation. Cells cultured in the presence of 100 U/ml rhIL-10 revealed an additional proliferative phase from day 4 to day 9, as shown by a complete reversal of any inhibitory effects (Fig. 1c). At day 6 the number of cells proliferating was higher than the control cell culture stimulated with anti-CD3 in the absence of rhIL-10.

Figure 1
(a). A proliferation time-course following stimulation of PBMN cells with either anti-CD3 immobilised (0·5 μg/ml) on plastic, PMA/CaIon or tissue culture media alone. (b). A dose–response curve showing the inhibitory effects of ...

Accumulative dose effect of IL-10 on cell proliferation

To investigate whether cells stimulated with anti-CD3 in the presence of 25 U/ml of IL-10, which resulted only in the primary inhibition (reduced initial phase proliferation), could be induced to proliferate following an additional exogenous dose of rhIL-10. At 96 hr poststimulation either a further 0, 1, 10 or 50 U/ml of IL-10 was added to each of the cultures (previously incubated with either 25 or 100 U/ml) and the cell proliferation measured 48 hr later. A dose-dependent inhibition was seen only in the cell cultures not previously incubated with rhIL-10 (Fig. 2). In contrast, cultures previously incubated with 25 U/ml rhIL-10 showed a significant dose dependent increase in cell proliferation following the addition of this second dose of rhIL-10. Interestingly, no effect on cell proliferation was observed following IL-10 addition at 4 days when cells had been stimulated with anti-CD3 in the presence of 100 U/ml rhIL-10. These experiments demonstrate that the primary inhibition of PBMN cells and the secondary cell proliferation were dependent upon the dose of IL-10.

Figure 2
Cells were stimulated in the presence of IL-10 at either 25 U/ml or 100 U/ml. At 96 hr post activation with anti-CD3 either 0, 1, 10, or 50 U/ml of IL-10 was added. Changes in cell prolifration following the addition of 25 U/ml rhIL-10 to cells not previously ...

Clonality studies of CD8 cells induced by IL-10

To investigate the possibility that IL-10 was acting as a polyclonal growth factor for CD8 cells the TCR-Vβ gene family usage was analysed. Studies were performed using CD8-enriched populations of cells prior to activation and at day 10 following culture in the absence and presence of rhIL-10 (100 U/ml). No change in the TCR-Vβ repertoire was demonstrated suggesting that IL-10 was acting as a non-specific growth factor for CD8 cells.

Is the IL-10 acting via monocyte signalling?

Using purified CD8 cells (>99%) we investigated whether the late increase in cell proliferation resulted from direct action of IL-10 on CD8 cells or via monocyte signalling. CD8 cell cultures stimulated with anti-CD3 in the presence of 100 U/ml of rhIL-10 showed the typical increase in cell number from day 4 to day 9 (Fig. 3a), suggesting that IL-10 was having a direct action on CD8 cells. To confirm that the CD8 populations of cells were not responding to endogenously produced IL-4 a duplicate set of cultures containing a neutralizing monoclonal antibody were included. No changes were observed when cells were stimulated and cultured in the presence of the neutralizing antibody (Fig. 3b).

Figure 3
Purified populations of CD8 cells were stimulated with anti-CD3 in the presence of IL-10 at 10 and 100 U/ml, respectively, and (a) thymidine incorporation determined. The results shown are the means and ranges of five individual experiments. A significant ...

Immunophenotype of cells proliferating to IL-10

To identify the immunophenotype of the cells induced to proliferate following the addition of 50 U/ml of rhIL-10; cells were analysed by flow cytometry. These studies revealed no change in the number of CD4 staining cells but a significant rise in CD8 positive cells in cultures containing high doses of rhIL-10 (data not shown). The IL-10 responsive CD8 positive cells were plated out and grown for a further 10 days in culture. At this point, FACS analysis of representative T-cell clones stained positive for CD3, TCR α/β, CD8 receptor (α/β heterodimer positive), CD11, CD18, CD95, CD45RO (data not shown). In addition, cells were stained for CD57 previously reported to be induced by IL-2 associated with NK-like activity and terminal differentiation. Interestingly, cells grown in high doses of IL-10 did not express CD57 (Fig. 3c) whereas those cell cultured in IL-2 (50 U/ml) showed a progressive increase in the number of CD57 positive cells. Recent reports suggest that expression of costimulatory molecules on CD8 cells may be an important pathway for T–T activation. To investigate this phenomenon CD8 clones were stained at the beginning of the cloning period (following 7–10 days in culture) and after the 10-day cloning period for CD80 and CD86 (Fig. 3d). At day 10 following the initial period of IL-10 incubation cells expressed low levels of CD80 and CD86. However, cells then grown on for a further 10-day period demonstrated no change in CD80 expression and a significant increase in the level of expression of CD86. Interestingly, incubation with IL-10 did not alter the level of expression at days 10 and 20 of culture consisting of 91% expressing CD28 cells, 50% expressing CTLA-4 cells and 45% demonstrating coexpression (Fig. 3e).

Effect of incubating cells with IL-10 (100 U/ml) prior to activation

To further explore the effect of rhIL-10 on activation, cells were incubated with rhIL-10 before anti-CD3 activation. CD8 enriched cells from normal donors were either incubated for 2 hr in the presence of 100 U/ml of IL-10 and then activated with anti-CD3 antibody or washed twice in HBSS and then resuspended in TCM to remove exogenous IL-10 prior to activation. Removal of exogenous rhIL-10 by washing the cells before activation did not reduce the secondary cell proliferation (Fig. 4).

Figure 4
Purified CD8 cells were either incubated for 2 hr before anti-CD3 stimulation in IL-10 (100 U/ml) or following incubation in IL-10 cells were washed prior to activation. No significant change in the number of CD8 was observed although maximal proliferation ...

Cytokine production

To investigate the pattern of CD8 cytokine release, tissue culture media was removed from cultures of eight representative clones 48 hr after stimulation with anti-CD3. These clones were maintained by weekly stimulation with anti-CD3 and 50 U/ml of rhIL-10. Typically high levels of IL-4 and GM-CSF could be demonstrated with an absence TNF-α and IFN-γ (Fig. 5). No individual clone exhibited consistent high production of Il-4 and GM-CSF. In addition, IL-2 was below the level of detection in each of culture supernatant from IL-10 stimulated clones in contrast to those activated in the absence of IL-10 which showed levels of greater than 400 pg/ml (data not shown). Analysis of cytokine production suggests a bias towards Tc2 types of cell response.

Figure 5
Tissue culture supernatant concentrations of IL-2, IFN-γ, IL-4, TNF-α and GM-CSF were measured in eight representative CD8 positive IL-10 responsive clones 48 hr after stimulation with anti-CD3.

Functional characterization of IL-10-induced CD8 cells

Following the demonstration that the CD8 clones failed to express CD57 an assessment of these cells to retain cytotoxic and NK activity was undertaken. Cells grown for a further period in IL-10 were harvested, washed and investigated for their ability to kill K562, Daudi, autologous and allogeneic cell targets in a 5-hr chromium release assay. Cells incubated in a high dose of IL-10 demonstrated a significant reduction in the level of allogeneic cytotoxicity, form 40 to 6%, and no killing of K562, Daudi and autologous target cells.

DISCUSSION

The presence of IL-10 in the microenvironment at the time of cell activation will have profound effects on the nature of any subsequent cellular response. Our studies both confirm the previously documented inhibitory effects of IL-10,13 which occur in a dose-dependent manner, and extend these by demonstrating selective activation and maintenance of a polyclonal population of CD8 positive cells. Furthermore, these cells were shown to have a Tc2 pattern of cytokine production and reduced cell cytotoxicity.

CD8 positive T cells have a regulatory role in certain circumstances and may not necessarily function purely as cytotoxic killers and it is suggested that differential cytokine production may be largely responsible for such an action.27 Seder et al. showed that anti-CD3 stimulated CD8 cells can be primed in vitro to secrete IL-4, with the production of this cytokine being dependent on prior exposure to IL-4 in a manner analogous to that shown in studies of CD4 cells.28,29 A more comprehensive knowledge of the properties of CD8 effector T cells generated under a wide variety of conditions, such as the concentration of IL-10 at the time of activation, would be of value in further efforts to interpret the regulatory role of these MHC class I-restricted T cells.

Interestingly, both the initial cell inhibition and secondary cell proliferation were only documented with anti-CD3 and not following PMA/CaIon activation. The addition of exogenous neutralizing antibody to IL-4 could not inhibit the proliferation observed with high doses of IL-10. Incubation of cells with high doses of IL-10 significantly altered the maximal CD8 cell proliferation by 24 hr. It is unclear at what stage of cell activation IL-10 has effected to bring about such a change but warrants further investigation. How IL-10 induces different cellular responses, such as inhibition of cell activation, prolonged viability and increased cell surface receptor expression is unclear, although the induction of differential signal transduction pathways may explain these observations. Recently, IL-10 has been shown to stimulate the differential formation of homo- and heterodimers of the signal transducers and activators of transcription STAT 1α and STAT330 in T cells and monocytes, respectively, by tyrosine phosphorylation. The expression of dimer formation is suggested to result in variable activation of signal transduction pathways. The dose-dependent cell response we report may therefore reflect differences in signal transduction following IL-10 signalling.

It has been shown that CD8 cells grown in the presence of IL-2 express CD57.23 This cell-surface marker is associated with ‘NK-cell-like activity’ and correlates with late stage terminal differentiation.31,32 The mechanism of IL-2 induced CD57 expression is not defined and suggests that IL-10 cell activation and maintenance may be acting via an alternative pathway. The failure of the of the IL-10-induced cells to recognize traditional NK targets but retain specific allogeneic cell cytotoxicity confirms a significant role of CD57 and the possibility of differentiating between these two potentially important mechanisms. In particular, a recent study reported by Albi and coworkers suggest that CD8 with NK-like activity retain graft-versus-leukaemia effect on the absence of graft-versus-host disease.33

The role of CD80 and CD86 expression by CD8 cells for T–T presentation has recently been highlighted.34 Results from our study show that IL-10 induces high levels of CD86 on the surface of CD8 in the absence of CD80. Intriguingly, recent work suggests that the ratio of CD80 and CD86 is important in determining the nature of cell activation or progression into a state of anergy. Furthermore, Chai and coworkers have demonstrated that, upon CD8 activation using peptides with high binding affinities, the ratio of CD80 to CD86 reverses from predominantly CD86 expression to at least five times the level of CD80.25 The potential differences in antigen presentation by IL-10-induced CD8 cells are currently being investigated.

Several studies have demonstrated plasma concentration in excess of 100 U/ml during episodes of infection and allograft rejection.35,36 The concentration of IL-10 used throughout these studies would therefore appear to be physiological at times of immune activation. The dose effects of IL-10 also reveal a suboptimal concentration of IL-10, which exhibits the initial inhibitory effects but is insufficient to cause the maintenance of the CD8 population of cells. The cellular response to the addition of exogenous IL-10 after 4 days in culture was shown to be dependent upon the concentration of IL-10 present at the time of cell activation. This may highlight important considerations for the therapeutic uses of IL-10 and its possible role at sites of tissue pathology. For instance, the presence of high doses of IL-10 during cell activation and expansion within a lymph node may affect any subsequent local response at the site of tissue injury.

We are currently investigating whether the population of CD8 cells induced by IL-10 represents a polarized effector population, which have originated either by avoiding selective downregulation or from general expansion of naive cells.

In conclusion, we have shown that the wide and diverse effects of IL-10 may be attributable to its concentration at the time of cell activation. Furthermore, this action is independent of antigen-presenting cells, which may have important implications for the clinical application of IL-10.

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