Mechanism of action of an antigen nonspecific inhibitory factor produced by human T cells stimulated by MPPS and PPD

CELLULAR IMMUNOLOGY 98,434-443 (1986)

Mechanism of Action of an Antigen Nonspecific Inhibitory Factor Produced by Human T Cells Stimulated by MPPS and PPD

GIOVANNA LOMBARDI,* ANNA MARIA DI MASSIMO,* FLAVIA DEL GALLO,* DANIELA VISMARA,* ENZA PICCOLELLA,* ORSOLA PUGLIESE,~ AND V. COLIZZI$

*Department of Cellular and Developmental Biology, University of Rome; tNationa1 Institute of Health, Rome; $Institute of Microbiology, University of Piss, Italy

Received August 26, 1985; accepted October 15, 1985

Human T lymphocytes cultured in vitro for 5 days with C. albicans purified polysaccharide (MPPS) and with purified protein derivative (PPD) from M. tuberculosis produce an antigen nonspecific inhibitory factor(s) (nslNH). nsINH blocks antigen-driven cell proliferation and the development of natural killer cells (NK) when added at the beginning of peripheral blood mononuclear cell culture. Analysis of the mechanism of action shows that nsINH inhibits the production of interleukin 2 (IL2), the expression of IL-2 receptor (Tat antigen), and the synthesis of immune interferon (IPN). The biochemical characterization of nsINH shows that the suppressive activity is acid (pH 2.5) and temperature (56’C) resistant. Gel filtration analysis indicates a molecular weight of 30-35K and 60-65K. These results suggest a role for nsINH in the down regulation of the lymphokine cascade. 0 1986 Academic PIZSS, hc.

INTRODUCTION

Interleukin 2 (IL2) and immune interferon (IFN) are lymphokines produced by activated T lymphocytes, both of which play unique roles in various immunological phenomena ( 1,2). IL-2 binds to a specific receptor (Tat antigen, 3) and allows T cells to pass from the early Gla phase to the late Glb phase and is the major signal for the proliferation and differentiation of T cells with antigen-specific and MHC-restricted cytolytic activity. Furthermore, IL-2 is directly involved in the production of IFN by T cells and in the activation of cells with natural killer activity [NK (4, 5)]. More recently, it has been shown that IFN may be required for IL-2 induction of NK cells suggesting a collaboration between IL-2 and IFN (6). According to this view, IL-2 stimulates T cells to produce IFN and in turn IFN would act as a differentiation signal in the IL-2-induced NK generation and proliferative response.

Although there is a large body of data describing the activation and the action of this lymphokine cascade, very little information is available on its down regulation. It has been reported that T suppressor cells and their soluble factors can block T-cell proliferation and differentiation by inhibiting the production and/or the action of different lymphokines. For example, IL- 1, IL-2, and MIF production has been shown to be influenced by T suppressor cells both in human and murine system (7- 10). Furthermore, more recent data indicate that the production/action of IL-l and IL2 is the target of soluble inhibitory molecules released by T suppressor cells ( 11, 12)

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REGULATION OF IL-2, IFN, AND NK ACTIVITY 435

suggesting a general function of the suppressive circuit in the regulation of the immune response. The finding that both cells and factors display antigen-nonspecific and MHC- unrestricted suppressive activity further supports their role as a relevant counterpart of the lymphokine pathway.

Recently, we have described a human model system in which a nonspecific inhibitor produced by Candida al&cans-activated T suppressor cells impairs cell proliferation by blocking IL- 1 production ( 11, 13). This paper analyzes the mechanism of action of such nonspecific inhibitory factor (nsINH) produced by culturing peripheral blood mononuclear cells (PBMC) with either PPD (protein purified derivative from Myco- bacterium tuberculosis) or MPPS (purified polysaccharide extract from C. albicans). The interaction of such nsINH with IL-2 and IFN production/action was investigated in relation to the inhibition of cytotoxic effector activity. The results show that nsINH interferes with the production of IL-2 and IFN, and the expression of IL-2 receptor, resulting in an inhibition of cell proliferation and development of NK cell activity.

MATERIALS AND METHODS

Cell preparation. Peripheral blood mononuclear cells (PBMC) from normal adult individuals were isolated on a gradient of Lymphoprep (Nyeegaard and Co.A/S, Oslo, Norway) according to the procedure of Boyum. The cells were suspended at 106/ml in RPM1 1640 medium (Flow Laboratories, Inc., McLean, Va.) supplemented with 10% autologous heat-inactivated serum, 15 mM glutamine (Eurobio, Paris), and 40 Kg/ml Gentalyn (Shering Corporation, Bloomfield, N.J.) (culture medium).

Microbial antigens. The polysaccharide fraction (MPPS) obtained from C. albicans (13) and purified protein derivative (PPD) from A.4. tuberculosis (Statens Seruminstitut, Copenhagen, Denmark) were both used at a final concentration of 10 Kg/ml.

Production of nsZAW. PBMC (5 X lO’/ml) were precultured in flasks (Nunc Inter Med) for 4 days in presence of MPPS or PPD in a humid chamber at 37°C in a mixture of 10% COZ, 7% 02, and 83% N2. The cells were then harvested, washed, resuspended in fresh medium with 5% of FCS, and cultured in new flasks for 2 days at 2.5 X 106/ml in the absence of antigens. The 48-hr supernatant was routinely used as source of nsINH (11). These supernatants obtained from MPPS- and PPD-stimulated cultures were designated, respectively, as nsINHMpps and nsINHppD.

Proliferation assay. Normal fresh PBMC were cultured ( lo5 cells/200 &well) in triplicate in flat-bottom microtiter plates (No. 3040, Falcon) in presence of MPPS or PPD. nsINH were added to culture in a volume of 100 &well. The plates were incubated for 6 days and [3H]thymidine ([3H]TdR, Amersham, England, sp act = 5 Ci/ mmol) was added to each well (0.5 &i) 20 hr before harvesting. The radioactivity was evaluated in a beta counter and expressed as counts per minute _+ standard de- viation (SD). The percentage of suppression was calculated according to the formula: ([cpm of control cultures - cpm of nsINH treated cultures]/[cpm of control culture]) x 100.

Production and assay of IL-Z. PBMC were resuspended in culture medium at a concentration of 5 X lO’/ml and cultured in tubes (No. 2054, Falcon) with MPPS or PPD in presence or absence of nsINH. The cell cultures were incubated for 48 hr and the supematants were collected, filtered through a 0:45qm Millipore filter (Millipore Corp., Bedford, Mass.), and stored at -20°C until tested. The supernatants were tested for IL-2 activity in a standard microassay by using an IL-2-dependent T-cell line

436 LOMBARD1 ET AL.

(CTLL) and evaluated the [3H]thymidine incorporation for 4 X lo3 cells in 200 ~1 of medium after 24 hr of culture (15). IL-2 activity is expressed in units per milliliter using a human recombinant IL-2 kindly provided by Biogen (16) as reference.

Expression of cell surface markers. An indirect immunofluorescence assay were performed on a FACS IV (Becton, Dickinson & Co, Mountain View, Calif.) for analysis of T-cell antigens. The monoclonal antibodies (mAbs) used were OKT3,OKT4,OKT8 (Ortho Pharmaceutical, Raritan, N.Y.) and anti-Tat kindly provided by Dr. T. Wald- mann (3). PBMC (5 X 10s/ml) were cultured with MPPS or PPD in presence or absence of nsINH. After 5 days the cells ( 106) were incubated with 100 ~1 of appropriate dilutions of mAbs for 40 min at 4”C, washed twice, and then incubated with a 1:40 dilution of affinity-purified goat anti-mouse IgG, F(ab’)z-fluorescein isothiocianate (FITC) (Cappel Laboratories, Cochranville, Pa.) in the same way. After washing, cells were brought up in 1 ml phosphate-buffered saline for FACS analysis. All incubation and centrifugation steps were done at 4°C. Background fluorescence was determined using diluted normal mouse serum instead of first antibody.

Production and assay of IFN. PBMC were cultured in the same condition used for the production of IL-2. The supematants were collected after 3 and 5 days of culture, filtered, and stored at -20°C until tested. The IFN present in the supematant was quantitated in human amnion WISH cells by a microassay which measures inhibition of cytopathic effect by Sindbis virus (17). The IFN species produced by MPPS/PPD- stimulated PBMC appeared to belong to gamma type since its biologic activity was largely destroyed by exposure to pH 2.0 and antisera prepared against IFN-a and IFN- B failed to neutralize IFN activity. IFN activity is expressed in laboratory units per milliliter. One IFN unit equals 3 IU by using the NIH international reference standard Gg 23-901-530 (IFN-7) supplied by the National Institute of Allergy and Infectious Diseases.

Induction and assay of cytotoxic cells. To study the effects of nsINH on the generation of cytotoxic cells, PBMC (106/ml) were cultured in tubes (No. 2052, Falcon) with MPPS and PPD in presence or absence of nsINH. In other cultures PBMC were stimulated with irradiated allogeneic Epstein-Barr virus transformed B cells. After 7 days of culture the cells were collected, washed, and resuspended at 2 X 106/ml in medium containing 5% of FCS. The cytotoxic activity was tested in a “Cr-release assay (18). K562 target cells were labeled with 5’Cr (NEN Research products, 1 Ci/ ml) for 1 hr at 37°C. Target cells were washed and added ( lo4 cells/well) to effector cells in round-bottom 96-well microtiter plates. At least three effector to target ratios were tested in triplicate. The cultures were incubated for 4 hr at 37°C in a CO2 incubator and the percentage specific 5’Cr release was determined by the formula: ([experimental cpm - spontaneous cpm]/[maximal cpm - spontaneous cpm]) X 100. Standard de- viation of triplicate wells was usually < 10%. Lytic units (LU) X 10’ celIs were calculated by extrapolating the number of effector cells required to lyse 45% of targets.

Molecular weight sizing of &NH. nsINH (20 ml) from either MPPS- or PPD- stimulated cultures was concentrated on a YMlO membrane to 4 ml. nsINHi was applied (4 ml) to a 3 X g-cm Sephadex G- 100 column that had been equilibrated in 10 mMPBS, pH 7.6, and calibrated with the following markers: bovine serum albumin, ovalbumin, chymotrypsinogen A, and soya trypsin inhibitor. The sample was eluted from the column at 10 ml/hr with PBS and fractions were pooled, concentrated by an YM 10 membrane, and finally dyalized against medium. nsINH2 was applied (200 ~1) to a Superose tm 12 column (Pharmacia) to estimate the molecular weight by fast


pressure liquid chromatography. The same markers were used to calibrate the column before and after sample elution. The sample was eluted by using 0.5 A4 acetic acid-l A4 pyridine buffer (pH 5.0) at a flow rate of 0.2 ml/min. The eluted fractions were pooled, concentrated, and then dyalized.

RESULTS

Inhibition of MPPS- and PPD-Driven Cell Proliferation by &NH

nsINH was produced by preculturing PBMC for 4 days in presence of MPPS or PPD. The cells were then washed and cultured for further 2 days with medium alone. The 48-hr supernatants were collected and tested for the inhibitory activity on the proliferation of fresh PBMC stimulated with either MPPS or PPD. Figure 1 shows that supernatants from both MPPS- and PPD-activated PBMC exerted a strong inhibitory activity on antigen-driven fresh PBMC proliferation. This inhibitory activity was clearly antigen nonspecific since both factors produced either in the MPPS (ns- INHMrPS) or PPD (nsINH& cultures impaired both MPPS- and PPD-induced proliferative response. Control supernatants produced by culturing unstimulated PBMC failed to exert any inhibitory activity in more than 10 experiments performed [data not shown, (1 l)].

Efect of &NH on the Development of Cytotoxic Activity

It has been reported that both cytotoxic T lymphocytes and lymphokine-activated killer cells may require a proliferative and differentiative phase to develop their function (10, 19, 20). We have then developed a system in which PBMC stimulated in vitro with MPPS or PPD kill NK-sensitive target cells (Piccolella, Cavallo, Lombardi, Vis- mara, Dolei, Fioravanti, Cochi, Mannella, and Colizzi, submitted for publication). This model system allowed us to investigate further the ability of nsINH to block the development of cytotoxic cell activity.

MPPS PPO

FIG. 1. Inhibitory effect of nsINH on MPPS- and PPD-stimulated PBMC. PBMC activated with MPPS and PPD were cultured for 6 days in presence of 50% of nsINH MPrs (Bl) and nsINHppD (I?#). The results are expressed as mean [3H]TdR incorporation + SD. The horizontal lines represent the SD of r3H]TdR incorporation of PBMC cultured in absence of nsINH (positive control). One typical experiment out of 10 performed is reported.

438 LOMBARDI ET AL.

PBMC cultured for 7 days with microbial antigen (MPPS or PPD) and nsINH were washed and then tested for NK activity on chromium-labeled K562 target cells. Figure 2 shows that a strong cytotoxic activity is induced by MPPS and PPD at all effector/ target used. When nsINH produced in MPPS or PPD systems were added to PBMC culture a significant inhibition of NK activity was observed. A similar inhibition was observed when an optimal dilution of mAbs anti-class II molecules [PT’F 29.12 kindly provided by Dr. A. Bargellesi, (21)] was added at the beginning of the culture.

To further investigate the effect of nsINH on the development of cytotoxic activity, PBMC were cultured in presence of allogeneic Epstein-Barr virus-transformed B cells. Six days later, effector cells were assessed on two different targets, namely EBV-transformed B cells (used as stimulator cells) and K562 cells. The results reported in Table 1 show that nsINHMrps inhibited significantly the induction of cytotoxic cells against both targets.

Efect of &NH on IL-2 and IFN Production

Since both NK and CTL activity has been reported to be dependent on various lymphokines, we then investigated whether the nsINH-mediated inhibition of cytotoxic activity was due, at least in part, to a defective production of IL-2 and/or IFN.

PBMC were cultured with MPPS or PPD in presence or absence of nsINH. At different times of culture, supernatants were collected and tested for IL-2 and IFN activity. The results reported in Fig. 3 show that nsINH produced both in the MPPS and PPD system inhibited IL-2 production evaluated by a classical CTLL proliferation assay. Figure 4 illustrates the production of IFN by MPPS- or PPD-stimulated PBMC

MPPS

5 10 20 5 10 m effector cells x to-4

PPO

FIG. 2. Inhibitory effect of nsINH on MPPS- and PPD-induced cytotoxicity by PBMC. PBMC isolated from two different individuals were cultured for 7 days in culture medium (0) or with MPPS or PPD (A). nsINHk(rrs (m) or nsINHppI, (0) or PTF 29.12 (0) were added at the beginning of the PBMC cultures. The cells were then washed and tested as effector cells against K562 target cells (104) in a 4-hr cytotoxicity assay. One typical experiment out of three performed is reported.


TABLE 1

Inhibitory Effect of nsINH on EBV-B-Induced Cytotoxicity by PBMC

Cytotoxicity ’

Culture conditions’ EBV-B K562

Medium EBV-B EBV-B + nsINHr,,r.r.s

0.5’ 8 26 50

5d 28

“PBMC were cultured for 5 days in culture medium or with irradiated-autologous EBV-B cells. The nsINH factor was added at the beginning of the culture.

b The cytotoxic activity of cultured PBMC was assayed in 4 hr %r release assay using EBV-B or K562 target cells.

’ LU/lO’ cells. d The underlying numbers mean a significance of inhibitory activity of at least P c 0.05 (Student T test).

at Day 3 and 5 of culture. As can be seen, the addition of nsINH to both MPPS and PPD stimulated cultures strongly blocked the release of IFN.

Eflect of nsINH on the Expression of IL-2 Receptor

The inhibitory effect of nsINH was also investigated on the expression of IL-2 receptor (Tat antigen). Fresh PBMC were cultured for 5 days with MPPS or PPD in presence or absence of nsINH. The cells were then harvested and incubated with anti- Tat antibodies as described under Materials and Methods. The fluorescence was evaluated by FACS IV analysis. Table 2 shows that when cells were cultured in presence of nsINH the percentage of Tat+ cells was reduced. When the expression of other surface markers was evaluated, no inhibition of T3, T4, and T8 antigens was observed

FIG. 3. Inhibitory effect of nsINH on IL-2 production by MPPS- and PPDstimuIated PBMC. Two-day culture supematants were collected from unstimulated or MPPS/PPD-stimulated PBMC in absence (0) or in presence of nsINHMrrs @II) or nsINH rpn &?I). One hundred microliters of culture supematants were added to 100 ~1 of CTLL cells which were cultured for 24 hr. Data are expressed as IL-2 units/ml.

440 LOMBARDI ET AL.

6000

I

OAY 5

medium MPPS PPO

FIG. 4. Inhibitory effect of nsINH on IFN production by MPPS- and PPDstimulated PBMC. Supematants were collected at Days 3 and 5 of culture in absence (medium) or in presence of MPPS/PPD. Control medium (O), nsINHwprs (LB), and nsINH rrn (I&l) were added at the beginning of culture. IFN activity is expressed in laboratory units. Shown are results of one out of three similar experiments.

in cultures containing nsINH, excluding a general inhibitory effect of nsINH on the surface markers expression.

Physicochemical Properties of &NH

nsINHMpps was further characterized in terms of physicochemical properties. Heating nsINH at 56°C for 30 min removed approximately 9% of the suppressive activity.

TABLE 2

Effect of nsINH on T-Cell Surface Marker Expression

Culture conditions” T3+ cells T4+ cells TS+ cells Tat+ cells

Medium 68' 33 21 1 MPPS 19 45 24 18 MPPS + nslNHMrrs 14 49 23 3d MPPS + nsINHppn 13 43 21 9 PPD 13 41 22 33 PPD + nsINHhnrrs 14 41 23 4 PPD + nsINHppn 15 49 25 11

‘PBMC were cultured for 5 days in culture medium or with MPPS or PPD. The nsINH factors were added at the beginning of the culture.

* The percentage of T3+, T4+, T8+, and Tat+ cells was determined by Fats IV analysis. ’ The results refer to a typical experiment out of five reported. d The underlying numbers mean a significance of inhibitory activity of at least P < 0.05 (Student T test).


nsINH also appeared to be stable in acid, since dialysis for 12 hr against pH 2.5 glycine buffer did not remove the suppressive effect (data not shown).

To estimate the molecular size of nsINH, two gel filtration experiments were performed and the pooled fractions were tested for the inhibitory activity on antigen- driven cell proliferation. As shown in Fig. 5, the biologically active material from nsINHhlpps (Fig. 5A) was detected at two MW, approximately 75-55,000 and 40- 30,000. Estimation of MW of nsINH rrn (Fig. 5B) by fast pressure liquid chromatography confirmed the previous results showing that the inhibitor occurred at two molecular weights of 65-60,000 and 35-30,000.

DISCUSSION

This paper analyzes the mechanism of action of a nonspecific inhibitory factor(s) produced by microbial antigen-stimulated PBMC. Three main findings are here described, namely (i) the inhibition of cell proliferation and cytotoxicity by nsINH, (ii) the failure of PBMC to produce IL-2 and express its receptor when cultured with nsINH, and (iii) the inhibition of IFN production by nsINH.

We have previously described an in vitro human model system in which a polysaccharide extract from C. albicans (MPPS) is able to induce the maturation of antigen- nonspecific T suppressor cells (22, 23) and the production of a soluble inhibitory factor(s) (11, 13). Although the production of nsINH requires the activation of T cells by antigen-presenting cells [FcR-adherent cells pulsed with MPPS ( 13)] the inhibitory activity of nsINH is antigen nonspecific and genetically unrestricted (11). Early information showed that nsINH can modulate the production of IL- 1 suggesting a direct control of the lymphokine cascade by nsINH (11). The involvement of nsINH in the control of the lymphokine pathway has been formally demonstrated by showing that PBMC cultured in presence of nsINH and stimulated either by MPPS or PPD fail to produce IL-2, to express IL-2 receptor, and to release IFN. The block of the lymphokine cascade exerted by nsINH is accompanied by a significant impairment of NK cell activity in the MPPS or PPD cultures. We would like to suggest that these two events

.Ll ” ‘1 ” ” ’ 1 <80 8/I 7/J 6p 6p 5,5 5? 40 3,O 20

70 65 60 55 50 40 30 20 i0

- ,. lC)O 90 8? 7$I s,5 6p 95 5,O 40 3,5 3,0 ~ 2, 90 80 70 65 60 55 50 40 35 30 20

MOLECULAR WEIGHT IKd)

FIG. 5. Molecular weight sizing of nsINH. (A) Shows a gel filtration experiment of nsINHMPm performed using a Sephadex G-100 column. (B) Shows a MW estimation of nsINH PPo by fast pressure liquid chromatography.

442 LOMBARD1 ET AL.

are strictly linked. In fact, it has been shown that mature NK cells resemble T cells in their ability to respond to IL-2 and to proliferate (20, 24). Moreover IL-2 released by antigen-activated T cells secondarily can induce NK cell to produce IFN which in turn might participate in inducing cell proliferation (6). However, formal experiments are needed to show a cause-effect relationship between the lack of IG2/1FN production and failure to develop NK activity.

There are several inhibitory molecules released by T suppressor cells which have been reported to down regulate the proliferative and cytotoxic response by inhibiting the lymphokine production. In mouse, antigen nonspecific factors which inhibits DNA synthesis and IL-2 production are released by T suppressor cells activated by M. bovis- BCG infection (12) and by alloantigen-stimulated spleen cells (8). In man, Con A- activated T suppressor cells which limit IL-2 production have been described (10) and this mechanism has been reported to be involved in the down regulation of cytotoxic T-cell responses (24, 26, 27).

Antigen nonspecific suppressor-T-cell products might also interfere with cell proliferation and cytotoxicity by an indirect mechanism. Suppressor activating factor [SAF, (28)] and suppressor cell inductive factor [SIF, (29)] both produced by T cells are able to induce other T cells to become effective suppressors of cell proliferation and cytotoxicity. However, we exclude this mechanism as responsible for the inhibition observed in our system since nsINH fails to induce suppressor cells from MPPS/PPD- stimulated PBMC (unpublished observations).

There are other factors induced by mitogens in human systems which share similar physicochemical properties. Concanavalin A induced suppressor factors have been described as a 30-45kDa (30) or a 60-90kDa (31) both of which are acid (pH 2.5) and temperature (56°C) stable. Further analysis at molecular level need to establish the relationship between Con A- and MPPS/PPD-induced suppressor factors.

In conclusion, what is the biological role of nsINH? It is conceivable that an antigen nonspecific and genetically unrestricted regulatory mechanism which balance the positive effect of the lymphokine cascade may be induced during microbial stimulation of the immune system (32). The presence of a counterpart of the lymphokine cascade is here supported by the finding that nsINH produced by PBMC stimulated with MPPS and PPD inhibits IL-l, IL-2, and IFN production as well as Tat antigen expression.

ACKNOWLEDGMENTS

We thank R. Morelli for preparation of MPPS antigen, A. Dolei for IFN titration, and E. Mannella for FACS analysis. We are indebted to Professor D. Guerritore for his continuous encouragement during this study. This work was supported by the Italian National Research Council (CNR), Grants 85.0219944 (Progetto Finalizzato Oncologia) and 84.02029.52 (Progetto Finalizzato Controllo Malattie da Infezione).

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Mechanism of action of an antigen nonspecific inhibitory factor produced by human T cells stimulated by MPPS and PPD