Eur. J. Immunol. 1984.14: 1057-1060 Recombinant interleukin 2 induces cytolytic cells 1057

Rene Devos', Geert Plaetinck Induction of cytolytic cells by pure recombinant human interleukin 2* and Walter Fiers

Laboratory of Molecular Biology, State University Ghent

-of Ghent and Biogent', Purified recombinant human interleukin 2, produced in Escherichia coli, was suffi- cient to generate cytolytic cells in concanavalin A-stimulated, T helper cell-depleted (Lyt-1.1:) or accessory cell-depleted (Ia-) murine spleen cell cultures. Moreover, recombinant interleukin 2 (rIL 2) was able (even at 0.5 ng/ml) to induce cytolytic cells in undepleted murine spleen cell cultures and undepleted human peripheral blood lymphocyte cultures in the absence of a mitogen. Purified recombinant human interferon-y, produced in Chinese hamster ovary cells, did not induce either cytolytic activity or IL 2 responsiveness in human peripheral blood lymphocyte cultures. Possi- bly, the rIL2-induced cytolytic cells are formed as a result of nonspecific secondary cytotoxic T lymphocyte activation.

1 Introduction

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are two cytotoxic systems which may play an important role in host defence, particularly against cancer and various infec- tions. NK cells are capable of cytotoxic activity without obvi- ous prior sensitization, while the induction of antigen-specific CTL involves cell-cell interactions and occurs by way of solu- ble mediators produced by antigen-triggered T helper cells and major histocompatibility complex class I1 antigen-restricted accessory cells. Grimm et al. [1-31 demonstrated the existence of a cytotoxic system which differed from the conventional CTL and NK cell systems since its activation was accomplished by way of lectin-free, purified interleukin 2 (IL 2) preparations alone. Here, we report that purified recombinant human IL2 (rIL 2) preparations produced in E. coli efficiently induce these activated killer cells in human peripheral blood lympho- cytes (PBL) as well as in murine spleen cell (SC) cultures. Recombinant glycosylated human interferon-y (IFN-y) neither activates cytolytic cells nor enhances their rIL 2-induced acti- vation.

2 Materials and methods

2.1 Cell cultures

Murine (C3H/HeJ, BALB/c, CBAnu) SC suspensions, treated with 0.83% NH4Cl to lyse erythrocytes, were prepared in RPMI 1640 medium (Gibco, Grand Island, NY) containing 10% fetal calf serum (FCS; Gibco) supplemented with L- glutamine (0.03%), HEPES buffer (20 mM), sodium hydrogen carbonate (2 g/l) and antibiotics (complete medium). The cell suspension was passed over nylon wool and then over Sephadex G-10. Human PBL were isolated from a buffy coat (Red Cross Blood Center, Ghent) by centrifugation over Lym-

[I 45651

* This work was supported by Biogen, N.V.

Correspondence: Walter Fiers, Laboratory of Molecular Biology, State University of Ghent, Ledeganckstraat 35, B-9000 Ghent, Bel- gium

Abbreviations: Con A Concanavalin A CTL: Cytotoxic T lym- phocyte(s) IFN-y: Interferon gamma IL 2: Interleukin 2 rIL 2 Recombinant IL 2 LPS: Lipopolysaccharide PBL: Peripheral blood lymphocytes PHA: Phytohemagglutinin NK: Natural killer SC: Spleen cell(s) C: Complement

phoprep (Nyegaard, Oslo) and plastic-adherent cells were removed by incubation at 2 x lo6 cells/ml for 2 h at 37°C in RPMI 1640 medium supplemented with 10% FCS in plastic flasks (Falcon Plastics, Oxnard, CA). Nonadherent cells were subsequently incubated with nylon wool for 1 h at 37°C and nylon wool-passed cells collected.

2.2 Preparation of Lyt-l.1'- and Ia+-depleted cell cultures

Two x lo7 cells from C3WHeJ mice were treated (30 min at 4°C) with a 1 : 200 dilution of a monoclonal anti-Lyt-1.1 anti- body (New England Nuclear, Boston, MA) or a 1 : 30 dilution of a monoclonal anti-Iak antibody (A.TH anti-A.TL, Cedar- lane Laboratories, Hornby, Ontario, Canada), and nontoxic rabbit complement (Lowtox M rabbit complement, Cedar- lane) diluted at 1 : 10 or 1 : 6, respectively. After 1 h at 37"C, dead cells were removed by centrifugation over Lymphopaque (Q = 1.086; Nyegaard, Oslo).

2.3 Assay for IL2 activity

IL2 was assayed as described [4]. Briefly, 2 x lo4 human phy- tohemagglutinin (PHA) splenocytes were distributed into mi- crotiter plate wells in 200 pl RPMI 1640 medium containing 10% fetal calf serum in the presence of logz dilutions of the test sample. After a 24-h incubation period at 37"C, the cells were pulsed during 6 h at 37°C with 0.5 pCi = 18.5 kBq [3H]thy- midine (Amersham, GB; 23 Ci/mmol). After harvesting, the incorporated radioactivity was measured in a liquid scintilla- tion counter. Our purified rIL 2 preparation contained 0.05 mg proteidml and 5 x lo5 unitdm1 relative to a PHA- induced human splenocyte supernatant [4], set at 100 Uiml.

2.4 Induction for cytolytic activity

Replicates of lo5 cells were seeded into flat-bottom micro- plates (Becton Dickinson, Sunnyvale, CA) in 200 p1 complete medium in the presence or absence of lymphokines (rIL2 preparations purified from E. coli and diluted 1000-fold in complete medium, i.e. 500 unitdml; 2000 units/ml human recombinant IFN-y produced in Chinese hamster ovary cells [13]*) plus mitogens 12.5 pgfml concanavalin A (Con A, Phar-

* R. Devos, C. Opsomer, S. J . Scahill, J. Van der Heyden and W. Fiers, J. Interferon Rex, in press.

0014-2980/84/1111-1057$02.50/0 0 Verlag Chemie GmbH, D-6940 Weinheim, 1984

1058 R. Devos, G. Plaetinck and W. Fiers Eur. J. Immunol. 1984.14: 1057-1060

macia, Uppsala, Sweden); 5 yg/ml lipopolysaccharide (LPS, Gibco), or 5 yglml PHA (Wellcome, Beckenham, GB)] as in- dicated.

2.5 Measurement of proliferative and cytotoxic activity

After 42 h (mouse SC cultures) or 5 days (human PBL cul- tures) at 3 7 T , triplicate wells were pulsed with 0.5 yCi [3H]thymidine and the proliferative response was assayed after 6 h at 37 "C. The cytolytic activity was measured after pooling cells from 6 wells, washing, and incubation of various numbers of cells in triplicate during 4 h at 37°C with 2 x lo3 'lCr- labeled target cells (P815 cells in the absence or presence of 10 pg/ml PHA, or Daudi cells in the absence of PHA) in 200 y1 medium in U-bottom microplates (Titertek, Irvine, Scotland). Aliquots of 100 ~1 were harvested and counted in a y-counter (LKB, Bromma, Sweden). Spontaneous release (without effector cells) and maximal release (in the presence of 1% Triton X-100) of 'lCr by the target cells were measured and the percent specific release calculated as follows:

Release effectors - spontaneous release Maximal release - spontaneous release

% Specific release = x 100

3 Results and discussion

3.1 Characterization of human rIL 2

Human rIL2 was purified to homogeneity from induced E. coli K514 cells harboring the plasmid pTrp-Hi1201 [4] and its biological activity was measured in a 24-h proliferation assay using IL 2-dependent human splenocytes. These preparations at a concentration of 0.05 rng proteidml contained approxi- mately 5000 times more biologically active IL2 per unit vol- ume than our standard PHA-induced human splenocyte super- natant (Fig. 1) and efficiently supported the long-term prolif- eration of PHA-activated human PBL, tonsillar or spleen- derived human T lymphocytes and a murine IL 2-dependent CTL line.

3.2 rIL2 induces cytolytic activity in Lyt-1.1'- or Ia+-depleted Con A-activated murine SC cultures

Recently, several laboratories [5 , 61 have demonstrated that the polyclonal induction of CTL by mitogens such as Con A, in T helper cell-depleted [anti-Lyt-1.1 plus complement (C)] or accessory cell-depleted (anti-Ia plus C) murine spleen or lymph node cultures is dependent on other soluble factors besides IL2 which are present in a mitogen-induced spleen culture supernatant. Since purified rIL 2 preparations are obviously devoid of other factors, we investigated whether rIL2 was sufficient to induce CTL activity in such depleted cultures using PHA-treated mastocytoma P815 cells as a target. Figs. 2a and b show that the proliferation and induction of CTL in nylon wool nonadherent and Sephadex G-10-passed Con A-activated SC from C3WHeJ (H-zk) mice was totally abolished by treatment with a monoclonal anti-Lyt-1.1 or anti- Iak and c. This was expected since the proliferation and induc- tion of CTL is dependent on the production of IL2, which itself is dependent on the presence of T helper and accessory cells. Expression of IL2 receptors on T cells in Con A-acti- vated SC cultures is not dependent on interactions between T

helper cells and accessory cells (antigen-presenting Ia- restricted cells) [7]. Addition of rIL2 to Lyt-1.1' or Ia+- depleted, Con A-activated cells, however, only slightly restored the proliferative capacity (Fig. 2a and b). On the other hand, the cytotoxic activity was completely restored by rIL 2 and even exceeded the cytotoxic activity found in total,

- -66 K - -45 K

-29 K

-21 K I - 1 8 4 K - - 14 3K

1

0 1 1 1 1 1 1 1 1 1 . . .

2 3 L 5 6 7 8 9 1011 1 2 1 3 m log dilution

Figure 1. Dose-response curve of IL 2 present in PHA-induced human splenocyte supernatant [4] (M) and of purified rIL2 (A-A) (diluted 1000-fold) on the [3H]thymidine incorporation by PHA-activated human splenocytes. The insert shows 2.5 yg rIL2 (A) electrophoresed in a reducing sodium dodecyl sulfate polyacrylamide gel (stained with Coomassie blue).

90 t

/

c o n t r o l

2 10 50 E f f e c t o r t a rge t Ef fector t a r g e t

Figure 2 . Induction of cytolytic activity by rIL2 in Lyt-l.l+- (a) and Ia' (b)-depleted, Con A-activated mouse SC cultures. Nylon wool- and Sephadex G-10-passed SC from C3WHeJ mice, depleted for Lyt- 1.1' cells, or Ia+ cells (see Sect. 2.2), or treated with rabbit C alone (diluted 1 : 6; control), were cultured for 42 h at 37°C in the presence of Con A or Con A plus rIL2 as indicated. Thereafter, the prolifera- tive response and cytolytic activity towards P815 target cells (in the presence of PHA) were measured.

Eur. J. Immunol. 1984.14: 1057-1060 Recombinant interleukin 2 induces cytolytic cells 1059

undepleted, Con A-treated cultures. (Incidentally, this experi- ment also shows that the cells which are converted to active CTL by rIL2 have not been eliminated by the anti-Lyt-1.1 treatment).

3.3 rIL 2 induces cytolytic activity in undepleted murine SC cultures and undepleted human PBL cultures in the absence of a mitogen

The finding that Con A was unnecessary as a mitogenic trigger for the induction of cytolytic activity by rIL2 in Lyt-1.1'- or Ia+-depleted (not shown), or even in total, undepleted, spleen culture (Fig. 3a), led to our presumption that rIL2 induced the differentiation of cytolytic cells different from antigen or mitogen-induced differentiation of CTL. It has been observed in the mouse system by Yron et al. [8] and in the human system by Lotze et al. [9] that lymphoid cells, from either normal or tumor-bearing individuals and expanded in IL2- containing preparations, are capable of lysing a wide variety of fresh malignant cells. Furthermore, Grimm et al. [l-31 demon- strated the generation of cytotoxic cells, termed lymphokine- activated killer cells, in fresh normal human lymphocyte popu- lations that are cytotoxic to tumor cells and only required purified IL 2 preparations for their activation. Our rIL 2 prep- arations were likewise very efficient in inducing cytolytic activ- ity in murine (C3H/HeJ, CBA/N, BALB/c) SC cultures (mea- sured against "Cr-labeled P815 cells) (Fig. 3a) and in fresh, human, nylon wool nonadherent PBL or tonsillar cell cultures

10

ms Effector target

Figure 3. rIL2 induces cytolytic activity in a total mouse SC culture and in a human PBL culture in the absence of a mitogenic stimulus. (a) lo5 SC from a BALB/c mouse were cultured in microtest plates in 200 p1 medium alone or in the presence of 5 pg/ml Con A , or 5 pg/ml LPS (Gibco) or 500 unitdm1 rIL 2. After 42 h at 37 "C, the proliferative response and cytolytic activity towards P815 cells (in the presence of PHA) were measured. (b) lo5 human, plastic nonadherent and nylon wool-passed PBL were seeded into microtest plates in 200 ~1 medium alone (control) or in the presence of 5 pglml PHA or 500 unitsiml rIL2 or 2000 unitdm1 IFN-y [13], or 500 units/ml rIL2 plus 2000 unitdm1 IFN-y. After 5 days at 37"C, the proliferative response and cytolytic activity against 5'Cr-labeled Daudi cells (in the absence of PHA) were measured.

10 50 100 200 300 LOO 500 m uni ts lml Inter leukin 2

Figure 4. Induction of cytolytic activity in a mouse SC culture in func- tion of the concentration of human rIL2. SC (lo5) from a C3H/HeJ mouse were seeded into microplates in 200 pl medium alone or in the presence of 1, 5 , 10, 50, 100 and 500 unitslml rIL2 (100 units rIL2 contain 10 ng of protein and correspond to the IL2 activity found in our standard PHA-induced human splenocyte supernatant). After 48 h at 37"C, the proliferation and cytolytic activity at an effector to target ratio of 50 : 1 (0) and 10 : 1 (A) towards "Cr-labeled P815 cells (without PHA) were measured.

(measured against 51Cr-labeled Daudi cells, Fig. 3b). Removal of adherent cells on nylon wool and a Sephadex G-10 column did not reduce the cytolytic activity (not shown) indicating that these cells are not essential in the generation of cytolytic cells by rIL2 in the absence of antigen. As shown in Fig. 4, a con- centration of rIL2, corresponding to only 5% of a PHA-stimu- lated human splenocyte supernatant, is already able to gener- ate these nonspecific killer cells.

3.4 rIFN-y does not induce cytolytic activity in human PBL cultures

In a recent report, Brooks 1101 implicated high concentrations of IL2 present in induced SC supernatant as responsible for the induction of an NK cell-like activity in a cloned murine CTL line. The NK cell activity can be potentiated by IL2 and IFN [ll, 121 Indeed, the presence of both purified IL2 and IFN enhanced the induction of the CTL-derived NK cell activ- ity [lo]. Hence, we tested whether the activation of cytotoxic activity by rIL2 in fresh human PBL was replacable or enhanced by the presence of purified recombinant glycosy- lated human IFN-y (produced in Chinese hamster ovary cells [13], see also footnote Sect. 2.4). Fig. 3b shows that pure human IFNy neither induced nor enhanced the induction of cytolytic activity caused by rIL 2. We conclude, therefore, that the rIL 2-induced cytotoxic cells are neither activated nor gen- erated by IFN-y, and therefore differ also in this respect from NK cells. It may be recalled that Grimm et al. [3] also reported that lymphokine-activated killer cells are not identical to NK cells. Furthermore, it has been shown that IL2 enhances NK cell activity through the induction of IFNy [14]. rIL 2-induced activation of killer cells, however, was not abrogated by rabbit anti-human IFN-y antiserum* (data not shown).

* Rabbit antiserum prepared against bacterially expressed recombi- nant human IFN-y was a gift of Dr. Liang, Biogen, S.A., Geneva.

1060 R. Devos, G. Plaetinck and W. Fiers Eur. J . Immunol. 1984.14: 1057-1060

3.5 Proliferative response induced by rIL 2 and rIFN-y

It is generally believed that fresh, nonactivated T lymphocytes do not respond to the growth-promoting activity of IL2 until IL 2-receptors are expressed on these cells following antigen or mitogen stimulation. Compared to Con A or PHA treatment, however, a weak but still significant proliferative response was obtained with high concentrations of rIL 2 using nonactivated mouse SC cultures or human PBL cultures (Fig. 3a and b). LPS induced a strong proliferation in these cultures but gener- ated no cytotoxicity (Fig. 3a); this observation can also be considered as a control to show that our rIL2 preparations do contain very little or no E. coli-derived LPS. Johnson and Farrar [15] recently presented experiments indicating that par- tially purified human IFN-y induced the expression of IL 2 receptors on human peripheral T cells in a similar manner to that of Con A. However, after a 5-day incubation of human nylon wool-passed PBL in the presence of high concentrations of both rIL2 and IFNy (2000 U/ml), we did not observe an enhanced proliferation of these cells compared to the response obtained with rIL2 alone (Fig. 3b).

Some T cells may be exposed to a form of foreign antigen during isolation and culturing and may therefore begin expres- sing IL2 receptors. This would explain the small but distinct proliferation response obtained with rIL2. It is possible but not proven that these proliferating cells are identical to the cytotoxic cells. Also, Grimm et al. [16] demonstrated the de novo appearance of the Tac antigen, corresponding to the IL2 receptor [17], during the development of IL 2-induced cytotoxic activity.

4 Concluding remarks

We have shown that pure rIL2, even at low concentrations in the absence of any other extrinsic factor, very efficiently induces the generation of cytolytic cells both in mouse SC and in human PBL cultures. Possibly, this phenomenon is related to the reactivation of memory CTL by IL2. Indeed, Lefran- cois et al. [18] showed that IL2 is capable of inducing re- expression of specific cytolytic activity from inactive, IL2 receptor-positive, memory CTL. Similarly, we propose that rIL2 can trigger memory CTL, leading to a nonspecific secon- dary CTL activation. Recently, Teh and Yu [19] described the generation of nonspecific CTL by IL 2 even in the presence of specific antigen stimulation. The generation of such non- specific cytotoxic cells by exogenous IL 2 should be carefully

considered in studies aimed at dissecting the differentiative signals required for an antigen-triggered CTL response and for analyzing the fine specificities and frequencies of antigen- specific CTL (as suggested by Teh and Yu; [19]). The high cytolytic activity towards tumor cells obtained with rIL 2 alone should be further explored with regard to its potential role in cancer immunotherapy.

Geert Plaetinck thanks the lWONL for a fellowship. We are grateful to Chris Opsomer and Wilma Burm for expert technical assistance and Fred Shapiro and Wim Drijvers for their editorial and artistic help, respectively.

Received May 10, 1984; in revised form July 12, 1984.

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