Immune interferon (IFN-γ) production in autologous mixed cultures

CELLULAR

IMMUNOLOGY

76, 196-199 (1983)

Immune interferon (IFN-y) Production Autologous Mixed Cultures SHMUEL

ARGOV,*

KARI

CANTELL,?

EVA KLEIN,*

in

AND GEORGE

KLEIN*

*Department of Tumor Biology, Karolinska Institute, S-104 01 Stockholm, Sweden, and fCentra1 Public Health Laboratory, Mannerheimintie 166, SF-00280 Helsinki, Finland Received September 29, 1982; accepted December 11. 1982 T cells were exposed in vitro to autologous B cells or monocytes. Tested on the seventh day, the cultured lymphocytes lysed K562, Daudi, autologous, and allogeneic phytohemaggiutinin (PHA) blasts. Autologous B blasts were not affected. The supematants contained gamma interferon (IFN-y). The quantity of IPN did not correlate with the strength of the proliferative response nor with the strength of the cytotoxic potential.

INTRODUCTION Immune interferon (type II, IFN-7) is produced by lymphocyte populations when they respond to mitogens (1) and antigens (2). In accordance, the supernatant of ahogeneic mixed lymphocyte cultures (MLC) contain IFN-y (3). T cells are activated in vitro when exposed to autologous B cells or monocytes (AMC) (4). The AMC system was shown to have the characteristics of an immune response with regard to specificity and memory (5). Blastogenesis in AMC is much weaker than in the MLC (4). In both systems, cytotoxic potential is generated, but stimulator-specific lysis appears only in the MLC (4). It has been shown, with the help of monoclonal reagents, that the T subsets which respond to autologous non-T cells and allogeneic lymphocytes do not overlap completely and the responding population in AMC is considerably smaller (6). The autoreactive T cells are predominantly OKT 4 positive, while alloreactive cells are in the OKT 4-, OKT 5, and OKT g-positive subsets (6). It was reported that IFN-7 is not produced in mixed cultures of cells derived from HLA identical siblings (7). In view of this claim and the effect of IFN on events which occur in mixed culture, i.e., generation of cytotoxicity and cell proliferation and its assumed regulatory role, we have assayed whether it is produced with autologous reactants. MATERIALS

Medium. In the cultures and heat-inactivated human serum cillin, 120 &ml) was used. Cell separation. Mononuclear donors by the Ficoll-Isopaque transferred into 250-ml Falcon

AND

cytotoxic assays, RPMI- 1640 supplemented with 10% and antibiotics (streptomycin, 100 /Ig/ml and penicells were obtained from heparinized blood of healthy method. The mononuclear cells, twice washed, were plastic flasks (10’ cells/ 15 ml medium) and kept in 196

0008-8749183 $3.00 Copyright 0 1983 by Academic Press Inc. All rigbls of reproduction in any form reserved

METHODS

SHORT

COMMUNICATIONS

197

5% CO2 at 37°C for 1 hr. Nonadherent cells were collected after careful shaking. The adherent cells were recovered by vigorous shaking of the flask after addition of phosphate-buffered saline (PBS, 2 mM) and EDTA. This population consisted mainly of monocytes and was used as a stimulator in the mixed autologous cultures. The nonadherent cells were placed on a nylon wool column (10’ cells/O.85 g nylon wool), and kept in 5% CO2 at 37°C for 1 hr. The passed cells were used as responders in AMC. This population consisted mainly of T cells, a few B cells, and monocytes (less than 3%). Mixed cultures, AMC, and MLC. Cells of the T responder populations (15 X 106) were mixed with 7.5-15 X lo6 mitomycin C-treated (25 &ml, at 37°C for 30 min) or X-ray-irradiated (4000 rad) stimulator cells, in 15 ml medium. To the AMC, either plastic-adherent or nylon-adherent cells were added; to the MLC, allogeneic lymphocytes were added. The cultures were maintained in a humidified 5% CO2 atmosphere at 37°C for 6-7 days. Cytotoxic assay. The lytic effect of the AMC and MLC effecters was tested in a “Cr-release assay as described previously (4). Daudi- (8) and PHA-induced blasts served as targets. Assessment of level of DNA synthesis. This was performed as described previously (4). Assay of interferon. This was done by the plaque reduction of vesicular stomatitis virus (VSV) in U (= HeLa) cells. All titers are expressed as units (u/ml) with the 69/19 preparation of IPN-cu as reference. Characterization of interferon-pH 2 sensitivity. The interferon samples were dialyzed against 100 vol of glycine-HCl buffer at pH 2 for 24 hr at 4°C and then diaiyzed against PBS back to neutrality. Antigenicity. Serum from a sheep immunized with human leukocyte interferon was used for neutralization of the interferon preparations (9). It contained 450,000 neutralizing units against human leukocyte interferon (IFN-(u) and 3000 neutralizing units against human fibroblast interferon (IPN-/3). The serum had no detectable activity against human gamma interferon (IFN-y). The serum was used at a final dilution of 1: 100 for neutralization of 10 units of the interferon preparations. Bovine cells. Some of the preparations which showed activity on human U cells were assayed by VW plaque reduction on NLB-1 bovine cells (10). RESULTS

AND

DISCUSSION

The separation procedure and the culture conditions did not activate the lymphocytes for interferon production except in rare cases (Table 1). The levels of IFN accumulated in the supernatant during the 6-7 days of cultivation in 15 AMCs varied considerably, between 3 and 200 u/ml. Variable amounts were detected in 16 MLCs as well, between 11 and 110 u/ml (Table 1). In six of nine experiments with the same responder population in AMC and MLC, higher amounts of interferon were produced in the latter. Previous reports with MLC- and mitogen-stimulated lymphocytes detected no correlation between the level of IPN produced and the proliferative and cytotoxic responses in the cultures (11). It has been shown that cell division is not obligatory for interferon production since it appeared when DNA synthesis was inhibited (12). The same is true for the

198

SHORT COMMUNICATIONS TABLE

1

Interferon Production, Blastogenesis, and Cytolytic Activity in Mixed Cultures” Autologous cultures: % speciIic 51Cr release

Interferon (u/ml) Expt’ 2 3 4 5 6 I 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Control <60 <50 <60 30 <3 20 11
MLC

60 35 20 11 35 11 110 110 20 20 20 11 110 60 110 60 <6

AMC 60 110 200 200 3 20 35 35

Reactivity index” 11 23 45 33 6 8

Daudi 11 40 56 46 5 26 12

Allo-PI-IA blast

11

26 45 95

6 10

22 12 4 5 23 9

11

23

38

43

11

38

35

18

47 47 15 6
2 5 48 4 42 26 95

68 72 19 4 4 18 84

9 16 5 61

’ [3H]Thymidine incorporation in the mixed cultures/[3H]thymidine incorporation in the control cultures X 100 = reactivity index. ‘In experiments 1-18 and 23-25, the nylon column-passed subset was the responder population; in 1922, the SRBC rosette cells sedimented in Ficoll were used.

activation for cytotoxic potential, which is not dependent on the enlargement of the specific clone, and affects certain cell lines such as Daudi (13). This seems to be the case in the AMC system also (Table 1). The cytotoxic potential was tested against Daudi and allogeneic PHA blasts. It has been proposed that in the AMC response to xenoantigens, provided by exposure of lymphocytes to fetal calf serum and SRBC, contributes to their activation (14, 15). This is excluded by our experiment. In order to see whether the methods of lymphocyte separation influence the functions of the responders, AMCs were established with cells separated by sedimentation as SRBC rosettes (ES) or by nylon column passage (NCp). These populations were confronted either with plastic-adherent cells (Mti), or with those which remain in the interphase after sedimentation of SRBC rosettes (E-). T cells separated with SRBC did not produce IFN when cultured alone (Table 1, experiments 19-22). In one experiment in which SRBCrosetted T cells or NCp cells were responders, similar amounts of IFN were produced (Table 2, experiment 5). The cells which remained in the interphase after SRBC

199

SHORT COMMUNICATIONS TABLE 2 Characterization of the Interferon Produced in AMC Expt

Responder

Stimulator

NCp’ NCP E+ E+ E+ E+

IFN u/ml”

Sensitivity topH2

II 11 <20 47 <20 47 15 15 22 60 6

NCP

NCP E+ E+ NCP

Neutralizing antibodies b

Activity on bovine cells

t + + +

Y Y

+ +

Y Y

+

’ The supematants of all control cultures were devoid of interferon. b Neutralization assay performed with antibodies directed against IFN-CX,IFN-/3. cNCp, Nylon column passed; E+, SRBC rosetted lymphocytes; E-, SRBC nonrosetted lymphocytes (non-T cells); M$, plastic adherent cells (macrophages and monocytes).

rosetting (E-) were more efficient stimulators than the macrophage-monocyte fraction (Table 2, experiments 3-5). The AMC supernatant contained IFN-7. This was determined by three properties (Table 2): (i) IFN-y is labile in pH 2.0; (ii) human INF-LY but not IIN-7 is active on bovine cells; and (iii) neutralization test with anti-RN serum. Since exposure to pH 2 left no antiviral activity in the supernatant (less than 6 u/ ml), other types of IFN were not produced in these cultures. REFERENCES 1. Stewart, W. E., II, “The Interferon System.” Springer-Verlag, New York, 1979. 2. Epstein, L. B., In “Biology of lymphokines” (S. Cohen, E. Pick, and J. J. Oppenheim, Eds.), p. 443. Academic Press, New York, 1979. 3. Perussia, B., Mangoni, L., Engers, H. D., and Trinchieri, Cl., J. Immunol. 125, 1589, 1980. 4. Argov, S., and Klein, E., &and. J. Immunol., in press. 5. Weksler, E. M., and Kozak, R., J. Exp. Med. 146, 1833, 1977. 6. Kozak, R. W., Moody, C. E., Staiano-Coico, L., and Weksler, M. E., J. Immunol. 128, 1723, 1982. 7. Andreotti, P. E., and Cresswell, P., Hum. Immunol. 3, 109, 1981. 8. Klein, E., Klein, G., Nadkami, J. S., Nadkami, J. J., Wigzell, H., and Clifford, P., Cancer Rex 28, 1300, 1968. 9. Mogensen, K. E., Pyhala, L., and Cantell, K., Acta Pathol. Microbial. Stand. 838, 443, 1975. 10. Wiranowska-Stewart, M., and Stewart, W. E., II, J. Interferon Res. 1, 233, 1981. 11. Klimpel, R. G., Day, K. D., and Lucas, D. D., Ceil. Immunol. 20, 187, 1975. 12. Gotleib, B. A., Fu, S. H., Yu, D. T. Y., Wang, C. Y., Halper, J. P., and Kunkel, H. G., J. Immunol. 123, 1497, 1979. 13. Wallen, W. C., Dean, J. H., and Lucas, D. O., Cell. Immunol. 6, 110, 1973. 14. Ortaldo, J. R., Bonnard, G. D., and Herberman, R. B., J. Immunol. 119, 1351, 1977. 15. Huber, C., Merkenschlager, M., Gattringer, C., Royston, I., Fink, U., and Braunsteiner, H., J. Exp. Med.

155, 1222,

1982.