Radiation leukemia virus-transformed immunocompetent T cells

CELLULAR

IMMUNOLOGY

102,89-98

(1986)

Radiation Leukemia Virus-Transformed II. Antigen-Induced

lmmunocompetent

T Cells

Macrophage Migration Inhibition Factor and Leukocyte Migration Inhibition Factor Production

R. SZIGETI,* K. KAGAN-HAION,~

E. KLEIN,* AND S. Z. BEN-SASSON?

*Department of Tumor Biology, Karolinska Institute, Stockholm, Sweden, and TLautenberg Centerfor General and Tumor Immunology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel Received August 14, 1985; accepted May I I, 1986 OVA-specific T cells were immortalized by infection with radiation leukemia virus (RadLV). Some clones derived from such population were shown to exhibit helper activity. We then tested clones without such function and found among them some that secreted macrophage migration inhibition factor (MIF) and leukocyte migration inhibition factor (LIF) upon exposure to the antigen in vitro. The lymphokine-producing clones, which were Thy- 1+, Ly- I+ and Ly-2-, did not secrete MIF and LIF constitutively. Like other antigen-specific T cells, the immortalized clones could not be stimulated by free soluble antigen but required macrophages for presentation and for triggering the lymphokine production. The antigen-activated clones exclusively produced MIF and LIF, but not interleukin 2 or colony-stimulating factor. They neither provided helper activity nor induced delayed-type hypersensitivity. The data suggestthat the T-cell clones carry the antigen receptors and that their antigen-inducible biological function is restricted to the migration inhibitory factor production. o 1986 Academic PBS, IX. INTRODUCTION

Recent technical developments in T-cell cloning and somatic cell hybridization considerably enlarged our knowledge of T-cell physiology and allowed their precise functional and molecular analysis (l-4). However, the hybridoma lines are frequently unstable and contain the genome of the tumor cell partner which may eclipse or alter the function and/or product(s) of the immunologically specific T-cell partner. The long term T-cell culture lines are generally dependent on periodic stimulation with the antigen and/or growth factor and may also change their characteristics (5, 6). A third approach for establishment of antigen-specific T-cell lines exploited the transforming capacity of murine radiation leukemia virus (RadLV)’ (7,8). As malignant transformation may arrest the cell at a certain stageof differentiation (9- 1l), it was expected that when T cells with immunological specificity were infected ’ Abbreviations used: BSS, balanced salt solution; CFA, complete Fruend’s adjuvant; CSF, colony-stimulating factor; DTH, delayed-type hypersensitivity; FCS, fetal calf serum; HBSS, Hank’s balanced salt solution; HS, horse serum; IL-2, interleukin 2, LIF, leukocyte migration inhibitory factor; MIF, macrophage migration inhibitory factor; OVA, ovalbumine; PEC, peritoneal exudate cells; PFC, plaque-forming cells; RadLV, radiation leukemia virus. 89 OOO8-8749/86$3.00 Copy&tit 0 1986 by Academic Pms, Inc. All right.5of reproduction in any form reserved.

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and transformed by RadLV, some will be “fixed” at the prestimulation phase. It was indeed found that immortalized clones responding to activation by antigen were present in such populations. The additional genetic material in the RadLV-transformed cell is relatively small (12). The molecular changes in the cell surface, products, and functions of such cells were therefore less extensive than those imposed by the partner in the hybridomas. The experiments showed that RadLV-transformed cells and their products were functionally corresponding to their normal counterparts. Thus, such tumor lines provide an adequate tool for in vivo and in vitro studies of antigen-specific T cells. So far, helper ( 13) and suppressor (14) T clones were obtained with this method. We posed the question as to whether clones that did not function as helper also exhibit antigen recognition and therefore looked for other functions that can be induced by antigen exposure such as lymphokine production-LIF, MIF, CFS, IL-2, and also DTH. MATERIALS AND METHODS RadL V-transformed T clones. Generation of RadLV-induced lymphomas and their subsequent cloning was described previously (8, 13). Enriched populations of ovalbumine (OVA)-primed T lymphocytes from C57BL mice were obtained as described elsewhere (15). The cells were infected in vitro with RadLV and inoculated intrathymically into irradiated (400 R) C57BL mice. Two to four months later, 5090% of the mice developed thymic lymphomas. The cells were explanted and maintained in tissue culture medium (RPM1 1640) containing 5% heat-inactivated fetal calf serum (Biolab, Jerusalem, Israel), 1 mMsodium pyruvate, 2 Mglutamine, l0 mM Hepes buffer, 5 X low5 A4 2-mercaptoethanol, 100 fig/ml streptomycin, 100 U/ ml penicillin, and 4 pg/ml gentamycin. The culture lines were cloned by limiting dilution (0.1 cell/microtiter well). Antigen-presenting cells. Peritoneal exudate cells (PEC) were harvested 14 days after the intraperitoneal injection of complete Freund’s adjuvant (CFA; 0.2 ml). The PEC (2 X lo6 cells) were incubated on 3-cm tissue culture dishes (Nunc, Copenhagen, Denmark) for 2 hr in 1 ml of culture medium with or without antigen (500 pg/ml OVA) at 37°C in a 5% CO;! humidified atmosphere. At the end of the incubation period, free antigen and nonadherent cells were removed and the plates were washed 3X with Hank’s balanced salt solution (HBSS). The antigen-pulsed adherent macrophages were used as antigen presenting cells. Activation of RadLV-transformed T clones by antigen-pulsed macrophages. Aliquots of lymphocytes were incubated on control or antigen-pulsed macrophages in 3 ml of tissue culture medium at a concentration of lo6 cells/ml. Antigen (100 pg/ml OVA) was added to the pulsed macrophage plates. After 24 hr at 37°C in a humidified 5% COz atmosphere, the medium was collected and centrifuged in order to remove free-floating cells and cellular debris. The samples were kept in -20°C until assayedfor lymphokine activity. Cell surface staining. The various clones were examined for surface expression of Thy- 1,2; Ly- 1,2, and Ly-2,2 by direct immunofluorescence. The cells ( 1 X 106)were incubated for 30 min at 4°C in 0.1 ml of HBSS + 5% FCS containing monoclonal anti-Thy- 1,2, anti-Ly- 1,2, or anti-Ly-2,2 (Cedar Line, Homby, Canada). The washed cells were further incubated for 30 min at 4°C with fluorescein isothioscyanate (FITC)-conjugated affinity-purified sheepanti-mouse IgG antibodies (Sigma Chemi-

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CLONES OF MIF- AND LIF-PRODUCING

T CELLS

91

cal Co., St. Louis, MO.) followed by appropriate washing. Control samples were incubated initially with HBSS + 5% FCS and then treated with the FITC conjugate. The cells were mounted in PBS and glycerol 1:1 and the frequency of stained cells was determined under the UV microscope. LZF assay. The indirect leukocyte migration inhibition technique has been performed as described elsewhere (16): granulocytes were separated from heparinized human blood. The dextran-sedimented cells were separated into subsets by FicollIsopaque gradient centrifugation. Erythrocytes and granulocytes appear in the pellet. Erythrocytes were removed by distilled water shock. The granulocytes were used as migrating cells. An agarose medium, containing 1% agarose, 10% inactivated horse serum, and antibiotics were transferred to plastic Petri dishes (48 X 8.5 mm, 6 ml medium each). After the gel had solidified, the dishes were incubated at 37°C in a humidified COZ atmosphere. Seven to nine wells of 2.3-mm diameter were punched in the dish. Granulocytes (15 X 106)were mixed with 90 ~1of test or control supernatants and aliquots in 9 ~1were placed in the wells. After 24 hr incubation and fixation with 5% glutaraldehyde, the agaroselayer was removed and the migration areaswere measured. MZF assay.PEC were induced in mice by injecting 0.75 ml of a 2.4% thioglycolate solution intraperitoneally 3 days before collection. The mice were killed and their peritoneal cavity was washed with cold, Ca-Mg-free BSS. The cells were washed three times with F13 medium containing 10% inactivated horse serum (17). Cells (20 X 106)were mixed with 135 ~1 nutrient agarose medium, containing equal volumes of 0.4% agaroseand two-fold concentrated RMPI 1640 medium supplemented with 20% inactivated horse serum and antibiotics. Droplets (2 ~1) of the cell-agarose suspension were placed into flat-bottom 96-well microplates. After the droplets had been solidified, test or control supematants were added to triplicate wells in a 150-~1 volume. The microplates were covered and incubated for 18-24 hr as above. Evaluation of the migration inhibition assays.Two diameters were measured (inner, droplet diameter; outer, cell migration circle diameter). The area ofthe migration was calculated according to Weeseet al. ( 18). Mean migration area of the test (Mt) and control (MC) triplicates was used to determine the percentage migration inhibition according to the formula

In this study, MI > 19 or 2 1% for LIF and MIF, respectively, was considered as positive migration inhibition. The horizontal dotted line in the graphs represents the borderline between negative and positive. Statistical analysis. Migration inhibition percentages obtained with antigen-free (control) supernatants were used as the statistical borderline between negative-positive responses.The mean and +2 SD (95% confidence limit) determined the negative range. Since these were 8.5 f 12.4% and 3.5 f 15.4% for MIF and LIF, respectively, the cut-off value for MIF was considered as 2 1% MI, whereas the same was 19% for LIF. For group statistics Wilcoxon’s nonparametrical test was used. IL-2 assay. Supematants from the T clones were tested for their ability to support the growth of an IL-Zdependent T-cell line (CTL-D) (19). Briefly, triplicate wells containing 1O5CTL-D cells were dispensed in 100-111 aliquots and incubated with an

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equal volume of various dilutions of the test samples for 48 hr. The cultures were pulsed with 1 &i [3H]thymidine during the final 6 hr of incubation. The cells were harvested with an automatic cell harvester and the IL-2 activity in the different samples was expressed as average cpm + SEM of the triplicate wells. Purified murine IL-2 was used as a standard. CSF assay. The presence of colony-stimulating factors (CSF) was determined by the bone marrow colony assayed in semisolid agar (20). Briefly, lo5 femoral bone marrow cells from 8- to 12-week-old C57BL mice were suspended in 1.Oml of warm (40°C) RPM1 medium containing 25% horse serum (HS), antibiotics, and 1.8%agar. The mixture was added to a 50-mm petri dish plate containing 1.2 ml of RPM1 1640 supplemented with 25% HS, antibiotics, 3.3% agar, and 40% (v/v) of the various dilutions of the test supematants. Conditioned media of Wehi/3B cell line were included in some of the bottom agar layers as CSF standard. The cultures were incubated at 37°C in a humidified 5% CO2 atmosphere for 6 to 7 days. The dishes were inspected with a dissecting microscope at 40X magnification, and proliferative centers (> 150 cells) were scored. DTH activity. The cell populations (5 X 106/ml) were incubated at 37°C for 3 hr together with PEC (105/ml) from CFA-boosted (0.2 ml/mouse, ip) mice as source of antigen-presenting cells, and 100 pg/ml of OVA in culture medium. Controls were incubated without antigen. Subsequently, the cells were centrifuged and resuspended in HBSS. Irradiated (1000 R) control or experimental cells ( 107)were injected in a volume of 50 ~1into the hind footpads of 4-6 mice. Footpad swelling was determined 24 hr later with an engineer’s micrometer (Mitutoyo, Japan). Assessmentof helper activity. Irradiated (3000 R) cloned cells (10’) were injected intraperitoneally together with DNP-OVA (100 pg) into mice preimmunized with DNP-KLH 45-60 days earlier. Control mice were injected with antigen alone. DNPspecific PFC in the spleen were determined 7 days following the inoculation according to Jeme et al. (2 1). Sheep red blood cells coupled with 2,4,6-trinitrobenzesulfonic acid (TNP-SRBC) served as indicator cells and normal guinea pig serum was used as a source of complement both for direct and indirect PFC. Purified rabbit antimouse Ig was employed as the developing antibody for the indirect plaques. RESULTS

Lymphokine Production by T-Cell Clones Five of seven clones derived from one tumor line responded with MIF production when exposed to OVA-pulsed macrophages. From another line, three clones were tested and two responded with lymphokine secretion (Fig. 1). This was not constitutive but induced by the antigen, as none of the supematants collected from the control cultures exhibited significant migration inhibition. The supematants of the antigenpresenting macrophages, and of the control RadLV-transformed T line ( 136.5), derived from unprimed T-cell population exposed to OVA, were also negative (Fig. 1). Therefore, the lymphokine production was an antigen-induced function. In the next experiment the supematants were tested both for MIF and LIF production and additional clones, derived from a third tumor line, were included in the assay.Ten of the clones produced both MIF and LIF while the supematants of two were negative in both assays.Thus the results were concordant (Fig. 2).

IMMORTALIZED

CLONES OF MIF- AND LIEPRODUCING

,!$136.5

5-3 5-4 5-5

5-6 5-6 CLONE

5-9 5-10 7-l

7-4

T CELLS

93

7-10

FIG. 1. Antigen-induced MIF production by immortalized T-cell clones. Cloned cells ( 106/ml) from two different lymphomas (designated 5 and 7) were incubated on either OVA (100 &ml) exposed or control macrophage monolayers. As control, C57BL thymoma cells (136.5) induced by intrathymical inoculation of RadLV were used. The supematants of the various cultures, including control macrophage monolayers without T clones, were collected after 24 hr and assayed for the presence of MIF. The data are presented as means k SD of the triplicate cultures. *Significant (P > 0.05 by nonparametrical Wilcoxon’s test) MIF production. The horizontal dotted line represents the borderline between negative and positive values.

The antigen had to be presented to the T cells by macrophages as free soluble antigen did not induce lymphokine production (Fig. 3). Subsequent experiments verified that antigen-pulsed macrophages alone provide the stimulatory signal to the MIF/ LIF-producing clones (data not shown).

OVA’-+-+-+-+ ayo$e I-I

l-2

1-4

l-5

CLONE

l-6

l-8

l-9

a$t$-2

5-4 5-6 CLONE

FIG. 2. Antigen-specific MIF and LIF production by immortalized clones of RadLV-transformed T cells. Cells (106/ml) of several clones from two tumors (a, 1; b, 5) were incubated for 24 hr on either OVA (100 &ml) containing macrophage monolayer or control unpulsed macrophage monolayers. At the end of the incubation period the supematants of the cultures, including those of macrophage monolayers without added T cells, were harvested and assayed for the presence of MIF and LIF. The data are presented as means + SD of the triplicate cultures. *Significant (P > 0.05 by nonparametrical Wilcoxon’s test) MIF or LIF production. The horizontal dotted line representsthe borderline between negative and positive values.

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SZIGETI ET AL.

O”i-’

-*

M#-

-t

-. -

+ l-2

+ 5-2

CLONE

FIG. 3. Free soluble antigen does not activate specific T clones to secrete lymphokine (LIF). Cells ( 106/ ml) of antigen-specific LIF producing T clones (l-2 or 5-2) were incubated with or without the stimulating antigen (100 rgjrnl OVA) in 3-cm tissue culture plates or in plates containing macrophage monolayer. After 24 hr the supematants of the various cultures were collected and assayedfor LIF activity. The data are presented as means + SD of the triplicate cultures. *Significant (P > 0.05 by nonparametrical Wilcoxon’s test) LIF production.

When MIF/LIF-producing clones (l-2, 1-4, 1-8, 5-2, 5-8, and 7-4) from three different lymphomas (1,5, and 7) were examined for their cell surface phenotype, it was found that more than 90% of the cells from each of these clones were positive for Thy- 1 and Ly- 1. Nevertheless all of the clones were negative for Ly-2.

The Kinetics of Lymphokine Production Detectable levels of lymphokines were present in the supernatants of the clones already 2 hr after antigen presentation (Fig. 4). The migration inhibitory capacity of the supematant increased with time, up to 24 hr after the stimulation.

The MIF/LIF-Producing Clonesare Restricted in Their Inducible Immune Function The supematants of the OVA-triggered clones were examined for other activities. Table 1 gives an example from these experiments: MIF/LIF-secreting clones were tested for IL-2 production. None of the supematants supported the growth of the IL 2-dependent T cells. Other MIF/LIF-secreting clones also gave negative results. The helper clone (41), which did not secrete MIF or LIF, did not produce IL-2 either. The supematants of the antigen-exposed helper and MIF/LIF-producing T cells were examined also for CSF production. These results were negative (data not shown). The immune activity of the clones was assayedin vivo as well. Table 2 shows the result of one of the screening experiments. Initially, the clones were selected on the basis of the lack of helper activity. These results were confirmed. The MIF/LIF-producing clones, as well as the helper clone, were also examined for ability to mediate DTH response in vivo. The antigen-activated clones did not induce DTH reaction (data not shown).

IMMORTALIZED

CLONES OF MIF- AND LIF-PRODUCING

T CELLS

95

ii v 4; TIME (Hr)

b

24 TIME (Hr)

FIG. 4. Kinetics of antigen-specific LIF production by immortalized clones of RadLV-transformed T cells. Cells ( 106/ml) from clone 1-2(a) or clone 5-2 (b) were incubated on antigen-pulsed (0) or unpulsed macrophage monolayer (0). The supematants of the control and the antigen-stimulated cultures were collected at various times after the initiation of the culture and assayedfor LIF activity.

DISCUSSION The results demonstrate the existence of clones present in RadLV-transformed antigen-specific T-cell populations that answer with lymphokine production upon exposure to the antigen. These clones, which have the cell surface phenotype of helper T cells (Thy-l+, Ly- I+, Ly-2-), do not have helper activity while the helper clone does not secreteMIF/LIF. The dichotomy between the migration inhibition factor production and the helper function is in agreement with the functional variety of T-cell subsetswith specialized functions. The production of MIF is considered to be the in vitro correlate of the DTH assay (22). Lack of DTH elicitation by the MIF-producing clones substantiates the view that DTH is a multistep process involving several cell types and products (23, 24). Although MIF is one of the components participating in the DTH reaction (22), other cells and/or factors may also be needed.

96

SZIGETI ET AL. TABLE 1 MIF-Producing T Clones Do Not Secrete IL-2 [‘H]Thymidine incorporatio$

Examined clone”

Activation by antigen

Medium control IGZstandard 5-8

+ + + + +

5-10 7-l 7-4 41

Supernatant dilution:

1:4

123

I:16

1:32

293 +61 160,561+ 2234 263 f 72 573 + 108 213k27 268 + 79 1,041 t 125 758 k 79 168k8 319*74 210+39 14423

321 + II 190,513f 21935 185+81 505k 115 162 f 7 137 + 14 870 f 26 625 f 80 195 + 16 309 + 28 184 k 26 200*21

408 * 37 82,188 + 5275 306k71 260 + 37 252 f 50 126 f 17 1,001 -+ 320 1,053 + 65 142+22 406*40 221+6 272f51

308 f 20 19,459 + 1729 310+83 268 f 66 130+ 10 135+5 4872 17 1,081 f 210 150+45 400+6 324 f 66 19Ok40

o Cells (106/ml) of the various MIF/LIF-producing clones as well as one hleper clone (4 1) were incubated on OVA ( 100 &ml) exposed (+) or control (-) macrophage monlayer. The supematants, including control macrophage monlayer without T clones, were collected after 24 hr and assayedfor the presenceof 1L2. ’ The data were expressedas cpm k SEM of the triplicate wells.

The absence of IL2 and CSF in the MIF- and LIF-containing supernatants of the antigen-stimulated clones reveals a functional restriction. One clone (5-2) was also tested for interferon production; the result was negative. It is not known whether each T cell has a defined precommited repertoire, limited to the production of one lymphokine or a restricted group of lymphokines. There are many examples of cloned T cells and T-cell hybridomas that produce more than one lymphokine upon stimulation with the specific antigen or mitogen (25-29). However, a restricted array of secretedlymphokines from activated T clones and hybridomas was also reported (22,25,30-32). Although it is possible that some

TABLE 2 MIF/LIF-Producing Clones Do Not Induce Helper Activity Injection” (clone)

5-2 5-4 5-5 5-8 41

IgG anti-DNP response (PFC/106 spleen cell~)~ 3+1 4fl 6+2 221 3+2 24+3

’ Cells of the various MIF/LIF-producing clones including positive control of helper clone (4 1) were irradiated (3000 R) and inoculated into unprimed C57BL mice (10’ cells/mouse) together with DNPOVA ( 100 &mouse); control mice were inoculated with the antigen alone. The number of anti-DNP PFC in the spleenswas determined 7 days later. ’ Mean PFC + SEM of the four mice used in each experimental point.

IMMORTALIZED

CLONES OF MIF- AND LIF-PRODUCING

T CELLS

97

of the apparent heterogenous lymphokine secretion is due to multiple biological activities of one molecule (33, 34), many T clones secrete several lymphokines proven to be different molecules (25-27, 29). The apparent linked secretion of some and segregation of other lymphokines (28) suggeststhat stimulation of a T clone may activate clusters of genesthat control the production of these different lymphokines. Among the clones analyzed, no IL-2- or CFS-producing ones were found. It remains to be seenwhether this was determined by the strategy of the experiment in that the selection of a particular functional T cell was favored or the viral transformation selectively acted on a particular subset. Since macrophage-leukocyte and PMN-leukocyte migration inhibitory activities are due to two different molecules, MIF and LIF, respectively, in the humans (35), we considered it worthwhile to test the supernatants for both activities. There are no available data on murine-LIF, probably due to the technical difficulties. Namely, the preparation of murine PMN-leukocytes is difficult and tedious. Therefore, we used human PMN-leukocytes as indicator (migrating) cells. Our expectation to have migration inhibition was based on the well-known species nonspecificity of most lymphokines. It was demonstrated that LIF could cross speciesbarriers, since, e.g., guinea pig PMN leukocytes were susceptible to human LIF action (36). Indeed, we found that supernatants of activated murine cells did reveal LIF-like activity, measured with human PMN granulocytes. Whether the same or two different lymphokines are responsible for the LIF and MIF activity has not been studied in this work. Although we do not know the functional potential of the LIF-producing T cells, they will allow the characterization of the early events in the activation process. LIF/ MIF production is one of the earliest inducible events, independent of and preceding cell proliferation (37). Presently it is not known whether MIF and LIF are coded by a separate cluster of genesthat are activated independently of other lymphokines or if the production of these inhibitory factors can be induced in cells secreting other lymphokines as well. ACKNOWLEDGMENTS The authors thank Ms. Suzanne Pomeranz and Ms. Ruth Stoll for their excellent secretarial work. Robert Szigeti is a recipient of a Research Fellowship from the Swedish Cancer Society. This investigation was supported by Public Health Service Grant SROl CA25250-5 awarded by the National Cancer Institute, Department of Health and Human Services, and the Swedish Cancer Society; and by the Herman and Sophia Taubmann Foundation and the Concern Foundation of Los Angeles.

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30. Palacios, R., J. Immunol. 132, 1833,1984. 31. Evequoz, V., Bettens, F., Kristensen, F., Trechsel, U., Stadler, B. M., Dayer, J. M., De Week, A. L., and Fleisch, H., Eur. J. Immunol. 14,490, 1984. 32. Butler, J. L., Falkoff, R. J. M., and Fauci, A. S., Proc. Natl. Acad. Sci. USA 81,2475, 1984. 33. Pace, J. L., Russell, S. W., Torres, B. A., Johnson, H. M., and Gray, P. W., J. Immunol. 130,2011, 1983. 34. Schreiber, R. D., Pace, J. L., Russell, S. W., Altman, A., and Katz, D. H., J. Zmmunol. 131,826, 1983. 35. Rocklin,R.E., J. Immunol. 112,1461, 1974. 36. Hoffman, P. M., Spitler, L. E., Hsu, M., and Fundenberg, H. H., Ceil. Zmmunol. l&21, 1975. 37. Gorsky, S. J., Dupont, B., Hansen, J. A., and Good, R. A., Proc. Natl. Acad. Sci. USA 72,3197, 1975.