48

P. Creemers

Eur. J. Immunol. 1977.7: 48-53

9 Rabbitts, T.H., Jarvis, J.M. and Milstein,C., Cell 1975. 6: 5. 10 Bishop, J.O., Rosbash, M. and Evans, D., J. Mol. Biol. 1974.85: 75. 11 Weber, H. and Khorana, H.C., J. Mol. Bwl. 1972. 72: 219. 12 Gillam, S., Waterman, K., Doel, M. and Smith, M., Nucleic Acid Res. 1974. 2: 1649. 13 Egan, B.Z. and Kelmers, A.D., Methods in Enzymology 1974. 29: 469.

17 Ryffel, G.U. and McCarthy, B.J., Biochemistry 1975. 14: 1379. 18 Rabbitts, T.H. and Milstein, C., Duns. Biochem. Soc. 1975. 3: 870. 19 Humphries, S., Windass, J. and Williamson, R., Cell 1976. 7: 267. 20 Bonner, T.I., Brenner, D.J., Neufeld, B.R. and Britten, R.J., J. Mol. Biol. 1973. 81: 123. 21 Ullman, J.S. and McCarthy, B.J., Biochim. Biophys. Acta 1973. 294: 416.

14 Rabbitts, T.H. and Milstein, C., Eur. J. Biochem 1975. 52: 125.

22 Storb, U., Hager, L., Putnam, D., Buck, L., Farin, F. and Clagett, J., Proc. Nat. Acad. Sci. US 1976. 73: 2461.

15 Milstein, C., Brownlee, C.C., Cheng, C.C., Hamlyn, P.H., Proudfoot, N.J. and Rabbitts, T.H., in 27. Colloquium-Mosbach 1976, SpringerVerlag, Berlin, Heidelberg 1977, p. 75.

24 Hammerling, U., Mack, C. and Pickel, H.C., Immunochemistry 1976.13: 525.

16 Bishop, J.O., Morton, J.C., Rosbash, M. and Richardson, M., Nature 1974.250: 199.

25 Hammerling, U., Pickel, H.C., Mack, C. and Masters, D., Immunochemistry 1976. 13: 533.

Paula Creerners* Radiobiological Institute TNO, R ijswij k

23 Boylston, A.W. and Mowbray, J.F., Immunology 1974. 2 7 855.

The role of leukocyte subpopulations in the indirect leukocyte adherence inhibition assay in the mammary tumor virus system A modification o f the leukocyte adherence inhibition test for the detection of cellular immunologic reactivity of mice t o the mammary tumor virus (MTV) has been described. It involves t h e transfer of t h e leukocyte adherence inhibition factor (LAIF) produced by spleen cells from immunized animals when cultured with antigen t o indicator cells, for which peritoneal exudate cells from normal mice are used. The method proves t o be sensitive a n d highly reproducible. By crude separation of leukocyte subpopulations it became established that for t h e production of LAIF the following sequence is needed: 1. incubation of adherent cells with MTV; 2. transfer of a soluble factor SFI produced by the adherent cells to T cells; 3. transfer of another soluble factor SF2 released by T cells t o the adherent cells.

1. Introduction The leukocyte adherence inhibition (LAI) assay for demonstrating immunologic cellular reactivity t o certain antigens has been developed by Halliday a n d Miller [ 11. They observed that the proportion of cells adhering to glass surface was reduced if antigen was added t o which the animals were sensitized. In this test system, the blocking and enhancing properties of serum could also be established. Later it has been reported that the LA1 assay may be regarded as a depend-

able in v i t r o test for determining immunologic reactivity t o tumor specific antigens in t h e case of melanoma [ 21 and breast cancer [3].

Holt [ 4 ] reported that the LA1 reaction can be inhibited by pretreatment of t h e cells with anti-@ serum and complement and t h a t t h e inhibition of adherence can be transferred t o normal cells by the supernatant of spleen cells incubated with t h e antigen. He therefore concluded that a soluble factor, the T cell-dependent leukocyte adherence inhibition factor (LAIF) is responsible for the inhibition of adherence. His results with the LA1 test did not parallel those of the leuko[I 15241 cyte migration inhibition assay; he therefore suggested that different mechanisms be involved in the two tests. We per* This study was performed under NIH Contract NO1 CP 43328 with formed the LA1 assay with peritoneal cells derived from mice the National Cancer Institute, Division of Cancer Cause and Prevention, Viral Oncology, Department of Health, Education and Welfare, Bethesda, bearing primary mammary tumor virus (MTVtinduced tumors Md., USA, to Dr.Peter Bentvelzen. as indicator cells [ 51 a n d found that the results parallel the MTV-specific proliferation of spleen and lymph node cells Correspondence: Paula Creemers, Radiobiological Institute TNO, which is stimulated by T cells [ 61. 151 Lange Kleiweg, Rijswijk, The Netherlands Abbreviations: LAI: Leukocyte adherence inhibition LAIF: Leukocyte adherence inhibition factor MTV: Mammary tumor virus R L V Rauscher leukemia virus SF,: Soluble factor produced by adherent cells upon contact with MTV SF2: Soluble factor produced by T cells upon contact with SFI PBS: Phosphate buffered saline SE: Standard error

In t h e indirect LA1 assay, which is described below, in vitroinduced reactivity t o MTV is transferred t o normal indicator cells. Evidence is presented that, for LAIF production, an interaction between T cells and adherent cells is necessary, but that direct cell t o cell contact is not required.

Mechanisms of the LA1 assay in the MTV system

Eur. J . Immunol. 1977. 7: 48-53

2. Materials and medrods

2.1. Animals DBA/2f mice which were immunized intraperitoneally with 1 pg of purified MTV precipitated on alum were used as positive control animals. Male DBA/Zf, BALB/c and C57BL mice of 10 weeks of age served as normals. The chimpanzees were 7-year-old males that have been in our colony (Primate Centre TNO, Rijswijk) for four years. 2.2. virus The standard strain of mouse mammary tumor virus (MTV-S) was isolated from BALB/cfC3H mammary tumors. Fifty grams of tumor tissue were homogenized in 500 ml phosphate buffered saline (PBS) in a Sorval omnimixer for 50 sec at 1 4 000 rpm. The homogenate was centrifuged for I5 min at 12 000 x g. To the supernatant 1 % 0.1 M ethylenediaminetetraacetate (EDTA) was added. This mixture was spun for 15 min at 12 000 x g. The supernatant was ultracentrifuged for 45 min at 75 000 x g (Beckman 35 N rotor; 35 000 rpm). The pellet was then resuspended in 90 ml 1.5 mM Tris-HCI buffer (pH 7.2) and spun for 15 min at 12 ooox g. The resulting supernatant was centrifuged on a discontinuous gradient of 4 ml 50 % sucrose (w/w), 12 m l 3 5 % sucrose (w/w) and 8 ml 20 % sucrose (w/w) in 1.5 mM Tris-HC1 for 180 min at 170 000 x g (Beckman SW 27.1 rotor, 27 000 rpm). Material collected from the interphase between the 50 % and 35 % sucrose layers was diluted with 1.5 mM TrisHCl buffer and centrifuged twice on a linear sucrose buffer (20-50 % w/w in 1.5 mM Tris-HC1) for 180 rnin at 17 000 x g in a SW 27.1 rotor. The bands at densities 1.16 t o 1.1 8 g/ml were collected and centrifuged for 40 min at 150 000 x g (Beckman 50 Ti rotor, 40 000 rpm). The pellets were resuspended in 0.025 M sucrose in 1.5 mM Tris-HC1 buffer. The purified virus was stored in liquid nitrogen. All isolation steps were carried out at 4 "C. Rauscher leukemia virus (RLV) was isolated from leukemic spleens of BALB/c mice in the same manner. Virus content was estimated on the basis of the amount of protein, as measured by the Folin method. Before use as antigen, the virus was disrupted by repeated freezing and thawing.

49

trol and test cells were alternating*. The plates were incubated in a humidified atmosphere with 5 % C02 for 2 h at 37 OC. Thereafter, the plates were washed three times in PBS and the adherent cells were fixed, stained and count'ed. 2.4. Indirect LA1 assay This test was performed under sterile conditions. Spleen cells were taken aseptically and teased apart by forceps in a petri dish containing medium. The debris was then allowed t o settle in a tube. The supernatant containing the cells was centrifuged twice at 26 x g for 10 min t o remove erythrocytes and afterwards centrifuged twice at 200 x g for 10 min. This was done to remove LAIF that could be attached t o the cells in v i v a After counting, the cells were resuspended t o a final concentration of 20 x 1O6 cells/ml in medium supplemented with 10 % fetal calf serum. The cultures ( 1 ml) were incubated in plastic tubes (Falcon, 12 x 75 mm) for 24 h with 0.1 j& antigen in a humidified atmosphere with 5 % COz. The cells were subsequently spun down and the supernatant was removed. Fresh peritoneal cells derived from normal animals (washed 4 times at 200 x g for 10 min) were then suspended in the supernatant t o a final concentration of approximately 20 x 1O3 cells/ml, and 20 @ were again pipetted into the wells of microtest plates. The plates were then treated as described for the direct LA1 test. To test if LAIF produced by spleen cells of mice could be transferred t o primate leukocytes, leukocytes from pooled peripheral blood of three chimpanzees were purified by Ficoll-Hypaque sedimentation and washed 4 times in medium. They were then suspended in the supernatant to a final concentration of 1 O6 cells/ml; 20 pl were used/well. In both the direct and indirect tests, at least eight wells for each antigen were counted. Normal animals sometimes show unspecific LAIF production [ 51; t o avoid unspecific reactions of the normal indicator cells, purified RLV was used as a specificity control throughout. Percent reduction was calculated according to the formula: ( I - x I00 % in which u is the average number of cells incubated with MTV (or the MTV-treated supernatant) and b that incubated with RLV (or the RLV-treated supernatant). Statistical significance was established by means of the two-tailed Students' t-test. The assays were always performed with pools of cells derived from at least five mice.

t)

2.5. Separation and testing of cell subpopulations 2.3. Direct LA1 assay Killed mice were injected intraperitoneally by means of a pasteur pipette with 5 ml of ice-cold medium (RPMI 1640 with 100 IU penicillin and 100 pg streptomycin/ml and 5 x I O-s M 2-mercaptoethanol) t o which heparin (5 IU/ml) was added. The fluid was withdrawn and reinjected several times and then collected into glass tubes. The cells were centrifuged once at 200 x g for 10 min. They were then resuspended in medium containing 7 % fetal calf serum (Flow, Irvin, Scotland). The final cell concentration was adjusted to approximately 20 x 1 03/ml. Then 0.1 pg antigenlml was added and 20 pl of the suspension were pipetted into the wells of Falcon microtest 3034 plates. The wells for the con-

I!;

Macrophag9 were removed by adherence of spleen cells 4 (4 x 1 06/ml)*tothe surface of Falcon tissue culture flasks (25 cm2) during 2-3 h, or with the iron-magnet method. B cell-enriched cell fractions were obtained by incubating macrophage-depleted spleen cells with mouse anti-@ serum and complement for 1 h. The anti-@ serum was made by injecting 2 x 1 O7 thymocytes derived from C3H mice intraperitoneally into AKR mice. This was done six times with

*

It was sometimes observed that more cells adhered in the wells at the edges of the plates than in the middle. Also plating efficiency was very low. This could be restored by incubation of the plates in saline for two days. Greiner microtest plates (Niirtingen, Cermany) did not have these disadvantages.

50

Eur. J. Immunol. 1977. 7: 48-53

P.Creemers

intervals of 14 days. Then the AKR mice were bled; t h e resulting anti-@ serum killed 7 0 % of C3H thymocytes u p t o a dilution of 1 / 16. As a source of complement guinea pig serum absorbed with agarose and stored in liquid nitrogen was used. The complement was active u p t o a dilution of 1 /8. After treatment the cells were washed and used f o r the LA1 assay. T cell-enriched cell populations were obtained by adherence on a nylon wool column [7]. After elution about 20 % of the original cell population was recovered. Sixty 88 % of the eluted cell population could be killed with mouse anti-@ serum and complement, whereas 10-30 % were Igpositive in membrane-fluorescence studies (tested 6 times). When adherent cells alone or in combination with T cellenriched cell fractions were tested, t h e adherent cells present in 20 x 1 O6 spleen cells were allowed t o adhere to the surface of the plastic tubes in which the test was performed; after three washings, medium containing 10 % fetal calf serum or T cell-enriched cell fractions respectively, were added.

direct assay were always washed four times. In t h e indirect test the highest response was obtained if t h e cell concentration was 20 x 1 06/ml (Fig. 1). On prolonging the incubation time of the spleen cells, LAIF production also increases; however, this increase in no longer noteworthy after 24 h (Fig. 2). If the same incubation time (2 h ) was used for both the indirect and t h e direct test, reduction was increased by about 30 % in the indirect method, but only if t h e supernatants were derived from spleen cells. This is illustrated in Table 1.

-" r

60-1

0

.

0

40-0

20

-

I 10

To determine whether cell-to-cell contact was necessary for LAIF production, macrophages were allowed t o adhere t o the bottom of plastic tubes (Falcon, 75 x 100 mm). Afterwards, sterilized Millipore filters with a diamter of 47 mm and a pore size o f 0.45 p (Millipore, Bedford, Mass.) were moistened in medium and pushed into the tubes in such a way that they became funnel-shaped. Only the point of the filter made contact with the underlying medium. One ml of a T cell-enriched cell population was then transferred into t h e f u n n e k h a p e d filter. The filters were removed and the remaining cells were spun down prior t o testing the supernatant. When supernatant transfer experiments were performed, the cells were spun down and the supernatant was removed; before adding a supernatant incubated with a different cell population, the cells were washed twice in medium. In all experiments the RLV control supernatant was prepared simultaneously and treated in the same manner as the MTV supernatant. 2.6. Clearance of

MTV

To remove MTV, supernatants were incubated for 1 h a t 3 7 OC with rabbit anti-MTV serum that was bound t o Sepharose 4B beads [ 81; thereafter the beads were spun down and the supernatant was used for the transfer experiments. For sterilization the beads were left overnight at 4 "C in a 0.02 % sodium azide solution in PBS, and then washed 4 times in medium. After this treatment the beads were still active in binding MTV, as was shown by immunofluorescence in which bound MTV was determined by treatment with fluorescein-conjugated rabbit anti-MTV serum. The amount of beads added t o 1 mi of supernatant could bind approximately 100 pg of MTV.

3. Results 3.1. Optimal conditions for the indirect LA1 assay When the direct assay was performed with peritoneal exudate cells, it was observed that LAlF could be removed by repeated washing: a significant reduction of 37, 26, 29 and 20 % that was measured after o n e washing, decreased t o zero if the cells were washed four times. Since we wanted t o measure the amount of LAIF produced in virro, the cells used for the in-

30

20 6

Spleen c e l l s x 10 / m l

m?l

Figure 1. MTV-specific LAIF production as measured by the indirect

LA1 assay at different spleen cell concentrations. Spleen cells were derived from positive control animals Vertical bars indicate SE of the percent reduction measured. The Figure represents one out of three experiments.

.

. 10

.

.

. 20

30

ma

-

.

40

50

Hours of incubation

Figure 2. MTV-specific LAIF production by immune spleen cells after varying incubation times as measured by the indirect LA1 assay. Vertical bars indicate SE of the percent reduction measured. This Figure represents one out of two experiments. Table 1. MTV-specific LAIF production by immune cells as measured by the direct and the indirect LA1 assay during a 2 h incubation period

Method and Incubation Incubation cell population with R L V with MTV Average no. of adherent cells

Direct LA1 Peritoneal cells 14.8 f 0.5 Indirect LA1 Pcritoneal cells 19.3 f 1.3 Spleen cells 15.8 f 0.6

f

Experiment 1

2

3

Reduction

SE

(5)

10.8 0.9

27"")

2OS

15"

21.0 f 0.9

28" 37x

18"

19s

3 1

27"

+_

10.0 f 1.0

a) x: p