Effect of interleukin 2 on cytotoxic effectors

CELLULAR

IMMUNOLOGY

73, 275-289 (1982)

Effect of lnterleukin II. Long-Term

2 on Cytotoxic

Effecters

Culture of NK Cells

CHOU-CHIKTING,STRINGNER S.YANG, AND MYRTHEL E. HARGROVE Laboratory

of Cell Biology, National

Cancer Institute, Bethesda, Maryland

20205

Received May 13. 1982; accepted August 15, 1982

The present study reports the establishment of an NK (natural killer) cell line, designated as ILZ-CEl This cell line was maintained in active growth in culture with the supplementation of interleukin 2 (IL2). The cells gave cytotoxicity against a variety of murine tumors. There was no detectable cytotoxicity against three human tumors tested. The reactivity obtained with the murine tumors was generally correlated with the sensitivity of these tumor cells to NK cytotoxicity. With few exceptions, the reactivity was much higher for NK-sensitive targets like YAC and RM 1 cells. There was very little cytotoxicity against NK-resistant targets like P8 15 and HFL/d. The reactivity of the effecters against YAC was susceptible to trypsin treatment and this effect was reversible. In determining the surface markers of ILZ-CEl cells, it was shown that over 90% of the cells had Thy- 1.2 antigen despite their resistance to the lytic effect of anti-Thy- 1.2 antibody. There were no detectable surface Ig or Fc receptors. Therefore, the ILZ-CEl cells were consistent with being NK cells. Although these cells gave high levels of cytotoxicity against murine tumors, in the tumor neutralization tests the ILZ-CE 1 cells failed to give any protection against an immunosensitive leukemic line, FBL-3. After the ILZ-CEl cells were cloned, it was found that different NK clones showed variations in their fine specificity. Our finding supports the presence of different populations or subsets of NK cells. Establishment of these NK clones in long-term culture should be helpful for the detailed characterization of the NK cells and aid in the determination of their significance in the immune surveillance against neoplasia.

INTRODUCTION The establishment of lymphoid cells in long-term cultures has proved to be very useful for the functional analysis of various lymphocyte populations. The development of the hybridoma technique has opened an exciting field for the study of B cells. In the past, studies on T cells have been limited by the lack of functional monoclonal T-cell lines. This problem was overcome by the recent discovery of interleukin 2 (IL2, or T-cell growth factor)’ (1). IL2 was found to be essential for the generation of cytotoxic T cells (2, 3). It also supports the growth of T cells in culture. Because of this unique feature, several functional T-cell lines have been established (e.g., 4-7). It was later found that, in addition to T cells, NK cells can also be maintained in active growth in medium with IL2 supplementation (8). In this paper, we describe the establishment of a long-term cultured cell line which has the characteristics of cytotoxic NK cells. ’ Abbreviations used: NK, natural killer cells; Ig, immunoglobulin; B, bone marrow derived, T, thymus dependent; IL2, interleukin 2; B6, C57BL/6; FMR, Friend, Moloney, Rauscher virus induced. 275 0008-8749/82/160275-15$02.00/O

276

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It has been established that in vivo NK cells are heterogenous in nature (9). Different populations of NK cells displayed different cell surface markers and different cytotoxic activities. Among the cloned NK lines, those established by Dennert (10) were quite different from those established by Nabel (11). They differed in the target specificities and their ability to mediate antibody-dependent cell-mediated cytotoxicity. At present the nature and function of NK cells are poorly understood. It appears that additional efforts are needed to obtain NK clones from various sources and then detailed comparative studies can be done with these different NK clones. Through this approach one may be able to understand the diverse nature of NK cells which leads to their diverse functions. Although many functional T-cell lines have been successfully established (4-7), there was limited successin establishing functional NK-cell lines. Only two documented reports have demonstrated such success(10, 11), suggesting that NK cells may be difficult to grow in long-term cultures. It is with this intention that we attempted to grow NK cells derived from an immune host of relatively low NK responder (C57 BL/6 mice). After these NK cells were successfully maintained in long-term cultures, they were further cloned. The issue of the heterogenous nature of NK cells has also been examined exhaustively. Different clones of NK cells were tested against a variety of tumor cells. The specificity of NK cytotoxicity was further verified by cold target inhibition experiments. Furthermore, the in vivo antitumor activity of the long-term cultured NK cells was also examined. MATERIALS

AND METHODS

Mice Female C57BL/6 mice (B6) at 2-5 months of age, CBA mice of 6-7 weeks of age, and BALB/c nude mice (nu/+ and nu/nu) of 6-8 weeks of age were obtained from the Veterinary Resources Branch, Division of Resource Services, National Institutes of Health, Bethesda, Maryland.

Tumor Cell Lines Eight murine tumor cell lines and three human tumor cell lines were grown in suspension culture in RPM1 1640 containing 5% fetal bovine serum. These cell lines are FBL3 (9), RBL-5 (9), EL-4 ( 12), YAC ( 13), RL$ 1 ( 14), L5 178 ( 15), HFL/d ( 16), P8 15 (17), K562 (18), Daudi (19), and Raji (20). The origin of these cell lines is shown in Table 1.

Growth of FBL-3 Cells and Immunization Intraperitoneal (ip) inoculation of as few as 10 FBL-3 cells into B6 mice produced progressive ascites tumor growth and killed the mice. Inoculation of 5-25 X lo6 FBL-3 cells subcutaneously (SC)produced transient tumor growth with subsequent complete regression (regressors).The regressorsbecame immune to FBL-3 and eventually resisted the ip challenge of at least 1 X lo6 FBL-3 cells. The immunization was usually achieved by SCinoculation of 5 X lo6 FBL-3 cells.

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TABLE 1 Cytotoxic Activity and Specificity of the Cytotoxic Reactions Obtained with Long-Term Cultured Cells ILZ-CE 1a Net percentage lysis obtained at E/T* Target FBL-3 RBL-3 EL-4 YAC RL61 L5178 HFL/d P815 K562 Daudi Raji

Species or strains

Origin

IO/l

3/l

l/l

l/3

Mouse (H-2b) Mouse (H-2b) Mouse (H-2”) Mouse (H-2”) Mouse (H-2d) Mouse (H-2d) Mouse (H-2d) Mouse (H-2d) Human Human Human

Friend virus Rauscher virus Benzpyrene Moloney virus Radiation leukemia virus Methylcholanthrene Friend virus Methylcholanthrene Acute myelogenous leukemia Burkitt lymphoma Burkitt lymphoma

53 54 20 37 35 29 I1 7 0 3 2

43 28 20 34 35 33 7 6 2 7 3

32 18 14 22 25 23 5 2 I 5 1

20 14 9 13 15 12 4 I I 4 I

’ This test was performed 3 months after starting the IL2-CEl cells. h Net percentage lysis was calculated by comparing to the total percentage lysis obtained with normal thymocytes testing at same E/T ratio.

Preparation of Interleukin 2 (ILZ) The IL2 was prepared according to Gillis et al. (2 1). W/Fu rat spleen cells at 5 x lo6 cells/ml were cultured with Con A (Calbiochem, San Diego, Calif.) at 5 pg/ ml for 48 hr. The supernatants were harvested and filtered through 0.20~pm Millipore filters (Nalge Co., Rochester, N.Y.), and stored at -70°C. The Con A-free preparation could be produced by culturing W/Fu rat spleen cells at 1 X 10’ cells/ ml in the presence of Con A at 10 #g/ml for 4-5 hr. The cells were washed twice in Hanks’ basal salt solution (HBSS) containing 5% FBS. They were then resuspended in RPM1 1640/5% FBS at 5 X 106/ml and cultured for an additional 44 hr. The supernatants were harvested as described above. Partial Purification of IL2 Crude IL2 was purified by 30-60% (w/v) ammonium sulfate cut. After extensive dialysis against four changes of 1 liter each of 0.25 M sodium phosphate (PB) buffer overnight, the IL2 was loaded onto a preequilibrated Sephadex G- 150 column. The protein profile was monitored by A 280absorption with an LKB Unicord II spectrophotometer. Fractions eluting with PB were assayed for IL2 activity. The major peak of IL2 activity centered at the 15,000 molecular weight (MW) fractions. A very minor peak of IL2 activity was also found at the 30,000 MW area. IL2 at the 15,000 MW peak was about lOO- to 150-fold purified when compared with the specific activity of the starting materials. This partially purified IL2 was essentially free of Con A. Activity of the IL2 The IL2 activity was determined by a method developed by Gillis et al. (1) with some modification. Ten- to 14-day Con A-stimulated and IL2-maintained normal B6 spleen cells were used as the target cell population. The optimal concentration

278

TING,

YANG,

AND

HARGROVE

of IL2 activity for promoting the T-cell growth was determined by its effect on maintaining and expanding the maximal growth of the target cell populations. It was found to be 5-10% and lo-20%, for the crude IL2 and purified IL2, respectively. This was determined by the growth of the target cells in 24-well (16-mm) culture plates (Costar, Cambridge, Mass.) or by the incorporation of “‘IUdR in Microtest II plates. Tumor Neutralization Test B6 Recipient mice were given an ip inoculation of either (1) 1 X lo6 immune lymphocytes, or (2) IL2 CEl cells. To prepare immune lymphocytes, B6 mice were first inoculated SCwith 5 X lo6 FBL-3 10 days prior to the experiment. Lymphocytes were obtained from the spleens and lymph nodes (cervical, axillary, and inguinal) of these immunized mice. To determine the in vivo antitumor activity of these lymphocyte preparations, all mice also received an ip challenge of 5 X lo4 FBL-3 cells. The development of tumor growth was observed for 60 days. Cell-Mediated Cytotoxicity Assay The ‘251UdR release assay (IRA) was used to measure cell-mediated cytotoxicity. The details of the technique have been described elsewhere (20). In brief, 0.05 ml of 1251UdR-labeledtarget cells at 1 X 1OS/ml and 0.15 ml of effector cells at an appropriate concentration were added to the wells of Microtest II plates. All effector cells were adjusted to contain the same number of viable cells for each effector:target ratio (E/T). Incubation was carried out at 37°C in a 5% CO2 atmosphere for 18-24 hr. The samples were then harvested by a MASH II (Microbiological Associates, Bethesda, Md.). The supematants and cell pellets were collected separately and their radioactivity was determined in a well-type gamma scintillation counter. The results were expressed as total percentage of lysis and net percentage of lysis according to the following formulas: cpm in supematants x 100, total % lysis = cpm in supematant + cpm in cells net % lysis = (total % lysis obtained with test effectors) - (total % lysis obtained with control effecters). Normal thymocytes were generally used as control effecters. The standard errors obtained with total percentage lysis were usually between 0.5 and 3%. Testing the SpeciJicity by Cold Target Inhibition To test the specificity of the cytotoxic reactions, various unlabeled cold inhibitor cells were added to determine their effect on the cytotoxic activity. Three doses of inhibitor cells were used in these experiments: 1.5 X 104, 5 X 104, and 1.5 X lo5 cells. The inhibitor to target cell ratios were 3/ 1, lO/ 1, and 30/ 1, respectively. Treatment with Anti-Thy-l.2

Antibody

Monoclonal anti-Thy-l .2 antibody was purchased from New England Nuclear (Boston, Mass.). Treatment of 1 X 10’ spleen cells with 0.1 ml of the anti-Thy-l.2

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antibody at 1:300 dilution and 0.1 ml of nontoxic rabbit complement at 1:10 dilution eliminated all the proliferative responses of the spleen cells to T-cell mitogens Con A or PHA with preservation or enhancement of proliferative response to a B-cell mitogen LPS. In the treatment of cytotoxic effecters, l-2 X 1O6cells were incubated with 0.1-0.2 ml anti-Thy-l.2 at 1:100 dilution for 30 min at room temperature. They were washed, followed by a second incubation with 0.1 ml of nontoxic rabbit complement at 1:10 dilution. They were then washed, counted, and resuspended to an appropriate concentration. Determination of the Surface Markers by a Rosette Assay The cell surface markers were determined by a rosette assayas described by Koo and Goldberg (23). The indicator sheep erythrocytes were coupled with Protein A (Pharmacia Fine Chemicals, Uppsala, Sweden) by the chromic chloride method (24). The indicator cells (PA-SRBC) were kept at 0.5% packed cell volume in phosphate-buffered saline with 0.1% NaNj. In testing, 25 ~1 of test cells at 1 X lo7 cells/ ml were incubated with an equal volume of antiserum at various dilutions for 30 min at 4°C. The cells were washed with Medium 199 containing 1% of h G-free fetal bovine serum. Then the cells were mixed with 300 ~1 of indicator cells (PASRC) and left to incubate at room temperature for 30 min. After completion of the assay, the cells were fixed with 0.1% glutaraldehyde for preservation and stained with Giemsa-Wright on a slide for reading. A cluster of more than 3-5 SRBC was considered to be a positive rosette. Antisera For surface marker determination, the anti-mouse Thy-l .2 antibody was purchased from Becton-Dickinson (Mountain View, CA). The goat anti-mouse Ig antiserum was kindly provided by Dr. Asofsky (NIAID, NIH). Trypsin Treatment Ten million effector cells were suspended in l-l 5 ml of 0.5% trypsin and were incubated at 37°C for 45 min. The cells were washed three times in Hanks’ balanced salt solution containing 5% FBS and were then resuspended in RPM1 1640/5% FBS at appropriate concentrations for testing. Cell Cloning The limiting dilution technique was used for cell cloning. Initial attempts to clone the cells in Microtest plates were not successful. The cells usually died after two or three transfers. Then we modified the technique by seeding 30 to 100 cells per well in the 24-well (16-mm) culture plates. Seeding 30 cells or less usually gave no cell growth. The plating efficiency in these larger-size wells was found to be only 1 to 3%; thus the clones obtained were likely to be derived from a single cell. The cells in the wells which showed active cell growth were transferred to a new 24-well culture plate and expanded to give a total cell number around 5- 15 X 1O6cells. The medium used for cloning was the same as that for maintaining the cell growth (RPM1 1640/5% FBS with 10% IL2).

280

TING, YANG, AND HARGROVE

Time After Each Subculture

(hr)

FIG. 1. Growth pattern of IL2-CE 1 cells. The cells were subcultured every 48 hr with a change of fresh RPM1 1640 medium containing 5% FBS and 10% IL2.

RESULTS Establishment of a Long-Term Cytotoxic Cell Line in Culture With the supplementation of IL2, T cells were maintained in active growth for 3-5 weeks. In most cases, a “crisis” developed and the T cells lost their responsiveness to IL2 and died. After repeated efforts, we obtained a cytotoxic cell line which passedthe crisis and emerged to grow. This cell line was originated from the spleen of a B6 mouse which had been immunized with a syngeneic leukemia FBL3 (9). In the first 6 months, the doubling time for the culture cell growth was approximately 24 hr. Then the doubling time gradually increased to 48 hr. A typical growth curve is depicted in Fig. 1. This cell line was designated IL2-CE 1. Cytotoxic Activity and SpeciJcity of the Cytotoxic Reactions Obtained with ILZ-CEI Cells In the development of ILZCEl cells, there was considerable change in their cytotoxic activity and specificity of the cytotoxic reactions. The results are summarized in Fig. 2. During the first 2 weeks in culture, the cytotoxic effecters showed considerable increase of the specific cytotoxicity against FBL-3 cells. There was no significant cytotoxicity against EL-4 cells, a chemically induced leukosis. When tested on Days 28 and 35, there was a significant increase of cytotoxicity against EL-4 cells. At this time, normal B6 spleen cells kept in culture in the same manner also showed increased levels of cytotoxicity against both FBL-3 and EL-4 cells. After culturing for 3 months, the ILZ-CEl cells were tested against a variety of murine tumors and three human leukemia/lymphoma lines. The results are summarized

10 2030 40

W-DAYS

10 +-DAYS

-MONTHS-I

TIME

20

30 40 +MONTHS4

IN CULTURE

FIG. 2. Cytotoxic activity of IL2-CEl cells. Starting at 11 days after initiating the culture, the IL2-CEI cells were tested at 7- to lo-day intervals against FBL-3 and EL-4 as targets. The E/T ratio was 30/l. After culturing for 3 months, they were repeatedly tested against these targets at E/T of 3/l.

0 7 7 8

2 19 17 13

100/Ih

3 13 10

30/l

5 11 6 9

-2 15 17 12

loo/l

I 15 14

30/l

RBL-5

1 8 4 7

i 17 16 12

loo/l

EL-4

3 12 9

30/l

4 25 21 8

4 33 29 I1

100/I

YAC

12 32 20

30/l

3 20 17 6

-2 20 22 12

100/l

RLdl

-3 16 19

30/I

2 8 4 6

-2 7 9 14

100/I

HFL/d

-1 7 8

30/l

1 3 23

-2

-2 6 8 18

100/l

P815

2 4 2

30/l

-2 -2 0 9

N.D.d N.D.

100/l

30/l

L5178

n In experiment I, the effecters were thymocytes or spleen cells from 7-week-old CBA mice. In experiment 2, the effecters were spleen cells from BALB/c nu/+ (heterozygous) or nu/nu (homozygous) mice. ’ E/T ratios. ’ NK = (net percentage lysis obtained with CBA or BALB/c nu/nu spleen cells) - (net percentage lysis obtained with CBA thymocytes or BALB/c nu/+ spleen cells). The net percentage lysis was calculated by comparing to the total percentage lysis of the medium control as shown in the parenthesis. d Not done.

Experiment 1 CBA Thymus CBA Spleen NK’ Medium Experiment 2 nu/+ Spleen nu/nu Spleen NKC Medium

Effecters”

FBL-3

Net percentage lysis obtained with

Susceptibility of Various Murine Tumors to NK Cytotoxicity

TABLE 2

282

TING, YANG, AND HARGROVE TABLE 3 Effect of Concanavalin A on the Cytotoxic Activity of the Effecters Net percentage lysis obtained at6 6 hr

Effecters kept” in medium with: Experiment 1 Con A Con A-free Experiment 2 Con A Con A-free

20 hr

FBL/3

YAC

FBL-3

YAC

lO/lC

3/l

l/l

10/l

3/l

l/l

10/l

3/l

l/l

10/l

3/l

l/l

18 15

II 8

6 3

23 24

11 15

6 6

46 44

33 30

18 13

67 66

37 41

34 35

10 9

4 5

3 2

9 8

2 3

7 2

31 33

28 25

19 20

49 51

38 40

27 2.5

“Twenty-four hours before cytotoxicity test, the effecters were washed three times with Hanks’ BBS/S% TBS. These cells were divided into two parts. One part was kept in 1640 medium with 10% IL2 (with Con A) and the other part was kept in Con A-free medium. The next day, these cells were counted, washed, and resuspended in test medium (1640/5% BBS without Con A) at appropriate concentrations. b Two sets of tests were performed with two targets (FBL-3 and YAC). One set was incubated for 6 hr; the other set was incubated for 20 hr. ’ E/T ratios.

in Table 1. It was found that these cytotoxic effecters only gave significant cytotoxicity against murine tumors. There was no significant cytotoxicity against the three human tumors tested. The levels of cytotoxicity were in general correlated with the sensitivity of the tumor targets to NK cytotoxicity (Table 2). This was shown by testing the NK activity of splenocytes from CBA mice or BALB/c nude mice (nu/nu). These two strains are high NK responders. For IL2-CEl cells (Table I), higher levels of lysis were seen with NK-sensitive targets like YAC and RL81 cells. Very low levels of lysis were seenwith NK-resistant targets like P8 15 and HFL/ d cells. The exception was that IL2XEl cells gave considerable cytotoxicity against EL-4 and L5 178 cells which were relatively NK resistant. In addition, the highest levels of cytotoxicity were obtained with FBL-3 and RBL-5 cells. These two leukemic lines were of FMR origin. These findings indicate that although the pattern of cytotoxicity resembled the NK reactivity, there were also some differences. This pattern of reactivity remained for at least 9-12 months with only minor variation. E$ect of Lectin on the Cytotoxic Activity The IL2-CEl cells were started and maintained in IL2 with traceable amounts of lectin (Con A). However, their cytotoxic activity did not appear to be lectin dependent. In the 20-hr lz51UdR assay, the effecters were washed and suspended in lectin-free medium. We have further kept these washed effecters in lectin-free medium for 24 hr, then tested in the cytotoxicity assay (Table 3). It was found that there was no change in their cytotoxic activity, both in 6-hr and 20-hr assays.We have also performed experiments with cells incubated with a-methyl mannoside or with cells cultured in lectin-free IL2 for at least 2 weeks. There was also no appreciable change in the cytotoxic activity of these cells.

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TABLE: 4 Effect of Anti-Thy- 1.2 Antibody on the Cytotoxic Effecters Net percentage lysis obtained with FBL-3C Treatment of effecters’ No treatment Anti-Thy- 1.2 +c c’ alone

RLbl’

YAC’

EL-4’

IO/lb

311

l/l

10/l

3/l

l/l

10/l

311

l/l

IO/l

3/l

111

45d

31

26

50

39

24

41

36

23

56

50

36

39 47

37 35

29 28

46 47

40 36

24 20

47 42

37 31

23 22

41 4-I

44 47

32 31

a Anti-Thy- I .2 monoclonal antibody from New England Nuclear was used. (See Materials and Methods). * E/T ratios. ’ The total percentage lysis for the medium control (spontaneous release) for FBL-3, EL-4, YAC, and RL6 1 cells were 7% 9% 15%, and 12% respectively. The thymocyte controls gave similar levels of total percentage lysis as medium control. d The total net percentage lysis was calculated by comparing to the total percentage lysis obtained with thymocytes testing at same E/T ratio.

Efect of Anti-Thy-l.2

Antibody on the Cytotoxic Eflectors

The freshly obtained splenic cytotoxic effecters from B6 mice immunized with FBL-3 have been characterized to be T cells (25). They gave specific cytotoxicity against targets of FMR origin. From the preceding experiment (Table 1) it was evident that the cytotoxic reaction obtained with the long-term cultured IL2-CEl cells was broadly reactive. Experiments were performed to determine their susceptibility to the lytic effect of anti-Thy- 1.2 antibody. The results are summarized in Table 4. Untreated and treated effecters were tested against four different targets. It was found that these cytotoxic effecters were resistant to the anti-Thy- 1.2 antibody lysis. In short, after anti-Thy- 1.2 antibody plus complement treatment, the cytotoxic activity of the effecters remained the same as the untreated effecters or effecters treated with complement alone. Even after two treatments of the effecters with the TABL.E S Characterization of the Cell Surface Markets on the Cultured Cells IL2-CE I Surface markers (%)* Cell type” Experiment 1 Normal spleen ILZ-CE 1 Experiment 2 Normal spleen ILZ-CE 1

Thy-l.2

k

Fc receptors

35 93

42 2

14 3

32 91

38 3

15

’ Normal spleen cells were obtained from B6 mice. ’ Percentage of ceils which gave rosette formation.

2

284

TING, YANG, AND HARGROVE TABLE 6 Effect of Trypsin Treatment on the Cytotoxic Activity of the Effecters Net percentage lysis obtained atb 6 hr

20 hr

FBL-3 Treatment of effectors~ Experiment 1 No treatment Trypsin Experiment 2 No treatment Trypsin Experiment 3 No treatment Trypsin Experiment 4 No treatment Trypsin

10/lC

3/l

YAC 111

10/l

3/l

FBL/3

YAC

111

10/l

3/l

l/l

10/l

3/l

l/l

11 13

8 9

4 3

12 Id

8 le

2 1

41 51

34 42

18 17

67 50d

58 36d

28 lgd

10 10

4 6

3 5

9 2e

2 0

7 0

31 34

28 28

19 22

49 37d

38 2gd

27 15d

18 17

11 9

6 3

23 9d

11 3d

6 Od

46 42

33 32

18 19

67 50’

37 27d

34 2od

22 15e

7 4

3 2

12 -Id

2 0

1 0

73 58’

27 19’

12 3

43 27d

16 10d

13 5’

a Effecters were washed three times with PBS solution without PBS, then l-2 X lo6 effecters were incubated with l-2 ml 0.5% trypsin for 45 min at 37°C. Then they were washed three times with Hanks BSS containing 10% PBS. The cells were counted and were resuspended in 1640/5% PBS at appropriate concentrations for testing in the cytotoxicity assay. b Two sets of tests were performed with two targets (PBL-3 and YAC). One set of tests was incubated for 6 hr and the other set was incubated for an additional 14 hr. ’ E/T ratios. dThe statistics were evaluated by the Welch Distribution t test by comparing the percentage lysis obtained with effecters treated with trypsin to that without treatment, P < 0.01. pP < 0.05.

anti-Thy-l.2 antibody plus complement, there was still no reduction in their cytotoxic activity (not shown). Characterization of the Cell Surface Markers on the ILZ-CEl

Cells

To characterize the cell surface markers on the IL2-CEl cells, a rosette assay(23) was employed to determine the presence of Thy-l .2 antigen, surface Ig, or Fc receptors. The results are summarized in Table 5. It was found that more than 90% of the cell population possessedThy- 1.2 antigen, but there were no detectable surface Ig or Fc receptors. Eflect of Trypsin Treatment on the Cytotoxic Activity of the Efectors From the preceding experiments (Tables 1, 4, 5), by determining the specificity and cell surface markers, it appeared that the IL2-CE 1 cells were of NK nature. One of the unique features of murine NK cells was their reversible susceptibility to trypsin treatment (26). That is, in a short-term 4- to 6-hr assay, the cytotoxic activity of NK cells was considerably reduced by trypsin treatment. However, this reactivity recovered after 18-24 hr. We have performed experiments to determine the sus-

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ceptibility of IL2-CEl cells to trypsin treatment. The results are summarized in Table 6. Two targets were used in these experiments (FBL-3 and YAC). In four separate experiments, it was found that in a 6-hr assay,the cytotoxic activity of IL2CE 1 cells against YAC cells was considerably reduced after trypsin treatment whereas the cytotoxic activity against FBL-3 cells was unaffected. After a 20-hr incubation, most of the cytotoxic activity of IL2-CE 1 cells against YAC recovered, whereas the activity against FBL-3 remained unaffected. Tumor Neutralization Test From the pattern of cytotoxic reactivity and the cell surface marker analysis (Table 6), the IL2-CEl cells could be characterized as NK cells. Due to their extremely high levels of cytotoxic activity (Table l), it would be of interest to test their in vivo antitumor activity. We elected to use FBL-3 cells, a Friend virus-induced leukemia in C57 BL/6 mice (9). This leukemia line has been well characterized by us and others and was shown to be very immunosensitive in the in vivo testing of tumor transplantation immunity (9, 25). The tumor neutralization test was performed in C57 BL/6 mice. The results are summarized in Table 7. It was found that recipients which were given lymphocytes from immune donors (FBL-3 immune) gave strong protection against the challenge of FBL-3 cells (Group 2). In contrast IL2-CE 1 cells failed to give any protective effect. Cloning of the Cytotoxic Effecters and Testing of the Specificity Reactions

qf the Cytotoxic

From the study performed with trypsin treatment, it appeared that there might be more than one population of cytotoxic effecters in IL2-CEl cells. One type of effecters was susceptible to trysin treatment whereas the other one was trypsin resistant. A direct approach to answer this question was to clone the cells and then test the activity and specificity of individual clone. Using the limiting dilution technique we have cloned the IL2-CEl cells. Four clones were obtained and were expanded to 5- 15 X lo6 cells. The cytotoxic activity and specificity of these clones TABLE Tumor Neutralization

Donor cells’ 1. None 2. Immune lymphocytes 3. ILZ-CEl

7

Test of IL2-CEl

Cells

Tumor incidenceb (Number of tumor takes/ number of mice inoculated) 14/14 2114 717

P’

co.01

a All recipients (normal B6 mice) received ip inoculation of 5 X IO4 FBL-3 cells. In Group I, no other cells were inoculated. In Group 2, 1 X lo6 immune lymphocytes were also inoculated ip. The immune lymphocytes were pooled lymph node and spleen cells from B6 mice which were immunized with sc inoculation of 5 X IO6 FBL-3 cells 10 days prior to the experiment. In Group 3, the recipients were also given ip inoculation of 1 X lo6 ILZ-CEI cells. * The tumor incidence was a cumulative incidence at 60 days after tumor cell inoculation. ’ The x2 test was used to compare the frequencies of tumor takes with the control group (Group I).

286

TING, YANG, AND HARGROVE FBL-3

RLbl

YAC

EL4

b-615

HFUd

Thy,,,.

FBL-3

YAC

RLbl

EL4

p815

HFUd

Thy",

m

d.UonaJ&ainURLdl u-lwu-l

' I

m b.CbmZA@mntFBL-3

u-luJl-uwwwl-u 1561.51651.51551.51551.51551.51551.51551.5

Inhibitor Cells ( x 10m4)

m c.CkmeZAgaimtYAC

15 51.515

51.515

51.51551.5

15 51.51551.51551.5

Inhibitor Cells I x 10-4)

FIG. 3. Testing the specificity by inhibition experiments. Three clones of ILZ-CEl cells were tested against various murine tumor targets as indicated. The E/T ratio was 3/l or 2/l. Three doses of cold inhibitor cells (from 1.5 X lo4 to 1.5 X lo5 cells) were added to determine their inhibitory activity on the cytotoxicity. Six murine tumors and a thymocyte preparation were used as inhibitors. The dotted lines represented the cytotoxic activity without inhibitors.

were then determined by testing against various targets with cold target inhibition experiments. The results are summarized in Table 7 and Fig. 3. Clone 1 only reacted with FBL-3 cells. In cold target inhibition experiments it was shown that FBL-3 cells inhibited only the cytotoxicity of Clone 1. All other clones (Nos. 2-4) reacted with a variety of targets and their cytotoxicity was also inhibited by a variety of cold target cells. As expected, the NK-resistant targets, the P815 and HFL/d cells, were marginally inhibitory, and thymocytes were not inhibitory. It should be noted that in direct testing (Table 8), there was some difference between Clone 2 and Clone TABLE 8 Reactivity of the ILZ-CEl Clones Net percentage lysis obtained with’ Effector

PBL-3

EL-4

YAC

RMl

Clone Clone Clone Clone

22 30 15 20

2 3 10

7 47 14 25

3 21 11 25

1 2 3 4

HPL/d

P815

3 18 14

2 3 7 3

(1Clones l-4 were grown at different times. Each clone was tested at different times against various targets. * E/T = 2/ 1 or 3/l. The net percentage lysis was obtained by comparing to thymocyte control testing at same E/T ratio.

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3. Clone 2 did not give significant lysis against EL-4 or I?815 cells whereas Clone 3 gave a relatively higher level of cytotoxicity against EL-4 cells. In the inhibition experiments, EL-4 cells were also more inhibitory for Clone 3 effecters than for Clone 2. It is interesting to note that within the same clone, the inhibitory activity of different tumors against different targets also varied. For Clone 2 effecters, the FBL-3 cells were more inhibitory when tested against FBL-3 target than against YAC target. In contrast, the YAC and RL8 1 cells were more inhibitory when tested against YAC target. It should be noted that except for Clone 1, the YAC cells were generally more susceptible to cytotoxicity than FBL-3 cells (e.g., Clones 2, 4). DISCUSSION There are four major classesof cytotoxic effector cells: cytotoxic T cells (CTL), natural killer cells (NK), macrophages, and K cells (mediating ADCC, antibodydependent cell-mediated cytotoxicity). Among them, cytotoxic T cells have been most thoroughly studied. With the recent discovery of interleukin 2 (IL2 or T-cell growth factor) in has become possible to establish functional, monoclonal T-cell lines (4-7). This new technology allows the study of various aspects of T cells in greater depth. Numerous efforts have been dedicated to the study of NK cells concerning their characteristics and biological functions (27,28). Yet there are still many unanswered questions. It has been suggestedin various experiments that NK cells may play an important role in the immune surveillance against neoplasia (27, 28). However, there is still a lack of concrete evidence to support this view. One of the limitations for studying the NK cells is the limited quantity available. It was estimated that there are only l-2% NK cells in the mm-me spleen (27). Therefore, if NK cells can be isolated and expanded in culture, the study of NK cells will be greatly facilitated. Although many functional, monoclonal-T-cell lines have been established (4-7), attempts to establish NK-cell lines have met with limited success,indicating difficulty in growing NK cells in long-term cultures. In literature there are two documented reports on the successful establishment of NK-cell lines ( 10, 11). IL2-CEl cells were characterized as NK cells on the basis of the following facts. First, they were broadly cytotoxic against a variety of murine tumors and the reactivity was in general parallel with the sensitivity of these tumor lines to NK cytotoxicity (Tables 1, 2). Second, despite their resistance to anti-Thy- 1.2 antibody lysis (Table 4), over 90% of the effecters were shown to have surface Thy-l .2 antigen (Table 5). These effecters lacked surface Ig and had no detectable Fc receptors as tested by the rosette assay (Table 5). Third, trypsin treatment greatly reduced the cytotoxic activity of the effecters against an NK-sensitive target YAC (Table 6). In addition, the cytotoxic activity was shown not to be lectin-dependent (Table 3). Despite the fact that IL2-CEI cells possessedextremely high levels of cytotoxic activity in vitro, they failed to provide any antitumor (FBL-3) activity in vivo (Table 7). These results suggested several possible explanations. First, not all cytotoxic lymphocytes possessin vivo transplantation-type immunity. Second, cytotoxic lymphocytes may act as “suppressor cells” to inhibit host immunity. Third, ILZ-CEI cells are of different populations of NK effecters, each demonstrating different or even opposite types of in vivo effects, and they may cancel out each other’s activity.

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Although the ILZCE 1 cells were consistent with being NK cells, there were other features which were not of typical NK nature. Cytotoxic activity was also seen with NK-resistant targets, EL-4 and L5 178 cells. The highest level of cytotoxicity was seen with two syngeneic tumors of FMR origin (FBL-3 and RBL-3). The FBL-3 cells were relatively resistant to NK cytotoxicity. The reactivity against FBL-3 cells was also not affected by trypsin treatment (Table 6). One explanation for these differences is that IL2-CEl cells could be a T-cell line with anti-MuLV (murine leukemic virus) activity. However, this possibility was ruled out by the resistance of ILZCEl cells to the lytic activity of anti-Thy-l .2 antibody (Table 4) and the susceptibility of some MuLV negative lines (EL-4 and L5 178) to their lysis. These cells could be classified as NIST cells as proposed by Minato et al. (9). An alternative explanation is that there might be more than one population of effecters in the IL2CE 1 cells. The heterogeneity of NK cells has been demonstrated in fresh spleen cells (9). This phenomenon was also present in the in vitro-cultured NK lines. Those established by Dennert (10) were quite different from Nabel’s (11). Therefore it was not surprising to seethe difference between our 112-CEl cells and conventional NK cells. The IL2-CE 1 cells differed in their cytotoxic specificity and trypsin sensitivity. The results obtained from cell cloning experiments will further verify this point (Table 8, Fig. 3). Four clones were obtained from IL2-CE 1 cells and their reactivity and specificity were thoroughly examined. One clone (No. 1) gave specific cytotoxicity against FBL3 cells (Table 8) and the reactivity was only inhibited by FBL-3 cells (Fig. 3). The other three clones gave much higher levels of cytotoxicity against NK-sensitive targets (YAC and RLa 1) than against NK-resistant targets (P8 15), indicating that they were NK cells. However, there was a fine difference in their reactivity. Clone 3 gave a slightly higher level of cytotoxicity against EL-4 than that obtained with Clone 2 (Table 8), and cold EL-4 cells were also more inhibitory to the reactivity of Clone 3 cells than Clone 2 cells. When comparing the inhibitory activity of various tumor cells or thymocytes, differences were also seen in the same clone when tested against different targets. These findings suggest that there might be different populations of NK cells in existence. The fine difference in specificity could only be demonstrated clearly by the inhibition experiment. These results differed from that of Dennert. The different NK clones of Dennert obtained from nude mice appeared to give the same pattern of reactivity (10). However, the specificity of his NK-cell reactivity had not been verified by the inhibition experiment. It was also possible that normal nude mice might give a more homogeneous population of NK cells. In our case, the source of the NK cells was from a conventional mouse which had been immunized with a syngeneic tumor (FBL-3) and thus it might have a more heterogeneous population of NK cells. In the development of IL2-CEl cell line, it was possible that the initial growth in the first 2-4 weeks was predominantly specific cytotoxic T cells (against FBL-3). After prolonged culturing, however, the NK cells were polyclonally expanded to give rise to various clones and these NK clones eventually outnumbered the cytotoxic T-cell clone. It should also be noted that our NK-cell clones were derived from B6 mice which were “low responders” for NK activity. After these cells were grown in established cultures, their cytotoxic activity become extremely high (Table 1). This finding suggested that there might be no difference in the cytotoxic activity of individual NK cell, regardless whether these cells were from high responders or low responders.

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The present study shows that NK cells can be maintained in continuous culture with IL2 supplementation. However, cloning of these cells proved to be a very difficult task in our hands. In our study, the heterogeneity of the NK cells are clearly presented. In view of this fact, there is a need to establish various NK clones from different sources to allow more meaningful and comparative studies. This approach should help us to understand the diverse nature and function of NK cells. In the tumor neutralization test our ILZ-CEl cells failed to give any protective effect in the effector phase of in vivo tumor immunity. However, this does not rule out other roles which the NK cells may play in the in viva tumor immunity. On the contrary, it stressesthe need to study more thoroughly the biological functions of various populations of NK cells. Development of in vivo transplantation immunity involves a series of complex reactions, therefore it is necessary to exhaustively test various NK cells in different phases of immune response. Only through these studies shall we be able to understand their role in immunoprevention or immunosurveillance. REFERENCES 1. Gillis, S., Ferni, M., Ou, W., and Smith, K. A., J. Immunol. 120, 2027, 1978. 2. Shaw, J., Caplan, B., Paetkau, V., Pilarski, L., Delovitch, T. L., and MacKenzie, 1. F. C., J Immunol. 124, 2232, 1980.

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