Natural immunity against ovarian tumors

Comp. Immun. Microbiol. infect. Dis. Vol. 9, No. 2/3, pp. 269-275, 1986 Printed in Great Britain. All rights reserved

0147-9571/86 $3.00+ 0.00 Copyright © 1986 Pergamon Journals Ltd

N A T U R A L I M M U N I T Y AGAINST OVARIAN TUMORS EVA LOTZOVA~, C. A. SAVARYl, R. FREEDMAN2 a n d J. M. BOWEN3 ~Section of Immunogenetics, Department of General Surgery, 2Department of Gynecology, 3Department of Tumor Biology, The University of Texas System Cancer Center, M.D. Anderson Hospital and Tumor Institute, 6723 Bertner--Box 18, Houston, TX 77030, U.S.A. Abstract--We have analysed natural killer (NK) cytotoxic activity in peripheral blood and ascitic fluids of patients with advanced stage of ovarian epithelial carcinoma. All patients displayed low NK activity in peripheral blood and virtually no cytotoxicity in ascitic fluids. NK activity in ascitic fluids could be substantially augmented after regional administration of virus-modifiedtumor cell extracts (VMTE), and that in peripheral blood after culture of effector cells with interleukin-2 (IL-2) in vitro. Activated NK cells displayed cytotoxic activity against NK-sensitive and NKresistant tumor cell lines as well as against fresh ovarian tumors. Parallelism was found between regional NK augmentation and regression of malignant ascites. The latter observation suggests possible NK cell role in defense against ovarian tumors. Key words: N K cells, ovarian cancer, interleukin-2, tumor immunity

IMMUNITE

NATURELLE

C O N T R E DES T U M E U R S

OVARIENNES

R6sum~-Nous avons analys6 l'activit6 cytotoxique "natural killer" (NK) dans le sang p6riph6rique et le liquide d'ascite de patients avec un stade avanc6 de carcinome 6pith61ial ovarien. Tousles patients ont montr6 une faible activit6 NK dans le sang p6riph6rique et pratiquement aucune cytotoxicit6 dans le liquide d'ascite. L'Activit6 NK dans le liquide d'ascite a pu 6tre r6ellement augmentbe apr6s administration locale d'extraits de cellules tumorales modifl6es par un virus (VMTE), de m6me que celle du sang p6riph6rique apr6s culture de cellules effectrices avec de l'interleukine-2 (IL-2) in vitro. Les cellules NK activ6es ont montr6 une activit6 cytotoxique contre des lign6es de cellules tumorales sensibles ou r6sistantes aux cellules NK ainsi que contre des tumeurs ovariennes fraiches. Une corr61ation a 6t6 trouv6e entre l'augmentation de l'activit6 NK locale et la r6gressiondes ascites malignes. Cette derni6re observation sugg6re un r61e possible des cellules NK dans la d6fense contre les tumeurs ovariennes. Mots-clefs: Cellules NK, cancer ovarien, interleukine-2, immunit6 antitumorale

INTRODUCTION It has become quite o b v i o u s in recent years that the i m m u n e system plays a n i m p o r t a n t role in surveillance against malignancies, a n d that its failure m a y be one o f the m e c h a n i s m s by which cancer originates a n d disseminates. I n fact, patients with malignancies a n d p r e m a l i g n a n t diseases have been s h o w n to exhibit i m p a i r m e n t of cell mediated i m m u n i t y [1]. We have been interested in the i m m u n o l o g i c profile of patients with o v a r i a n epithelial c a r c i n o m a , a n d showed previously that this g r o u p o f patients experience low n a t u r a l killer ( N K ) cell-mediated cytotoxicity [2]. Since N K cells appear to represent a n i m p o r t a n t a r m of defense against p r i m a r y a n d metastatic t u m o r s [3], we investigated the possibilities leading to a u g m e n t a t i o n o f N K activity o f o v a r i a n cancer patients. 269

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MATERIALS AND METHODS Patients with advanced ovarian epithelial carcinoma were tested in these studies. Patients receiving virus-modified allogeneic tumor cell extract (VMTE) therapy had malignant ascites with or without palpable abdominal pelvic masses, and had received previous conventional therapy (primarily cis-platinum) that had failed [4]. VMTE was administered i.p. in the dose and schedule indicated in the legends of the tables. All patients were treatment-free for at least 1 month before the NK cell studies. Heparinized venous blood from normal donors and cancer patients was obtained by venipuncture; ascitic fluids (AF) of ovarian cancer patients were collected by paracentesis. Mononuclear cells from peripheral blood (PB) and AF were prepared by centrifugation on Ficoll-Hypaque density gradients [5]. Mononuclear cells were in some patients further separated from AF by centrifugation on two-step Ficoll-Hypaque gradients [6]. In some experiments, normal donor PB cells were further separated on nylon wool (NW) columns and fractionated on Percoll density gradients as previously described [5, 7]. Large granular lymphocytes (LGL)-enriched fractions were depleted of T cells by high-affinity rosettes forming technique [5, 7]. Cultures of PB mononuclear cells were performed by culturing 106/ml, with 103 u/ml of recombinant interleukin-2 (IL-2) (Biogen, Boston, MA), for 3-7 days at 37°C in a 5% CO2 humidified atmosphere, in RPMI 1640 medium supplemented with 10% human AB serum, antibiotics and HEPES buffer. For long-term cultures, the medium was further supplemented with 2 mM sodium pyruvate, 0.1 mM nonessential amino acids and 0.2 mM 2-mercaptoethanol. Cultures were supplemented with fresh medium every 3 days, and subcultured when the cell density reached > 106/ml. Effector cells were depleted of cells expressing CD5 or CD16 antigens in a two-step complement-dependent lysis technique [5, 7], using the monoclonal antibodies Leu-1 and Leu-1 l b, respectively, and rabbit complement (RC). The antibody and RC treatments were repeated twice, to ensure removal of all antigen-positive cells (based on immunofluorescence). The NK sensitive K-562 cell line was derived from a patient with chronic myelogenous leukemia in blast crisis, and was maintained as a continuous culture in S-RPMI. OV-2774, an ovarian carcinoma cell line, originating from ovarian ascites of patient with ovarian carcinoma [8] was maintained as a continuous culture in Leibovitz's L-15 medium supplemented with 10% fetal calf serum and 40 pg pg/ml gentamycin. When fresh ovarian solid tumors were used as targets, these were teased apart and minced in Hanks' balanced salt solution (HBSS) containing the following enzymes: 5 U/ml hyaluronidase, 2 mg/ml collagenase and 0.3 mg/ml deoxyribonuclease. The tissue was then exposed for 10 min to 0.25% trypsin in phosphate-buffered saline. The cells were collected into human AB serum, washed, and cryopreserved at -70°C in 90% human AB serum and 10% dimethyl sulfoxide. This method was found to result in recovery of tumor cells with 80% viability. NK cytotoxicity was measured in a [5~Cr]release assay as described previously [5,7]. The percentage of cytotoxicity was calculated by the following formula; CPM experimental- CPM spontaneous x 100 Total CPM incorporated into the cells Spontaneous [5~Cr]release, determined by incubating the target cells in medium alone, ranged from 4 to 8% for tissue culture cell lines and 8 36% for fresh tumor cells.

N K Activity in ovarian cancer patients

271

Table 1. N K lytic activity of normal donors and ovarian cancer patients against K-562 Lytic units20/lO 7 effector cellsa

Normal donors Cancer patients

Peripheral blood

Ascites

88.0 + 15.6(20) 29.4 + 7.9 (17)

-< 1 (9)

aNK activity was measured in a 3 h 5aCr-release assay. Values represent mean + SE; the number of individuals tested is shown in parentheses. Lytic units were calculated by linear regression analysis using 1: 50, 1 : 25 and 1 : 12 T: E ratios. N K activity of cancer patients was significantly decreased (P < 0.001).

In some experiments the data were expressed in lytic units (LU), where one LU represents the number of effector cells required for lysis of 20% of target cells. LU were determined from log-linear plots of the data points (obtained using 3 different T: E ratios), and are reported as LU20/107 effector cells. Tumor-binding cells (TBC) and cytotoxic-TBC (C-TBC) were determined in a single cell assay [5, 7]. A slide conjugate assay was used to determine the morphology of TBC [5, 7].

Statistical analysis The data were analyzed statistically using a Student's t-test. RESULTS

Potentiation of NK activity by VMTE As indicated in Table l, PB NK cell cytotoxicity against K-562 is low in patients with ovarian carcinoma. Furthermore, no cytotoxicity is displayed by patients' AF mononuclear cells. Based on these observations and in the light of possible NK cell importance in cancer defense, we investigated the effect of VMTE and IL-2 on NK activity of these patients. First, we tested the effect of regionally (intraperitoneally, i.p.) administered VMTE on N K cell lytic potential. PB and AF mononuclear cells of cancer patients were tested before and post VMTE treatment. NK activity in ascitic fluids was of most interest, since this anatomical location coincides with the location of the tumor. Table 2. Effect of VMTE on NK activity of ovarian cancer patients Lytic units20/107 effector cells Patient no.

Effector cells a

Prior treatment

Post treatment

1

AF PB AF PB AF PB AF PB AF AF PB

1.7 30.3 < 1 4.6 < 1 20.4 < 1 16.9 < I < 1 1.9

62.5 (2) b 40.0 (2) 126.0 (2) 10.3 (2) 139.1 (2) 62.5 (2) 200.2 (6) 47.6 (6) 58.8 (3) 40.0 (4) 3.4 (2)

2 3 4 5 6 7

aFicoll-Hypaqued ascitic fluid (AF) and peripheral blood (PB) effector cells were tested for cytotoxivity to K-562 in a 3 h [5~Cr]release assay. Cytotoxicity was evaluated prior to and 24-48 b after injection of 4.5-9 mg of VMTE, with the exception of patient 3, who was tested 3 weeks after injection. ~ Values in parentheses indicate number of V M T E injections. C.I,M.ID. 9/2-3--L

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Table 3. Generation of cytotoxicity in peripheral blood cultures of ovarian cancer patients

Table 4. Lysis of solid ovarian tumors by cultured peripheral blood effector cells

Percent of cytotoxicity~ Donor no. 1 2 3 4 5

Target

Prior culture

Post cultureb

K-562 K-562 OV-2774 K-562 OV-2774 K-562 OV-2774 K-562 OV-2774

9.5 4.2 0.2 7.2 0.7 21.9 1. I 18.6 0.3

26.0 44.9 29.4 78.0 69.4 81.3 88.1 80.9 82.3

Percent of cytotoxicity Target no. 1 2 3

3 4

~Ficoll-Hypaque separated effector cells were tested for cytotoxicity in a 3 h [5~Cr]release assay; T : E ratio was 1 : 12 (patient 1), I : 25 (patients 2-4) and 1: 50 (patient 5). bEffector cells were cultured for 3-11 days with IL-2.

Effector cellsa Patients 1 2¢ 3 Normal donor I I

Prior culture

Post culture b

5.2 0.3 2.0

16.8 19.5 16.1

0.4 0.5

14.3 14.2

aFicoll-Hypaque separated effector cells were tested against fresh ovarian tumor cells in a 3 h [51Cr]release assay. T : E ratio was 1:12 (patient 1), 1:50 (patient 2) and 1:100 (ptient 3 and normal donor 1). bCells were cultured for 6 days (patients 1 and 2), or 15 days (patient 3 and normal donor 1) with IL-2. CAutologous tumor.

As shown in Table 2, no NK cytotoxicity was manifested by AF mononuclear cells of 7 ovarian cancer patients prior VMTE treatment. However, high levels of cytotoxicity were induced after several injections of this biological response modifier; the NK augmentation ranged from 37 to 200-fold. It is important to note that such NK potentiation was of long duration, since the NK cytotoxicity remained elevated for 7-21 days after the treatment [2]. NK cell cytotoxicity was also potentiated, even though to a significantly lower degree, in PB of some, but not all, patients (Table 2). Since VMTE did not reproducibly augment PB NK cytotoxicity of ovarian cancer patients, we tested the stimulatory effect of IL-2 on NK activity in the latter tissue. Table 3 shows that IL-2 not only augmented NK activity against K-562 target, which is highly sensitive to NK lysis, but also induced lytic activity against NK resistant ovarian carcinoma cell line, OV-2774. Importantly, PB cytotoxic activity induced by IL-2 was displayed also against fresh solid ovarian tumors of both autologous and allogeneic origin (Table 4), suggesting that IL-2 activated N K cells have an ability to kill solid ovarian tumours.

Analysis of cytotoxic cells against ovarian tumours To characterize the effector cell population mediating cytotoxicity against ovarian tumors, we separated PB lymphocytes on Percoll density gradient into LGL-enriched and

Table 5. Cytotoxicity against malignant ascites is mediated by LGL Percent of eytotoxicity Effector cellsa

Ib

2

3

4

NW-filtered LGL-enriched LGL-depleted

8.1 43.2 1.2

2.3 24.0 4.6

-1.7 50.0 5.9

16.3 58.2 2.1

aLGL-enriched (59-81% L G L ) a n d L G L - d e p l e t e d ( < 2 % LGL) effector cells were obtained by separation of lymphocytes on Percoll density gradients, and tested for cytotoxicity to OV-2774 in a 3 h (test 1 and 2) or 14h (test 3 and 4) [SICr]release assay, bTest number.

Table 6. Evidence for N K cell involvement in cytotoxic reactivity against malignant ascites Percent of cytotoxicity Treatment ~

Donor 1

Donor 2

Donor 3

Untreated RC Leu-I and RC Leu-ll and RC

43.2 57.5 nt b 2.8

59.8 40.8 49.0 17.0

24.3 16.2 nt 8.0

aLGL of 2 normal donors (1 and 2) and Ficoll-Hypaqued PB of VMTE-injected ovarian cancer patient (donor 3) were treated with rabbit complement (RC), Leu-1 and RC, or Leu-ll and RC before cytotoxicity assay against OV-2774 in a 14h [~lCr]release assay, bNot tested.

NK Activity in ovarian cancer patients )~-

70-

X

60-

~

50-

0

40"

273

A i:i:

Z

u. 0

30"

iiii:

20"

~ 100

L--DONOR l--J

6o-~

t--DONOR 2.-J

L-DONOR 3,-I

B

50~

z

40-

301

:i:! :~:~:~:~

2o-J ~ 100

L-DONOR 1.~t

~DONOR 2 -J I--DONOR 3 "--I

Fig. I. Effect of Leu-I and Leu-I 1 antibodies on cytotoxic activity of IL-2 activated peripheral blood cells. NW-filtered effector cells of 3 normal donors were tested fresh (I-q) and 3-5 (donors 2 and 3) or 20 days (donor 1) after culture with IL-2 (D) for cytotoxicity in a 3 h [SICr] release assay. Panel A: NK activity to K-562 (T:E ratios were 1:12). Panel B: NK activity to OV-2774 (T: E ratios were 1 : 12 for donor 1, and 1:25 for donors 2 and 3). Significant decrease was observed following Leu-ll antibody treatment (P values <0.001 to <0.02). L G L depleted fractions, and tested both fractions for N K cytotoxicity. PB o f normal donors (instead o f patients) was used in these studies, to ensure fully functional N K cells. Table 5 s h o w s that LGL-enriched but not LGL-depleted fraction o f lymphocytes displayed cytotoxicity against OV-2774. In another set o f experiments we investigated the effect o f N K cell-associated antibody, Leu-1 lb, on activity o f LGL. Table 6 shows that PB cytotoxic activity was abolished by this antibody. Similarly, the lytic potential o f cytotoxic cells present in A F o f the patient treated with V M T E was significantly decreased after treatment with Leu-I lb antibody. These two observations indicate that N K cells with L G L m o r p h o l o g y display ability to kill ovarian tumors. W e also investigated the effect o f Leu-1 lb antibody on activity o f IL-2 generated PB cytotoxic cells against K-562 and OV-2774. The depletion o f cytotoxic activity with this antibody indicates the N K nature o f OV-2774-directed cytotoxic cells (Fig. 1). In contrast, treatment o f efiector cells with Leu-1 antibody directed against T cells, did not s h o w any effect on cytotoxic cells. These observations demonstrate that IL-2 activated or generated cytotoxic cells are not T cells.

Correlation between augmentation of NK activity and regression of malignant ascites One o f the m o s t clinically important questions which we asked was whether regional potentiation o f lytic activity was reflected by regression o f malignant ascites. To answer

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274

Table 7. Correlation between augmentation of NK cell cytotoxicity and regression of malignant ascites NK cytotoxicity against K-562(%)a Patient no. I

2 3 4 5

- VMTE 3.5 1.6 3.8 6.2 - 1.6

+ VMTE

Reduction of malignant ascites(%)b

65.1 69.5 55.2 43.3 34.0

100 87 67 75 58

~Ficoll-Hypaque separated effector cells from AF of ovarian cancer patients were tested before and 24h to 21 days after i.p. injection(s) of VMTE for NK activity in a 3h [5~Cr]release assay (1:50 T:E ratio). Patients had received 2 ~ injections of 6-9 mg of VMTE. bValues represent the percent reduction in malignant ascitic cells after VMTE treatment, as determined by cytological examination of AF samples.

this question, the AF were analyzed cytologically for the presence of tumor cells and correlated with NK activity. These results showed that reduction of malignant ascites paralleled NK augmentation after VMTE treatment (Table 7). Such reduction ranged from 58 to 100%. These data suggest that NK cells may be one of the components of the immune system playing a role in resistance to ovarian ascitic tumors. Furthermore, the observation that IL-2 activated NK cells mediated lysis of solid ovarian tumors indicates NK cells lytic activity also against solid ovarian tumors. Subsequently, NK cell regional augmentation may represent an effective approach to the destruction of malignant ovarian cells. Alternatively, adoptive transfer of IL-2 activated NK cells may represent another approach to the treatment of ovarian tumors. DISCUSSION We have shown that low NK activity in PB and AF of ovarian cancer patients can be augmented by IL-2 or VMTE, respectively. Morphologic and phenotypic analysis of NK cells showed that they were of LGL morphology and Leu-11 phenotype. Such activated NK cells mediated lytic activity against NK susceptible, as well as NK resistant tumor cell lines. Importantly, these cells also acquired ability to kill fresh ovarian tumors. This observation, together with the parallelism between NK potentiation in AF and regression of malignant ascites at this anatomical site, suggests possible NK cell role in destruction of malignant cells in vivo. Consequently, NK cells may represent an important counterpart of immunity against ovarian cancer. Acknowledgement--This work was supported by the grants CA 39632 and CA 31394 from National Cancer Institute. The expert secretarial assistance of Ann Childers in preparation of the manuscript is greatly appreciated.

REFERENCES 1. Herberman R. B. and Holden H. T. Natural cell-mediated immunity. Adv. Cancer Res. 27, 305-378 (1978). 2. Lotzov~i E., Savary C. A., Freedman R. S. and Bowen J. M. Natural killer cell cytotoxic potential of patients with ovarian carcinoma and its modulation with virus-modified tumor cell extract. 17, 124-129 (1984). 3. Lotzov~ E., Savary C. A., Freedman R. S., and Bowen J. M. N K cell antitumor activity of patients with ovarian carcinoma. Induction of cytotoxicity by viral oncolysates and interleukin-2. In Diagnosis and Treatment Strategy for Gynecological Cancer. UT Press, Austin (1986). In press.

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4. Freedman R. S., Bowen J. M., Lotzov~i E., Edwards C. L., Lewis E. and Katz R. Virus augmentation as a biological modifier approach: Experience with intracavitary virus augmentation therapy. In Diagnosis and Treatment Strategy for Gynecological Cancer. UT Press, Austin (1986). In press. 5. Lotzov~. E., Savary C. A., Keating M. J. and Hester J. P. Defective NK cell mechanism in patients with leukemia. In Mechanism for Cytotoxicity by N K Cells (Edited by Herberman R. B. and Callewaert D. M.), pp. 507-519. Academic Press, New York (1985). 6. Allavena P., Introna M., Mangioni C. and Mantovani A. Inhibition of natural killer activity by tumorassociated lymphoid cells from ascites ovarian carcinoma. J. natn. Cancer Inst. 67, 319 325 (1981). 7. Lotzov~ E., Savary C. A. and Herberman R. B. Antileukemia reactivity of endogenous and IL-2 activated NK cells. In N K Cells, Cancer and Other Diseases (Edited by Lotzov~ E. and Herberman R. B.). Karger AG, Basel (1986). In press. 8. Freedman R. S., Pihl E., Kusyk C., Gallager H. S. and Rutledge F. Characterization of an ovarian carcinoma cell line. Cancer 42, 2352-2359 (1978).