Interleukin-2 corrects defective NK activity of patients with leukemia

Comp. Immun. Microbiol. infect. Dis. Vol. 9, No. 2/3, pp. 169-175, 1986 Printed in Great Britain. All rights reserved

0147-9571/86 $3.00+0.00 Copyright 5?) 1986 Pergamon Journals Ltd

INTERLEUKIN-2 CORRECTS DEFECTIVE NK ACTIVITY OF PATIENTS WITH LEUKEMIA EVA LOTZOVA a n d C. A. SAVARY Section of Immunogenetics, Department of General Surgery, University of Texas System Cancer Center, M. D. Anderson Hospital and Tumor Institute, 6723 Bertner Avenue, Houston, TX 77030, U.S.A. Abstraet--NK cells of patients with leukemia display low cytotoxic potential. Since the NK cells have been suggested to play a role in natural resistance to leukemia, we considered it of importance to investigate the approaches leading to the correction of NK defect of leukemic patients. Our studies demonstrate that culture of effector cells with interleukin-2 (IL-2) resulted in restoration of cytotoxic defect. This was indicated by normalization of tumor-binding as well as lytic NK activity, by normal frequency of cytotoxic cells and their ability to recycle. The NK cell nature of cytotoxic cells was shown by abrogation or depletion of cytotoxicity by antibody directed against NK cell-associated, but not T cell-associated antigen. The generation of NK cell activity against fresh leukemic cells suggests that adoptive transfer of IL-2 activated NK cells may be a new approach to leukemic treatment. Key words: Natural immunity, NK cells, leukemia, interleukin-2, large granular lymphocytes, tumor immunity

L'INTERLEUKINE-2 CORRIGE DE PATIENTS

L'ACTIVITE NK DEFECTUEUSE LEUCEMIQUES

R6sum~-Les cellules NK de patients leuc6miques pr6sentent un potentiel cytotoxique r6duit. Puisque les cellules NK sont pr6sum6es avoir un r61e dans la r6sistance naturelle contre la leuc6mie, nous avons jug6 important d'explorer les moyens de corriger le d6ficit NK des patients leuc6miques. Nos essais montrent que la culture des cellules effectrices en pr6sence d'Interleukine-2 (IL-2) a conduit ~iune restauration du d6ficit cytotoxique. Ceci a 6t6 attest6 par la normalisation de l'activit6 NK de liaison 5. la tumeur et de lyse, par des cellules cytotoxiques en proportions normales et capables de recirculer. La nature cellulaire NK des cellules cytotoxiques a 6t6 montr6e par l'annulation ou la diminution de la cytotoxicit6 par des anticorps dirig6s contre des antig6nes associ6s aux cellules NK mais non aux cellules T. La production d'une activit6 cellulaire NK contre des cellules leuc6miques frai'ches sugg6re que le transfert adoptif de cellules NK activ6es par I'lL-2 pourra~t &re une nouvelle approche du traitement des leuc6mies. Mots-clefs: Immunit6 naturelle, cellules NK, leuc6mie, interleukine-2, grands lymphocytes granuleux, immunit6 antitumorale

INTRODUCTION N a t u r a l killer ( N K ) cells m a y r e p r e s e n t an i m p o r t a n t c o m p o n e n t o f n a t u r a l i m m u n i t y a g a i n s t c a n c e r a n d m i c r o b i a l i n f e c t i o n s [1-3]. C o n s e q u e n t l y , the defect in N K cell a c t i v i t y m a y p r e d i s p o s e to the d e v e l o p m e n t o f c a n c e r a n d i n f e c t i o u s diseases. I n d e e d , this p o s s i b i l i t y is s u p p o r t e d by h i g h i n c i d e n c e o f p r i m a r y a n d s e c o n d a r y t u m o r s in i n d i v i d u a l s d i s p l a y i n g d e f e c t i v e N K cell a c t i v i t y [for r e v i e w see 1]. M a l i g n a n c i e s o f h e m o p o i e t i c o r i g i n h a v e b e e n s h o w n to be p a r t i c u l a r l y sensitive to N K cells. T h i s o b s e r v a t i o n is c o m p a t i b l e w i t h l o w N K a c t i v i t y in p a t i e n t s w i t h l e u k e m i a a n d 169

170

EVA LOTZOVX and C. A. SAVARY

preleukemic disorders [1, 4-6], and susceptibility of patients with nonmalignant diseases and low NK activity, to leukemia [1]. Further support comes from experimental animal studies which showed that resistance to leukemia in leukemia-susceptible animals could be transferred by in vitro cloned NK cells [1]. Since natural immunity appears to be important in resistance to leukemia, we investigated the possibilities leading to the induction of NK activity of leukemic patients who are severely NK-compromized.

MATERIALS AND METHODS Peripheral blood of normal donors and untreated leukemic patients was obtained by venipuncture or following leukophoresis. Mononuclear cell preparation was obtained by Ficoll-Hypaque centrifugation [4]; in some experiments, the effector cells were further separated by passage through nylon wool (NW) columns. Large granular lymphocytes (LGL) were obtained by fractionation on Percoll gradient, followed by removal of high affinity sheep erythrocyte rosette-forming cells [6]. The K-562 cell line, obtained from a patient with chronic myelogenous leukemia (CML) in blast crisis was maintained as a continuous culture in supplemented RPMI 1640 medium [6]. In some studies, the leukemic cells used as targets were cryopreserved [7]; the viability of these cells ranged from 80-92%. NK cell cytotoxicity was measured in [5~Cr] release assay as described in detail previously [6]. Briefly, 50/11 of target cells and 100/d of effector cells were plated in quadruplicate in microtiter plates. The target-to-effector (T:E) ratio ranged from 1:12 to 1:100 (see Results). The plates were incubated for 3 h at 37°C in 5% CO2 humidified atmosphere, and released radioactivity was measured in a gamma scintillation counter. The percentage of cytotoxicity was calculated as described earlier [6]. Spontaneous release (determined by incubating leukemic cells alone) ranged from 7-9% and 17-26%, for K-562 and fresh leukemia, respectively. Tumor binding cells (TBC) and cytotoxic-TBC (C-TBC) were evaluated in a single cell assay, and the morphology of TBC in a slide conjugate assay [6]. For preparation of cytotoxic factor, NW-filtered effector cells were cultured with unlabelled K-562 (T:E, 1:50) for 24h at 37°C in a 5% CO2 humidified atmosphere. The supernatant was removed, filtered and stored at -20°C. Cytotoxic activity was tested in a microsupernatant assay

[8]. Effector cells were depleted of Leu-1 or Leu-ll-positive cells, using monoclonal antibodies and rabbit complement (RC) in a two-step complement dependent lysis technique [6]. The antibody treatments were repeated twice, to ensure removal of all positive cells (based on immunofluorescence). Active cytotoxic cells were prepared by culturing the effector cells at the concentration of 106/ml in RPMI 1640, supplemented with 10% human AB serum, antibiotics, HEPES buffer, and 10% human IL-2 conditioned medium (Cellular Products, Buffalo, N.Y.), for 3-7 days at 37°C in a 5% CO, humidified atmosphere. 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 maximum cell density was reached. The data were analyzed statistically using a Student's or paired t-test.

Correction of NK defect with IL-2

171

30i

Z 26-

.-r

22u~ +1 Z18I

TBC

C-TBC

Vmax

[~

E~l

Ix 103]

MRC

NKCF

['% ''isl

Fig. 1. Defect in cytotoxic mechanism of peripheral blood NK cells of leukemic patients. Bars represent mean + SE of 5-21 patients (1~) and 5-20 normal individuals (I-q). TBC, tumor binding cells; C-TBC, cytotoxic-TBC; Vmax,maximum rate of lysis; MRC, maximal recycling capacity; NKCF refers to lytic activity of NK cytotoxic factor. All parameters were significantly decreased in leukemic patients (P values ranged from <0.001 to <0.05).

RESULTS We have shown earlier that patients with various types of leukemia and preleukemia display low NK cytotoxicity [1, 4-6]. In these studies, we demonstrate that low NK activity of leukemic patients is due to several defects in NK cell cytotoxic mechanism (Fig. 1). This is illustrated by low NK capacity to recognize and bind to the tumor cells, low kinetics I

AML

I

CML

I

P5

~. 8 0 I p-70 ll

P3

X

0 0 ~. 0

1

60" 501 401

Z

P4

301 Z

UJ

20 I

.~" I 101

012

023

0123

0123

023

0234

WEEKS IN CULTURE

Fig. 2. IL-2 dependent generation of peripheral blood NK cytotoxicity in leukemic patients. Ficoll-Hypaqued mononuclear cells (patients 1, 3 and 5) or LGL (patients 2, 4 and 6) were tested against K-562 (1:12 T:E ratio); fresh (1), cultured ([]). CML, chronic myelogenous leukemia; AML, acute myelogenous leukemia.

172

EVA LOTZOVAand C. A. SAVARY AML

30-

CML

ALL

mO25" Iu. 2 0 O ~_ 1 5 z

t~ 1 0 -

0 70 60 ~ 50

du. 40

O ~ 30 -

0

1

2

3

4

5

6

7

8

PATIENT NUMBER

Fig. 3. Correction of the cytotoxic defects after culture of peripheral blood of leukemic patients

with IL-2. NW-filtered effector cells (patient I) or LGL (patients 2 8) were tested before ([-q) and after ([]) culture, using a single cell assay. ALL, acute lymphocytic leukemia. There was a significant increase in all parameters following culture of effector cells with IL-2 (P ranged from <0.001 to <0.05). o f lysis (Vm~x), low lytic potential, virtually no recycling capacity, and finally, the lack o f ability to p r o d u c e N K cell cytotoxic factor ( N K C F ) . The latter factor has been implicated in the mechanism o f lysis mediated by N K cells [8].

30

ANt.'4~3

°2 I

1

i

L ~

2

I

NORMAL DONORS

Fig. 4. Lysis of fresh leukemia cells by cultured peripheral blood of normal donors. NW-filtered effector cells of 2 normal donors were tested for cytotoxicity against fresh leukemic cells before (IS]) and 34i days after culture with IL-2 (Ira). Peripheral blood (PB) and bone marrow (BM) of leukemic patients were used as targets in a 3 h [~Cr] release assay (T:E l : 100). Signifcant increase in cytotoxicity was observed after culture of effector cells with IL-2 (P < 0.001).

Correction o f N K defect with I L - 2

173

Table 1. Rate of lysis and recycling capacity of fresh and cultured peripheral blood lymphocytes of leukemic patients Mean + SE a Parameter tested Vm,x ( X 103) MRC

Prior culture

Post cultureb

1.9 + 1.2 1.2 + 0.5

30.0 + 1.7 4.0 _ 0.1

aValues represent mean + SE of 3 patients (1 A M L , I C M L and 1 preleukemia), bCells were cultured 3-5 weeks with IL-2. Significant increase in Vmax and MRC was observed after culture of effector cells with IL-2.

In an attempt to potentiate NK antileukemia reactivity, we tested the effect of IL-2 on NK function. As illustrated in Fig. 2, defective NK cytotoxicity of 6 representative patients was corrected after culture of effector cells with IL-2. Differences, however, were observed in the time of generation of cytotoxic activity; specifically, lytic activity was acquired in some patients early (within 1-2 weeks), whereas in other patients 3-4 weeks were necessary for generation of lytic potential. Analysis of various steps of the mechanism of action of IL-2 generated cytotoxic cells demonstrated that all facets of cytotoxic mechanism were restored (Fig. 3, Table 1). It is important to note that IL-2 activated killer cells acquired an ability to lyse both cultured and fresh leukemic cells (Fig. 4). Characterization of killer cells indicated that they were of LGL morphology (Table 2), and expressed NK cell-associated antigen CD16, and not T cell-associated antigen CD5. The latter statement is based on the observation that Leu-1 lb, but not Leu-1 antibody (recognizing CD16 and CD5 antigen, respectively) abolished or significantly decreased cytotoxic function of IL-2 activated killer cells (Table 3). These data indicate that not all IL-2 activated killer cells are T cells, as has been indicated previously [9]. Table 2. Correlation between increase in cytotoxicity and percent of LGL in peripheral blood cultures of leukemic patients Patient no? I AML 2 AML 3 AML 4 CML 5 Preleukemic

Percent Weeks in cultureb LGL Cytotoxicity 0 2 0 2 0 2 0 7 0 4

39.0 84.0 0.5 58.0 1.5 33.0 17.0 82.0 41.0 93.0

3.9 28.6 - 1.4 45.3 0.5 64.9 1.7 36.3 13.2 59.9

~FicolI-Hypaqued mononuclear cells (patient 2 and 3) or LGL (patients 1, 4 and 5) were tested for LGL content and NK cytotoxicity to K-562 (1:12 T : E ratio), bPercent of L G L and cytotoxicity was significantly increased following culture of effector cells with IL-2 (P < 0.001). C.I M I.D 9/2-3--F

174

EVA LOTZOVAand C. A. SAVARY Table 3. Effect o f monoclonal antibodies on lytic activity of IL-2 generated effector cells Percent o f cytotoxicity c Donor A

2

Effector cells Fresh cultured

Fresh cultured

Treatment S --RC Leu-I and L e u - l l and --RC Leu-I and L e u - l l and

RC RC

RC RC

K-562

ALL-BM

AML-BM

19.0 35.4 36.1 47.9 13.0 26.3 51.0 52.2 44.5 10.7

4.5 29.7 20.6 nt b 10.2 0.4 13.7 8.2 nt 4.3

-0.8 14.0 11.2 nt 7.9 1.3 10.1 6.9 nt 3.5

aGL were tested before and 14 days after culture with IL-2 against K-562 (1:12 T : E ratio); NW-filtered cells were tested before and 3-7 days after culture against fresh leukemic blasts (1:100 T : E ratio), bnt = not tested. ¢Significant decrease in cytotoxicity was observed after treatment with Leu-11 antibody and R C ( P <0.001 to <0.005).

DISCUSSION We have shown that patients with various types of leukemia displayed low N K cytotoxicity even against K-562 cell line, which is highly sensitive to killing by N K cells. Even though in some patients inferior N K cytotoxicity could be attributed to low numbers of N K cells in the tested sample [6], in others the low cytotoxic profile was caused by the defect in N K cell lytic mechanism. This was shown by using population of L G L enriched on Percoll density gradient. These studies showed that N K cells of leukemic patients displayed defective tumor-binding and tumor-lytic activity and low rate of lysis. Furthermore, whereas the N K cell of normal donor recycles several times (kills more than one tumor), N K cells of leukemic patients do not possess recycling activity. Importantly though, the cytotoxic defect of N K cells was correctable after culture of effector cells with IL-2. The cultured effector cells, that acquired cytotoxic potential, demonstrated increased tumor-binding and lytic activity, more rapid rate of lysis, and recycling ability. The N K cell nature of effector cells was indicated by significant decrease and/or total abrogation of cytotoxic activity after treatment of these cells with antibody directed against NK-associated antigen. On the contrary, the antibody directed against T cells had no effect on cytotoxic function. This observation corrects the previously presented contention that all IL-2 activated killers, designated LAK, are T cells [9]. In summary, we have shown that defective N K activity of leukemic patients can be repaired in culture of effector cells with IL-2. Since such treated effector cells also acquire lytic activity against fresh leukemic cells, it is reasonable to suggest that treatment of leukemic patients with IL-2 or adoptive trasnfer of in vitro IL-2 activated autologous N K cells, may represent a new approach to the treatment of leukemia. Acknowledgements--The authors wish to acknowledge the Department of Hematology for blood samples of

leukemicpatients and leukopheresedblood of normal donors. This work was supported by Grant CA 39632from National Cancer Institute. REFERENCES 1. Lotzov~iE. The role of natural killer cells in immune surveillanceagainst malignancies. Cancer Bull. 36, 215-226 (1984).

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2. Lotzov~ E. Natural immunity and biological response: Introduction. Nat. lmmun. Cell Growth Reguln 4, 233-234 (1985). 3. Herberman R. B. and Ortaldo J. R. Natural killer cells: their role in defense against disease. Science 214, 24-30 (1981). 4. Lotzovfi E., Savary C. A. and Keating M. J. Leukemia diseased patients exhibit multiple defects in natural killer cell lytic machinery. Exp. Hemat. 10, 83-95 (1983). 5. Lotzovfi E., Savary C. A., Keating M. J. and Hester J. P. Defective NK cell mechanism in patients with leukemia. In Mechanism of Cytotoxicity o f N K cells (Edited by Herberman R. B.). Academic Press, New York. pp. 507-519. (1985). 6. Lotzovfi E., Savary C. A. and Herberman R. B. Impaired NK cell profile in leukemia-diseased patients. In Immunobiology of Natural Killer Cells (Edited by Lotzovfi E.). Herberman R. B. (Assoc. ed.), CRC Press. 1986. In press. 7. Pross H. F. and Maroun J. A. The standardization of NK cell assays for use in studies of biological response modifiers. J. Immun. Meth. 68, 235-249 (1984). 8. Wright S. G., Weitzen M. I., Kahle R., Granger G. A. and Bonavida B. Studies on the mechanism of natural killer cytotoxicity. II. Coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells. J. lmmun. 130, 2479-2483 (1983). 9. Grimm E. A., Mazumder A., Zhang H. Z. and Rosenberg S. A. Lymphokine-activated killer cell phenomenon. Lysis of natural killer-resistant fresh solid tumor cells by interleukin 2-activated autologous human peripheral blood lymphocytes. J. exp. Med. 155, 1823 1841 (1982).