Immunology Letters, 32 (1992) 209 - 214

Elsevier IMLET 01782

Defect in generation of LAK cell activity under oxygen-limited conditions S h i g e a k i I s h i z a k a , M a k o t o K i m o t o a n d T a d a s u Tsujii Third Department of lnternal Medicine, Nara Medical University, Kashihara, Japan

(Received 21 November 1991; revision received 25 February 1992; accepted 28 February 1992)

I. Summary

2. Introduction

In general, the in vitro induction of lymphokine activated killer (LAK) cell activities by interleukin 2 (IL-2) in h u m a n peripheral blood mononuclear cells (PMNC) has been performed in an atmosphere of 5°7o CO 2 in air (20o70 O2), whereas IL-2induced LAK cell activities are considerably reduced under concentrations of 5 °7o O 2 equal to arterial blood oxygen tension (100 m m H g ) and 2°7o 0 2 equal to venous blood oxygen tension (40 m m H g ) . Cultured cell viability, IL-2 receptor-/3 expression on large granular lymphocytes (LGL), the percentage of IL-2 receptor-13 positive LGLs and cell proliferation were not affected by oxygen-limited conditions. LAK cells were induced by IL-2 over 5 days at 20°70 O 2, at which time the LAK cells were further stimulated by IL-2 in 2070 0 2 and 20070 O 2. Under these conditions the activity of LAK cells in 2°7o 0 2 decreased day by day, while that of L A K cells induced in 2007o 0 2 was maintained at least until day 10 of the original culture. L A K effector cell-mediated lysis was not influenced by oxygen-limited conditions. These results point to more successful applications of the combination of oxygen therapy and adoptive cellular immunotherapy in the clinic.

Most studies of adoptive immunotherapy have employed the use of LAK cells with broad antitumor activity. Adoptive immunotherapy using L A K cells and IL-2 was most effective in patients with renal cell carcinoma, melanoma or nonHodgkin's lymphomas [1, 2]. However, other malignant tumors did not show a marked response to the adoptive immunotherapy [1 - 5 ] . Although in vitro induction of LAK cell activities is routinely performed at atmospheric oxygen tension (20°70 O2), the activity of LAK cells induced by IL-2 in vivo was lower than that observed in vitro [6, 7]. One factor possibly accounting for the difference between LAK cell activity in vivo and in vitro is oxygen tension. We have investigated the effects of oxygen concentration (5o70 0 2 = arterial blood oxygen tension (100 m m H g ) and 20/o 0 2 = venous blood and tissue oxygen tension (40 m m H g ) ) [8] in vivo on the in vitro induction of LAK cell activities using an oxygen concentration regulatory incubator.

3. Materials and Methods 3.1.

Cell culture

Key words: LAK cell; Oxygen; IL-2; Human Correspondence to: Dr. Shigeaki Ishizaka, Third Department

of Internal Medicine, Nara Medical University, 840 Shijo-Cho, Kashihara, Nara 634, Japan. Abbreviations: PMNC, peripheral blood mononuclear cells;

IL-2, interleukin 2; LAK, lymphokine activated killer; LGL, large granular lymphocyte.

H u m a n P M N C from healthy adult donors were separated by Ficoll-Hypaque density centrifugation. The P M N C (106 cells/ml) suspended in 1 ml of R P M I 1640 medium (Nisui Pharmaceutical, Tokyo) containing 60 m g / l of kanamycin and 10% fetal bovine serum were cultured with 1000 U / m l of recombinant IL-2 (Takeda Chemical Industries,

0165-2478 / 92 / $ 5.00 © 1992 Elsevier Science Publishers B.V. All rights reserved

209

Osaka, Japan) in 24-well flat-bottomed plates (Corning, NY) at 20%, 5% and 2% oxygen concentrations using an oxygen regulation incubator (Tabai Espec, Osaka) for 3 days. Oxygen tensions in the culture medium were determined with a blood gas analyzer to be 150+5 m m H g , 100+5 m m H g and 4 0 + 2 m m H g , which correspond to oxygen concentrations of 20%, 5% and 2% in the incubator atmosphere, respectively.

antibodies (Mik-/31: Nichire Co., Tokyo) and fluorescein isothiocyanate (FITC)-conjugated antimouse IgG antibodies (Cappel, PA). After washing three times in phosphate-buffered saline (PBS), the cells were fixed with 1°7o formaldehyde in PBS at 4 °C. 104 washed cells were analyzed on a FACStar. The mean fluorescence intensity per cell was calculated by use of the formula Enc/Ec in which n is the channel number and c the number of cells counted in the channel.

3.2. Assay for L A K cell activity

3.5. Cell proliferation Daudi target cells (2 × 106/0.6 ml medium) were labeled with 150/zCi Na25~CrO 4 (540.83 m C i / m g , New England Nuclear) for 60 min at 37 °C. The cells were washed three times and 1 x 104 cells were used in each well. Effector cells at the appropriate dilutions for E:T ratios of 100:1, 30:1, 10:1 and 3:1 were mixed in triplicate with 5~Cr-labeled Daudi target cells (1 × 104) in flat-bottomed 96-well microtiter plates (Corning, NY) for 4 h. The percentage specific release was calculated as follows: percent specific 51Cr release = (mean cpm released from test well - mean cpm released from target cells only)/(mean total cpm - mean cpm released from target cells only)× 100. Spontaneous release represents the 51Cr release from target cells in medium only, and the total release control was estimated by adding 100 #1 of 1% Triton X-100 to target cells (100 #1) only. 3.3. Cell viability Dead cells were determined by staining with propidium iodide (PI: Sigma Chemical Co., St. Louis, MO) as described by Yokoyama [9]. PI was added to the cultured cell suspension to a final concentration of 0.5 /zg/ml. The mixed samples were analyzed immediately by flow cytometry (FACStar, Becton Dickinson) using an exciting wavelength 488 nm. 3.4. Quantification o f surface IL-2 receptor ex-

pression by flow cytometry Enrichment fraction for L G L from peripheral blood was separated by Percoll density gradients [10]. L G L were incubated for 30 min at 4 ° C with optimally diluted anti-IL-2 receptor-/3 monoclonal 210

LGLs (1 × 106 cells/ml) were cultured with or without 1000 U / m l of IL-2 in 96-well round-bottomed plates (Nunc, Denmark). [3H]thymidine (29.6 kBq) was added to each well 5 h before harvesting the cells. The cells were harvested with a Titertek harvester (Flow Laboratories) after adding cold 5 °7o trichloroacetic acid. The radioactivity remaining on the glass filter was measured in a liquid scintillation counter. 4. Results

4.1. Induction o f L A K activity in P M N C under oxygen-limited conditions H u m a n P M N C were cultured with 1000 U / m l of recombinant IL-2 at 20%, 5% and 2% oxygen concentrations for three days. To determine the LAK activity of these cells, the effector cells at the appropriate dilutions for E:T ratios of 100:1, 30:1, 10:1 and 3:1 were mixed with 51Cr-labeled Daudi target cells at 20°7o oxygen for 4 h. The results obtained show that induction of LAK cell activities at three days after cultivation was markedly reduced at O 2 concentrations of 5°7o or 2% as compared with 20% (Fig. 1). H u m a n P M N C were cultured with IL-2 at concentrations of 20% O 2 or 2% O 2 for seven days. At 20% O 2, LAK cell activities were rapidly enhanced during the first three days of cultivation and then remained stable for at least three days. In contrast, LAK cell activities at 2% O 2 gradually increased during the first three days and subsequently decreased rapidly (Fig. 2A). LAK cell activities generated from h u m a n P M N C cultured with IL-2 at 2% O 2 concentration were significantly lower than those at 20% O 2 concentration. No difference in

I00

100 o 20%0~

o 20%0~

•

8C

8o

~_ 6o 40 0_

0_

2O

20

I

I

I

,

I

,

100:1

30:1 10:1 3:1 100:1 30:1 101 3:1 E " T ratio E : T ratio Fig. 1. Induction of LAK cell activity under oxygen-limited conditions. LAK cell activity was determined in four separate experiments in a 5~Cr release assay. Student's t-test: *, P < 0.05; **, P < 0.005; ***, P < 0.001; ****, P < 0.0005. Each mark and vertical bar represents the mean and SD of four experiments (ten healthy men per experiment). 100 -

14

0 20%02

A

•

-s

T~

o 20%0~

2%02

__12

80

% .o_

x 1o

60

u)

40

._~ >

**

0_

6

20

6

i

I

3

;

J

7

Day

Day

Fig. 2. (A) Kinetics of LAK cell activity induced by IL-2 under 20070 and 2°70 oxygen conditions. PMNC (106 cells/ml) were cultured with recombinant IL-2 (1000 U/ml) at 20070 ( o ) and 2070 ( • ) oxygen concentrations. The LAK cell activity at an E:T ratio of 30:1 was determined in a 5tCr-release assay as described in Materials and Methods. Cytotoxic activities of unstimulated PMNC against Daudi cells at 20070 and 207o oxygen concentrations were 4 - 10°70 and 0 - 8070, respectively. Student's t-test: *, P < 0.05; **, P < 0.005; ***, P < 0.001. (B) The viability of cultured PMNC. Each mark and vertical bar represents the mean and SD of four experiments (ten healthy men per experiment).

cell viability of the cultured P M N C was observed for at least seven days at 0 2 concentrations of 20070 and 207o (Fig. 2B). 4.2. L A K activity by restimulation of lL-2 under

oxygen-limited conditions

with IL-2 at 20070 oxygen for 5 days. IL-2-restimulated LAK cell activities were consistently maintained at least until day 10 of the initial culture at 20070 oxygen concentration, but were reduced immediately at 2070 oxygen concentration after restimulation of IL-2 (Fig. 3).

P M N C were activated again by IL-2 under 20% or 2070 oxygen concentrations after stimulation 211

100

o 20%0~

• o ¢o a)

80

._o

60

4.3.

2%02

L A K cell-mediated lysis under oxygen-limited conditions

IL-2 (lOU/m~)

L A K e f f e c t o r cells in P M N C w e r e i n d u c e d by IL-2 at 20°7o O 2. W h e n t h e c y t o t o x i c a c t i v i t y o f the L A K cells a g a i n s t D a u d i was m e a s u r e d at 20°7o a n d 2°7o O 2 c o n c e n t r a t i o n , c e l l - m e d i a t e d lysis o f L A K e f f e c t o r cells was n o t i n f l u e n c e d by o x y g e n - l i m i t e d c o n d i t i o n s for t h e d u r a t i o n o f the lytic a s s a y (Fig. 4).

40 o

o.

4.4. 20

I

I

I

I

5

6

8

10

W h e n L G L s w e r e c u l t u r e d w i t h o r w i t h o u t IL-2 for 2 d a y s u n d e r e i t h e r 20°70 o r 2°70 o x y g e n , r e d u c e d o x y g e n c o n c e n t r a t i o n s f r o m 20°70 to 2°70 did n o t i n f l u e n c e s u r f a c e IL-2 receptor-/3 e x p r e s s i o n o n L G L o r t h e p e r c e n t a g e o f I L - 2 receptor-/3-positive cells in L G L s ( T a b l e 1). L G L s w e r e f u r t h e r c u l t u r e d w i t h I L - 2 for 6 d a y s at 20°70 a n d 2 % 0 2. A s s h o w n in Fig. 5, r e d u c e d o x y g e n c o n c e n t r a t i o n did not c a u s e a n y c h a n g e in I L - 2 - i n d u c e d p r o l i f e r a t i o n responses.

Day

Fig. 3. Kinetics of LAK activities in PMNC induced by the secondary stimulation of IL-2 under oxygen-limited conditions 5 days after stimulation with IL-2 at 2007o oxygen. PMNC (106 cells/ml) were cultured with IL-2 (1000 U/ml) at 20°7ooxygen for 5 days. The LAK cell activity in PMNC elicited by the re-stimulation of IL-2 at 20070 ( o ) or 2070 ( 2 ) oxygen 5 days after the initiated culture was determined by a 5tCr release assay. Each mark and vertical bar represent the mean and SD of five experiments (six healthy men per experiment). Student's t-test: *, P