< 1991 S. Karger AG. Basel 0030 2414 91 0485 0372 S 2.75 0
Oncology 1991;48:372-376
Influence of Serum Derived from Patients with Head and Neck Cancer on Natural Killer Cell Activity Hirobumi Kumazawa, Markus Hess Division of Surgical Oncology. Department of Otorhinolaryngology. University of Düsseldorf. FRG
Key Words. Head and Neck cancer • Natural killer cell activity • Clonogenic assay Abstract. The influence of serum from 23 patients with head and neck cancer on natural killer (NK) cell activity was analyzed using a double-layer agar assay, in which NK cells show an activity to inhibit the colony growth of K562 cells. In contrast to normal serum (or no treatment), serum from cancer patients reduced the inhibitory activity of NK cells, not only of autologous NK cells but also of allogeneic NK cells. These results suggest that the serum of patients with head and neck cancer may have some inhibitory factors on NK cell activity and may be involved in host resistance to tumor growth in vivo.
Natural killer (NK) cells play an important role in many immune functions including the lysis of tumor or virus-infected cells [1], Decreased NK cell activity has often been reported in advanced cancer patients with or without a decreased number of NK cells [2, 3]. What factors are decisive for the reduction of NK cell activity in cancer patients is not clearly understood. It is of importance to investigate the possibility that humoral factors in cancer patients are involved in the suppression on NK cell activity. In this study, we assessed whether the serum from cancer patients influences NK cell activity. NK cell activity can be expressed as the inhibitory effect of NK cells on the colony growth of K562 cells in double layer agar system [4]. We assessed the change of NK cell activity by the additional incubation of serum from cancer patients, comparing it with that after incuba tion with serum from normal individuals or without human serum in the double-layer colony assay.
Materials and Methods Preparation o f Lymphocytes and Serum Heparinized blood samples were obtained from healthy volun teers and 16 patients with head and neck malignant tumors (table 1). Two (No. 5. 13) of 16 patients with previous surgery showed
local recurrence of tumors. Peripheral blood lymphocytes were isolated on Ficoll-Hypaquc gradients from heparinized samples. The cells were washed twice with phosphate-buffered saline and resuspended in RPMI medium 1640 (Flow Laboratories. Irvine, UK) containing 10% fetal calf serum (FCS; Gibco. Paisley. UK) and incubated at 37 C for 60 min in Petri dishes for separation of monocytes. Nonadherent cells recovered from Petri dishes w'crc used for the clonogenic assay. Sera from cither normal volunteers or tumor patients were obtained from whole blood after clot formation, followed by cen trifugation. and were inactivated at 56 C for 30 min. Aliquots of serum were kept frozen at —80 C. Clonogenic Assay The clonogenic assay was performed according to the method described by Hamburger and Salmon [5] with slight modification. The under layer consisted of 1 ml of McCoy's 5a medium (Flow Laboratories) in 0.5% Bacto agar (Difco Laboratories, Detroit. Mich. USA) with 10% FCS in Petri dishes. K562 cells with or w'ilhout lymphocytes were suspended in CMRL1066 medium (Gib co) containing 20% FCS. penicillin/streptomycin and other nu trients in 0.3% agar as the over layer. Prior to cultivation in Petri dishes. K562 cells and lymphocytes were centrifuged at 25 g for 5 min, resuspended in 2.5 ml of medium, incubated at 37 C for 4 h and suspended in agar. Each Petri dish received 5 x 104 K562 cells without or with various numbers of lymphocytes resulting in several different effector:target (E:T) ratios. Thereafter, the agar culture was incubated at 37 C in humidified air with 5% CO 2 for 10-14 days. Colonics w'crc defined as cell aggregates at least 60 mm in diameter. The number of colonies was determined by counting the colonics with an inverted microscope. Every experiment was done in triplicate, and the results of each patient were calculated as NK cell activity by the following formula:
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Introduction
373
Influence of Head and Neck Cancer on NK Cell Activity
Patient
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23
Age years
Sex
Stage (TNM)
80 62 63 53 71 64 62 25
M M M M M M M M M M M M M M M M F F M F F M M
T j, no SCC hypopharynx T j. no SCC floor of mouth T 2. no SCC tonsil T 2. no SCC floor of mouth local recurrence tonsil (SCC) T i. no SCC skin of face maxillary sinus T 4 . no SCC T ,. N, UDC nasopharynx T2. no SCC hypopharynx T 2, no SCC hypopharynx t 2, n 2 SCC pyriform sinus larynx T 4 . no SCC local recurrence larynx (SCC) T i, N , SCC retromolar trigone t 2, n 2 SCC rctromolar trigone t 2, n 2 SCC tonsil nose T i. No ADC T 2. N 2 ADC parotis T 2, N, SCC tongue submandibular T 2. N o ADC local recurrence submandibular (ADC) larynx T 4 . N2 SCC T j, N2 UDC nasopharynx
66
64 62 88
50 57 69 65 57 61 72 61 48 69 22
Site
SCC = Squamous cell carcinoma; UDC = undifferentiated carcinoma; ADC = adenocarcinoma.
number of colonics at various E:T ratios per dish ----------------------------------------------------------------- x 100. number of colonics without effector cells per dish Treatment o f Hitman Serum in Clonogenic Assay Lymphocytes were incubated with K562 cells at 37 C for 4 h. and these effector and target cell suspensions w'ere divided into three groups: (A) incubation with heat-inactivated healthy human scrum; (B) incubation with heat-inactivated autologous scrum from tumor patients; (C) incubation without human serum. In groups A and B each. 25% scrum (0.5 ml in 2.5 ml) was added to the effector and target cell suspension. In group C, the same volume of complete medium was added as a control. The clonogenic assay was per formed in parallel in three groups. NK Cell Depletion with Anti-LeuUb Monoclonal Antibody + Complement To prove the functional importance of NK cells in the lym phocyte population used in this assay, lymphocytes were treated with monoclonal antibody (MoAb) to NK cells and complement. Prior to cultivation, lymphocytes were incubated with anti-Lculls MoAb (Bccton Dickinson, Mountain View, Calif., USA) at 4 C for 1 h. The antibody treatment was 40 pi of 100 gg ml antibody for 5 x 106 lymphocytes. Then, the cells were pelleted, and low-toxicity rabbit complement (Ccdarlane Laboratories. Ontario. Canada) was added at a final dilution of 1/10. The incubation was performed at 37 C for 1 h.
Statistical A Italysis The two-tailed Student's t test was used to calculate the signifi cance of differences in NK cell activities.
Results Influence o f Human Serum on NK Cell Activity Table 2 shows a summary of means ± standard deviations (SD) of NK cell activities in three ex perimental groups. In the group without human serum treatment (group C), an increase in the number of effector cells led to a substantial increase in NK cell activity. However, the addition of autologous patient serum to the lymphocyte and K562 cell suspensions (group B) resulted in inhibition of the NK cell activity at the 1:1, 5:1 and 10:1 E:T ratios as compared with those in the group without human serum treatment as well as that treated with healthy human serum (p < 0.01). When compared within each experiment, 2 out of 6 patients at the 1:1 E:T ratio (No. 2, 5), 11 out of 16 patients at the 5:1 E:T ratio (No. 1-3. 6. 8, 10, 13. 17, 19-21) and 9 out of 12 patients at the 10:1 E:T ratio (No. 11, 12, 15, 17-22) showed a significant depression of NK cell activities by the treatment of their autologous serum. Effect o f Tumor Patient Serum on Allogeneic Lymphocytes We studied whether the serum capable of decreas ing autologous NK cell activities can interfere with allogeneic NK cell activities derived from other pa tients as well as from healthy donors. The addition of patient serum (No. 1, 10. 11) to the suspension of allogeneic lymphocytes from No. 15 with K562 cells resulted in a significant increase (p < 0.01) in colonies of K562 cells compared with those of K562 cells with lymphocytes alone or lymphocytes plus normal hu man AB serum (fig. la). When using lymphocytes from healthy donors, the results seen in figure lb were similar. The NK cell activity of allogeneic normal lymphocytes was interfered with by the addition of tumor patient serum (No. 1, 2, 3, 8; p < 0.01). Effect o f Depletion o f Lymphocytes with Ant i-Leu IN MoAb -I- Complement When NK cells were depleted with anti-Leullb MoAb + complement, the number of colonies of K562 cells at the 0:1, 1:1 and 10:1 F.:T ratios was
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Table 1. Characteristics of patients with head and neck cancer
Kumazawa Hess
374 Table 2. Influence of serum on NK cell activity (means ± SD) at different E:T ratios (percent change)
1
2
3
4
5
6
7
8
9
10
11
12
13
NK cell activity at 0:1 n/dish
1:1 %
5:1 %
A B C
688.66 ± 32.82 749.00 ± 73.48 743.33 ± 46.07
72.6 92.21 90.21
55.12 93.63* 71.60
A B C
874.66 ± 32.78 624.66 ± 46.99 620.00 ±23.15
85.09 94.87* 85.59
65.85 92.05* 65.16
A B C
804.66 + 50.14 723.33 ± 16.13 608.66 ± 62.09
87.81 88.64 87.5
63.91 80.95* 38.33
—
A B C
810.66 ±30.13 850.66 ±37.91 839.00 ± 37.58
89.75 85.30 104.0
71.11 70.88 64.99
—
A B C
1.019.33 ±32.06 829.33 ± 44.27 945.00 ± 10.61
84.27 89.52* 77.54
69.26 74.79 76.43
_
64.14 69.96* 62.78
A B C
643.33 ±79.81 855.66 ± 141.54 567.00 ±46.18
-
A B C
696.33 ± 62.93 569.33 ± 64.45 662.00 ± 74.62
-
A B C
400.33 ± 17.32 359.00 ±11.57 291.33 ± 12.11
A B C A B C
587.66 ± 65.83 396.00 ± 44.95 489.33 ±62.51 544.66 ± 45.69 402.00 ±71.53 470.33 ± 24.07
-
-
— -
61.06 61.94 67.11 68.25 99.16* 53.66 75.21 77.27 63.21 67.25 86.98* 75.76
A B C
659.33 ± 47.59 741.66 ±47.30 698.66 ± 16.93
-
A B C
588.00 ± 54.35 702.00 ± 25.80 676.66 ± 73.07
-
-
-
-
-
-
A B C
829.66 ± 25.62 874.00 ± 19.40 896.66 ± 17.55
92 89.16 90.55
80.79 94.05* 77.39
-
-
-
-
-
10:1 %
Patient
NK cell activity at 0:1 n/dish
_
14
A B C
879.00 ±80.51 798.00 ± 76.80 773.66 ± 79.75
—
_
-
-
-
-
A B C
881.00 ± 105.01 875.33 ± 89.65 950.00 ± 34.02
_
_
-
-
-
-
-
_
15
-
-
16
-
17
-
18
_ -
19
_ -
20
-
_
1:1 %
5:1 %
10:1 % 53.73 46.53 45.01 31.48 57.42* 31.15
A B C
1,010.00 ±60.33 1.060.00 ±31.82 1.056.66 ±42.89
—
—
-
-
-
-
50.72 54.87 53.75
A B C
865.33 ±71.33 908.00 ±87.75 807.66 ± 64.43
—
75.12 84.66* 74.31
43.64 63.42* 51.35
A B C
755.66 ±39.08 857.00 ± 64.85 811.00 ±45.96
—
83.33 86.78 79.35
57.98 69.75* 54.23
A B C
597.66 ± 45.93 655.00 ±51.22 605.00 ±45.78
_
46.86 71.34* 56.93
39.54 55.33* 42.76
A B C
984.33 ± 79.75 875.00 ±87.66 887.66 ± 92.95
-
83.42 94.17* 79.81
61.44 75.62* 59.34
A B C
796.00 ±81.55 901.33 ±89.61 933.00 ± 97.87
—
-
77.12 86.22* 75.85
59.37 70.05* 61.37
A B C
649.00 ±57.21 721.33 ±68.73 688.33 ±52.67
-
-
-
-
-
-
47.46 59.07* 51.13
A B C
986.33 ± 88.65 952.33 ±71.52 891.33 ±87.92
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
21
_
-
-
22
_ -
54.27 74.42* 59.11 61.11 83.33* 67.73 — -
-
almost identical. In contrast, the treatment of comple ment alone showed the same inhibitory effects as in the control group without antibody and complement (fig. 2). The results suggest that the inhibitory effect on colony growth in the clonogenic assay is mediated by NK cells.
23
64.53 68.12 59.73
NK cell activity at the E:T ratio of 0:1 was considered to be 100%. A = Incubation with heat-inactivated healthy human serum; B = incubation with heat-inactivated autologous tumor patient serum; C = incubation without human scrum. *p < 0.01: significantly different from the corresponding values obtained from healthy human serum treatment (group A) and no serum treatment (group C).
Discussion Our purpose was to see whether the serum from cancer patients influences NK cell activity. Our data of patients with head and neck cancer showed that serum derived from patients reduced the inhibitory
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Patient
375
Influence of Head and Neck Cancer on NK Cell Activity
PATIENTS LYMPHOCYTES
NO
OF C O L O N I E S / D I S H
200
40 0
60 0
800
1000
12 00
20 0
40 0
60 0
80 0
1 0 00
1200
CONTROL W ITHOUT L Y M P H O C Y T E S
W I T H L Y M P H O C Y T E S ( N O 15) W I T H L Y M P H O C Y T E S ( N O . 1 5) ♦N O R M A L H U M A N A B S E R U M W I T H L Y M P H O C Y T E S ( N O 15) ♦ TU MO R P A T I E N T S S E R U M ( N O . 1 W I T H L Y M P H O C Y T E S ( N O . 1 5) ♦T UMO R P A T I E N T S S E R U M ( N O . 10) W I T H L Y M P H O C Y T E S ( N O . 15) ♦TUMOR ----------------------P A T I E ------------------------N T S S E R U M 3— ( NO ►..1 115
CONTROL W ITHOUT
»
LYM PHOCYTES
E : T RATI O
W ITH N O R M A L L Y M P H O C Y T E S ♦ N O R M A L HU M AN A B S E R U M W ITH NORM AL L Y M P H O C Y T E S ♦TUMOR P A T I E N T S S E R U M ( N O I ! W ITH NO RM A L L Y M P H O C Y T E S ♦T UMO R P A T I E N T S S E R U M (NO 2 W ITH NO RM A L L Y M P H O C Y T E S ♦ TU MO R P A T I E N T S S E R U M ( N O 3] W ITH NO RM A L L Y M P H O C Y T E S ♦T UMO R P A T I E N T S S E R U M ( N O 8)
H
Fig. 2. Inhibition of NK cell activity by anti-Lcul lb MoAb + complement. The depletion of NK cells by anti-Leul U MoAb + complement in the colony assay did not alter the number of colonics at the 0:1. 1:1 and 10:1 E:T ratios in the clonogenic assay.
H ________ H
Fig. I. Decrease in the inhibitory effect of allogeneic NK cells on the number of K562 cell colonies by treatment of serum derived from several tumor patients. When the tumor patient scrum (No. I, 10. 11) was pretreated in the suspension of K562 cells with allo geneic lymphocytes from No. 15. an increase in the number of colonies was observed compared to the treatment with normal human AB serum or with no treatment (a). When normal lym phocytes were used, the tumor patient serum (No. 1. 2. 3. 8) showed the same effect (b). Values are means + SD of triplicate cultures.
effect of NK cells on the colony growth of K562 cells. In 9 of 16 patients, the addition of autologous serum resulted in an increase in the number of colonies com pared with those of the control groups. Furthermore, when the serum capable of suppressing NK cell activ ity was incubated in the suspension of K562 cells and other allogeneic NK cells from cancer patients or healthy individuals, we also observed the same inhibit ory effect. Since the depletion of NK cells by Leullb MoAb -l- complement produced no inhibition of col ony growth of K562 cells seeded in Petri dishes, the clonogenic assay in this system is considered to detect NK cell activity. Thus, the results of the present ex periments demonstrate that the serum from patients with head and neck cancer has an inhibitory effect on NK cell activity.
The mechanism of regulation of NK cell activity is not clearly understood. Some soluble factors of serum, w'hich regulate NK cell activity have been reported in various patients. Autoantibodies in serum of autoimmune disease patients were considered to be suppressive to NK cell activity [6, 7]. Prostaglandin E2 produced by tumor cells can be a possible mechanism for the depression of the NK cell activity [8]. In our study, the inhibition of NK cell activity was not due to an influence of complement, since the serum was inac tivated by heating at 56 C for 30 min. Furthermore, the effect of FCS appears to be ruled out from the results obtained in our experiments, since it w'as used in each Petri dish. Pislarasu et al. [9] examined the modulation of NK cell activity by serum from human lymphoid and nonlymphoid tumors, indicating that NK cell activity was regulated by serum in both positive and negative ways. Their longitudinal follow-up study suggested that the presence of up-regulating NK cell activity by serum related to the patients with good clinical course. As previously described in their studies [10], mono meric immunoglobulin isolated from serum from healthy donors and cancer patients may be an attri bute of the inhibitory effect of NK cell activity in our study, although we did not delect such a suppression by the serum of healthy donors.
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NORMAL DONORS LYMPHOCYTES
Kumazawa/Hess
376
Acknowledgements We thank Dr. Koldovsky for the critical reading of the manu script, and all of the clinical staff in the Department of Otorhino laryngology for their cooperation.
References 1 Herberman RB. Ortaldo JR: Natural killer cells: Their role in defense against disease. Science 1981;214:24 27. 2 Steinhaucr EH, Doyle AT. Reed J, ct al: Defective natural cytotoxicity in patients with cancer: Normal number of effector cells but decreased recycling capacity in patients with advanced disease. J Immunol 1982;129:2255-2259. 3 Mickcl RA. Kessler DJ. Taylcr JM G . et al: Natural killer cell cytotoxicity in the peripheral blood, cervical lymph nodes, and tumor of head and neck cancer patients. Cancer Res 1988:48: 5017-5022. 4 Nomori H. Saijo N. Fujita J, ct al: Detection of NK activity and antibody-dependent cellular cytotoxicity of lymphocytes by human tumor clonogcnic assay - its correlation with 5lCrrclcasc assay. Int J Cancer 1985:35.449-455.
5 Hamburger AW. Salmon AE: Primary bioassay of human tu mor stem cell. Science 1977:197:461 463. 6 Goto M. Tanimoto K. Horiuchi Y: Natural cell mediated cyto toxicity in systemic lupus erythematosus. Arthritis Rheum 1980; 23:1274-1281. 7 Miyasaka N. Seaman W. Bakashi A. et al: Natural killing activity in Sjogren’s syndrome. Arthritis Rheum 1983;26: 954-960. 8 Balch CM . Dougherty PA. Cloud GA. et al: Prostaglandin F.2 -mediated suppression of cellular immunity in colon cancer patients. Surgery 1984:95:71 77. 9 Pislarasu M, Taranu AOD. Herberman RB. ct al: Modulation of natural killer cell activity by scrum from cancer patients: Preliminary results of a study of patients with colorectal adeno carcinoma or other types of cancer. Cancer Res 1988:48: 2596-2603. 10 Sulica A, Gherman M. Galatiuc C, et al: Inhibition of human natural killer cell activity by cytophilic immunoglobulin G. J Immunol 1982;128:1031-1036. II Whitehead JS, Kim YS: An inhibitor of lymphocyte prolifera tion produced by a human colonic adenocarcinoma cell line in culture. Cancer Res 1980:40:29-35. 12 Roth JA, Osborne BA, Ames RS. et al: Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochcmical characterization of a human sar coma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin related determinants. J Immunol 1983; 130:303 308. 13 Hersey P, Bindon C, Czerniecki M. et al: Inhibition of inter leukin 2 production by factors released from tumor cells. J Immunol 1983:131:2837 2842. 14 Guillou P. Scdman PC. Ramsden CW, et al: Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro. Cancer Immunol Immunother 1989;28:43-53. 15 Welsh RM. Karrc K, Hansson M, et al: Interferon-mediated protection of normal and tumor target cells against lysis by mouse natural killer cells. J Immunol 1981:126:219-225. 16 Uchida A. Vanky F. Klein E: Natural cytotoxicity of human blood lymphocytes and monocytes and their cytotoxic factors: Effect of interferon on target cell susceptibility. J Natl Cancer Inst 1985:75:849-857. 17 De Fries RU, Golub SH: Interferon-gamma reduces the sen sitivity of cultured and fresh human tumor cells to lysis by lymphokine-activated killer cells. Nat Immun Cell Growth Regul 1988:7:65-76. 18 Groenberg A. Ferm M. Tsai Li. et al: Interferon able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells. Cell Immunol 1989:118:10 21.
Dr. Hirobumi Kumazawa Division of Surgical Oncology Department of Otorhinolaryngology Kansai Medical University Fumizonocho-1, Moriguchi city Osaka 570 (Japan)
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It is, however, not easy to simply evaluate our results, comparing it with their [10] results, since the experimental systems used were considerably dif ferent. In their studies, lymphocytes alone were in cubated with serum, whereas in our study the serum was exposed to both tumor cells and lymphocytes to see the influence of serum on NK cell activity before 2 weeks of cultivation in Petri dishes. It is known that extracts or supernatants from human tumors have inhibitory effects on cellular functions including NK cells and lymphokine-activated killer cells [11-14], In addition, recent studies have suggested that pretreat ment of tumor cells with some cytokines or soluble factors leads to a change in the susceptibility of target cells of effector cells in vitro [15-18], In this respect, the experimental system used in this study seems to be better to obtain the information of serum, which may influence not only effector cells but also tumor cells during 2 weeks of cultivation. In conclusion, we observed that the culture con dition containing serum from cancer patients with head and neck cancer affected the results as to NK cell activity. These findings may provide the argument that some soluble factors capable of inhibiting NK cell activity should be considered in analyzing host resis tance to tumor growth.
Oncology 1991;48:372-376
Influence of Serum Derived from Patients with Head and Neck Cancer on Natural Killer Cell Activity Hirobumi Kumazawa, Markus Hess Division of Surgical Oncology. Department of Otorhinolaryngology. University of Düsseldorf. FRG
Key Words. Head and Neck cancer • Natural killer cell activity • Clonogenic assay Abstract. The influence of serum from 23 patients with head and neck cancer on natural killer (NK) cell activity was analyzed using a double-layer agar assay, in which NK cells show an activity to inhibit the colony growth of K562 cells. In contrast to normal serum (or no treatment), serum from cancer patients reduced the inhibitory activity of NK cells, not only of autologous NK cells but also of allogeneic NK cells. These results suggest that the serum of patients with head and neck cancer may have some inhibitory factors on NK cell activity and may be involved in host resistance to tumor growth in vivo.
Natural killer (NK) cells play an important role in many immune functions including the lysis of tumor or virus-infected cells [1], Decreased NK cell activity has often been reported in advanced cancer patients with or without a decreased number of NK cells [2, 3]. What factors are decisive for the reduction of NK cell activity in cancer patients is not clearly understood. It is of importance to investigate the possibility that humoral factors in cancer patients are involved in the suppression on NK cell activity. In this study, we assessed whether the serum from cancer patients influences NK cell activity. NK cell activity can be expressed as the inhibitory effect of NK cells on the colony growth of K562 cells in double layer agar system [4]. We assessed the change of NK cell activity by the additional incubation of serum from cancer patients, comparing it with that after incuba tion with serum from normal individuals or without human serum in the double-layer colony assay.
Materials and Methods Preparation o f Lymphocytes and Serum Heparinized blood samples were obtained from healthy volun teers and 16 patients with head and neck malignant tumors (table 1). Two (No. 5. 13) of 16 patients with previous surgery showed
local recurrence of tumors. Peripheral blood lymphocytes were isolated on Ficoll-Hypaquc gradients from heparinized samples. The cells were washed twice with phosphate-buffered saline and resuspended in RPMI medium 1640 (Flow Laboratories. Irvine, UK) containing 10% fetal calf serum (FCS; Gibco. Paisley. UK) and incubated at 37 C for 60 min in Petri dishes for separation of monocytes. Nonadherent cells recovered from Petri dishes w'crc used for the clonogenic assay. Sera from cither normal volunteers or tumor patients were obtained from whole blood after clot formation, followed by cen trifugation. and were inactivated at 56 C for 30 min. Aliquots of serum were kept frozen at —80 C. Clonogenic Assay The clonogenic assay was performed according to the method described by Hamburger and Salmon [5] with slight modification. The under layer consisted of 1 ml of McCoy's 5a medium (Flow Laboratories) in 0.5% Bacto agar (Difco Laboratories, Detroit. Mich. USA) with 10% FCS in Petri dishes. K562 cells with or w'ilhout lymphocytes were suspended in CMRL1066 medium (Gib co) containing 20% FCS. penicillin/streptomycin and other nu trients in 0.3% agar as the over layer. Prior to cultivation in Petri dishes. K562 cells and lymphocytes were centrifuged at 25 g for 5 min, resuspended in 2.5 ml of medium, incubated at 37 C for 4 h and suspended in agar. Each Petri dish received 5 x 104 K562 cells without or with various numbers of lymphocytes resulting in several different effector:target (E:T) ratios. Thereafter, the agar culture was incubated at 37 C in humidified air with 5% CO 2 for 10-14 days. Colonics w'crc defined as cell aggregates at least 60 mm in diameter. The number of colonies was determined by counting the colonics with an inverted microscope. Every experiment was done in triplicate, and the results of each patient were calculated as NK cell activity by the following formula:
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Introduction
373
Influence of Head and Neck Cancer on NK Cell Activity
Patient
1 2 3 4 5 6 7 8 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23
Age years
Sex
Stage (TNM)
80 62 63 53 71 64 62 25
M M M M M M M M M M M M M M M M F F M F F M M
T j, no SCC hypopharynx T j. no SCC floor of mouth T 2. no SCC tonsil T 2. no SCC floor of mouth local recurrence tonsil (SCC) T i. no SCC skin of face maxillary sinus T 4 . no SCC T ,. N, UDC nasopharynx T2. no SCC hypopharynx T 2, no SCC hypopharynx t 2, n 2 SCC pyriform sinus larynx T 4 . no SCC local recurrence larynx (SCC) T i, N , SCC retromolar trigone t 2, n 2 SCC rctromolar trigone t 2, n 2 SCC tonsil nose T i. No ADC T 2. N 2 ADC parotis T 2, N, SCC tongue submandibular T 2. N o ADC local recurrence submandibular (ADC) larynx T 4 . N2 SCC T j, N2 UDC nasopharynx
66
64 62 88
50 57 69 65 57 61 72 61 48 69 22
Site
SCC = Squamous cell carcinoma; UDC = undifferentiated carcinoma; ADC = adenocarcinoma.
number of colonics at various E:T ratios per dish ----------------------------------------------------------------- x 100. number of colonics without effector cells per dish Treatment o f Hitman Serum in Clonogenic Assay Lymphocytes were incubated with K562 cells at 37 C for 4 h. and these effector and target cell suspensions w'ere divided into three groups: (A) incubation with heat-inactivated healthy human scrum; (B) incubation with heat-inactivated autologous scrum from tumor patients; (C) incubation without human serum. In groups A and B each. 25% scrum (0.5 ml in 2.5 ml) was added to the effector and target cell suspension. In group C, the same volume of complete medium was added as a control. The clonogenic assay was per formed in parallel in three groups. NK Cell Depletion with Anti-LeuUb Monoclonal Antibody + Complement To prove the functional importance of NK cells in the lym phocyte population used in this assay, lymphocytes were treated with monoclonal antibody (MoAb) to NK cells and complement. Prior to cultivation, lymphocytes were incubated with anti-Lculls MoAb (Bccton Dickinson, Mountain View, Calif., USA) at 4 C for 1 h. The antibody treatment was 40 pi of 100 gg ml antibody for 5 x 106 lymphocytes. Then, the cells were pelleted, and low-toxicity rabbit complement (Ccdarlane Laboratories. Ontario. Canada) was added at a final dilution of 1/10. The incubation was performed at 37 C for 1 h.
Statistical A Italysis The two-tailed Student's t test was used to calculate the signifi cance of differences in NK cell activities.
Results Influence o f Human Serum on NK Cell Activity Table 2 shows a summary of means ± standard deviations (SD) of NK cell activities in three ex perimental groups. In the group without human serum treatment (group C), an increase in the number of effector cells led to a substantial increase in NK cell activity. However, the addition of autologous patient serum to the lymphocyte and K562 cell suspensions (group B) resulted in inhibition of the NK cell activity at the 1:1, 5:1 and 10:1 E:T ratios as compared with those in the group without human serum treatment as well as that treated with healthy human serum (p < 0.01). When compared within each experiment, 2 out of 6 patients at the 1:1 E:T ratio (No. 2, 5), 11 out of 16 patients at the 5:1 E:T ratio (No. 1-3. 6. 8, 10, 13. 17, 19-21) and 9 out of 12 patients at the 10:1 E:T ratio (No. 11, 12, 15, 17-22) showed a significant depression of NK cell activities by the treatment of their autologous serum. Effect o f Tumor Patient Serum on Allogeneic Lymphocytes We studied whether the serum capable of decreas ing autologous NK cell activities can interfere with allogeneic NK cell activities derived from other pa tients as well as from healthy donors. The addition of patient serum (No. 1, 10. 11) to the suspension of allogeneic lymphocytes from No. 15 with K562 cells resulted in a significant increase (p < 0.01) in colonies of K562 cells compared with those of K562 cells with lymphocytes alone or lymphocytes plus normal hu man AB serum (fig. la). When using lymphocytes from healthy donors, the results seen in figure lb were similar. The NK cell activity of allogeneic normal lymphocytes was interfered with by the addition of tumor patient serum (No. 1, 2, 3, 8; p < 0.01). Effect o f Depletion o f Lymphocytes with Ant i-Leu IN MoAb -I- Complement When NK cells were depleted with anti-Leullb MoAb + complement, the number of colonies of K562 cells at the 0:1, 1:1 and 10:1 F.:T ratios was
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Table 1. Characteristics of patients with head and neck cancer
Kumazawa Hess
374 Table 2. Influence of serum on NK cell activity (means ± SD) at different E:T ratios (percent change)
1
2
3
4
5
6
7
8
9
10
11
12
13
NK cell activity at 0:1 n/dish
1:1 %
5:1 %
A B C
688.66 ± 32.82 749.00 ± 73.48 743.33 ± 46.07
72.6 92.21 90.21
55.12 93.63* 71.60
A B C
874.66 ± 32.78 624.66 ± 46.99 620.00 ±23.15
85.09 94.87* 85.59
65.85 92.05* 65.16
A B C
804.66 + 50.14 723.33 ± 16.13 608.66 ± 62.09
87.81 88.64 87.5
63.91 80.95* 38.33
—
A B C
810.66 ±30.13 850.66 ±37.91 839.00 ± 37.58
89.75 85.30 104.0
71.11 70.88 64.99
—
A B C
1.019.33 ±32.06 829.33 ± 44.27 945.00 ± 10.61
84.27 89.52* 77.54
69.26 74.79 76.43
_
64.14 69.96* 62.78
A B C
643.33 ±79.81 855.66 ± 141.54 567.00 ±46.18
-
A B C
696.33 ± 62.93 569.33 ± 64.45 662.00 ± 74.62
-
A B C
400.33 ± 17.32 359.00 ±11.57 291.33 ± 12.11
A B C A B C
587.66 ± 65.83 396.00 ± 44.95 489.33 ±62.51 544.66 ± 45.69 402.00 ±71.53 470.33 ± 24.07
-
-
— -
61.06 61.94 67.11 68.25 99.16* 53.66 75.21 77.27 63.21 67.25 86.98* 75.76
A B C
659.33 ± 47.59 741.66 ±47.30 698.66 ± 16.93
-
A B C
588.00 ± 54.35 702.00 ± 25.80 676.66 ± 73.07
-
-
-
-
-
-
A B C
829.66 ± 25.62 874.00 ± 19.40 896.66 ± 17.55
92 89.16 90.55
80.79 94.05* 77.39
-
-
-
-
-
10:1 %
Patient
NK cell activity at 0:1 n/dish
_
14
A B C
879.00 ±80.51 798.00 ± 76.80 773.66 ± 79.75
—
_
-
-
-
-
A B C
881.00 ± 105.01 875.33 ± 89.65 950.00 ± 34.02
_
_
-
-
-
-
-
_
15
-
-
16
-
17
-
18
_ -
19
_ -
20
-
_
1:1 %
5:1 %
10:1 % 53.73 46.53 45.01 31.48 57.42* 31.15
A B C
1,010.00 ±60.33 1.060.00 ±31.82 1.056.66 ±42.89
—
—
-
-
-
-
50.72 54.87 53.75
A B C
865.33 ±71.33 908.00 ±87.75 807.66 ± 64.43
—
75.12 84.66* 74.31
43.64 63.42* 51.35
A B C
755.66 ±39.08 857.00 ± 64.85 811.00 ±45.96
—
83.33 86.78 79.35
57.98 69.75* 54.23
A B C
597.66 ± 45.93 655.00 ±51.22 605.00 ±45.78
_
46.86 71.34* 56.93
39.54 55.33* 42.76
A B C
984.33 ± 79.75 875.00 ±87.66 887.66 ± 92.95
-
83.42 94.17* 79.81
61.44 75.62* 59.34
A B C
796.00 ±81.55 901.33 ±89.61 933.00 ± 97.87
—
-
77.12 86.22* 75.85
59.37 70.05* 61.37
A B C
649.00 ±57.21 721.33 ±68.73 688.33 ±52.67
-
-
-
-
-
-
47.46 59.07* 51.13
A B C
986.33 ± 88.65 952.33 ±71.52 891.33 ±87.92
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
21
_
-
-
22
_ -
54.27 74.42* 59.11 61.11 83.33* 67.73 — -
-
almost identical. In contrast, the treatment of comple ment alone showed the same inhibitory effects as in the control group without antibody and complement (fig. 2). The results suggest that the inhibitory effect on colony growth in the clonogenic assay is mediated by NK cells.
23
64.53 68.12 59.73
NK cell activity at the E:T ratio of 0:1 was considered to be 100%. A = Incubation with heat-inactivated healthy human serum; B = incubation with heat-inactivated autologous tumor patient serum; C = incubation without human scrum. *p < 0.01: significantly different from the corresponding values obtained from healthy human serum treatment (group A) and no serum treatment (group C).
Discussion Our purpose was to see whether the serum from cancer patients influences NK cell activity. Our data of patients with head and neck cancer showed that serum derived from patients reduced the inhibitory
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Patient
375
Influence of Head and Neck Cancer on NK Cell Activity
PATIENTS LYMPHOCYTES
NO
OF C O L O N I E S / D I S H
200
40 0
60 0
800
1000
12 00
20 0
40 0
60 0
80 0
1 0 00
1200
CONTROL W ITHOUT L Y M P H O C Y T E S
W I T H L Y M P H O C Y T E S ( N O 15) W I T H L Y M P H O C Y T E S ( N O . 1 5) ♦N O R M A L H U M A N A B S E R U M W I T H L Y M P H O C Y T E S ( N O 15) ♦ TU MO R P A T I E N T S S E R U M ( N O . 1 W I T H L Y M P H O C Y T E S ( N O . 1 5) ♦T UMO R P A T I E N T S S E R U M ( N O . 10) W I T H L Y M P H O C Y T E S ( N O . 15) ♦TUMOR ----------------------P A T I E ------------------------N T S S E R U M 3— ( NO ►..1 115
CONTROL W ITHOUT
»
LYM PHOCYTES
E : T RATI O
W ITH N O R M A L L Y M P H O C Y T E S ♦ N O R M A L HU M AN A B S E R U M W ITH NORM AL L Y M P H O C Y T E S ♦TUMOR P A T I E N T S S E R U M ( N O I ! W ITH NO RM A L L Y M P H O C Y T E S ♦T UMO R P A T I E N T S S E R U M (NO 2 W ITH NO RM A L L Y M P H O C Y T E S ♦ TU MO R P A T I E N T S S E R U M ( N O 3] W ITH NO RM A L L Y M P H O C Y T E S ♦T UMO R P A T I E N T S S E R U M ( N O 8)
H
Fig. 2. Inhibition of NK cell activity by anti-Lcul lb MoAb + complement. The depletion of NK cells by anti-Leul U MoAb + complement in the colony assay did not alter the number of colonics at the 0:1. 1:1 and 10:1 E:T ratios in the clonogenic assay.
H ________ H
Fig. I. Decrease in the inhibitory effect of allogeneic NK cells on the number of K562 cell colonies by treatment of serum derived from several tumor patients. When the tumor patient scrum (No. I, 10. 11) was pretreated in the suspension of K562 cells with allo geneic lymphocytes from No. 15. an increase in the number of colonies was observed compared to the treatment with normal human AB serum or with no treatment (a). When normal lym phocytes were used, the tumor patient serum (No. 1. 2. 3. 8) showed the same effect (b). Values are means + SD of triplicate cultures.
effect of NK cells on the colony growth of K562 cells. In 9 of 16 patients, the addition of autologous serum resulted in an increase in the number of colonies com pared with those of the control groups. Furthermore, when the serum capable of suppressing NK cell activ ity was incubated in the suspension of K562 cells and other allogeneic NK cells from cancer patients or healthy individuals, we also observed the same inhibit ory effect. Since the depletion of NK cells by Leullb MoAb -l- complement produced no inhibition of col ony growth of K562 cells seeded in Petri dishes, the clonogenic assay in this system is considered to detect NK cell activity. Thus, the results of the present ex periments demonstrate that the serum from patients with head and neck cancer has an inhibitory effect on NK cell activity.
The mechanism of regulation of NK cell activity is not clearly understood. Some soluble factors of serum, w'hich regulate NK cell activity have been reported in various patients. Autoantibodies in serum of autoimmune disease patients were considered to be suppressive to NK cell activity [6, 7]. Prostaglandin E2 produced by tumor cells can be a possible mechanism for the depression of the NK cell activity [8]. In our study, the inhibition of NK cell activity was not due to an influence of complement, since the serum was inac tivated by heating at 56 C for 30 min. Furthermore, the effect of FCS appears to be ruled out from the results obtained in our experiments, since it w'as used in each Petri dish. Pislarasu et al. [9] examined the modulation of NK cell activity by serum from human lymphoid and nonlymphoid tumors, indicating that NK cell activity was regulated by serum in both positive and negative ways. Their longitudinal follow-up study suggested that the presence of up-regulating NK cell activity by serum related to the patients with good clinical course. As previously described in their studies [10], mono meric immunoglobulin isolated from serum from healthy donors and cancer patients may be an attri bute of the inhibitory effect of NK cell activity in our study, although we did not delect such a suppression by the serum of healthy donors.
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NORMAL DONORS LYMPHOCYTES
Kumazawa/Hess
376
Acknowledgements We thank Dr. Koldovsky for the critical reading of the manu script, and all of the clinical staff in the Department of Otorhino laryngology for their cooperation.
References 1 Herberman RB. Ortaldo JR: Natural killer cells: Their role in defense against disease. Science 1981;214:24 27. 2 Steinhaucr EH, Doyle AT. Reed J, ct al: Defective natural cytotoxicity in patients with cancer: Normal number of effector cells but decreased recycling capacity in patients with advanced disease. J Immunol 1982;129:2255-2259. 3 Mickcl RA. Kessler DJ. Taylcr JM G . et al: Natural killer cell cytotoxicity in the peripheral blood, cervical lymph nodes, and tumor of head and neck cancer patients. Cancer Res 1988:48: 5017-5022. 4 Nomori H. Saijo N. Fujita J, ct al: Detection of NK activity and antibody-dependent cellular cytotoxicity of lymphocytes by human tumor clonogcnic assay - its correlation with 5lCrrclcasc assay. Int J Cancer 1985:35.449-455.
5 Hamburger AW. Salmon AE: Primary bioassay of human tu mor stem cell. Science 1977:197:461 463. 6 Goto M. Tanimoto K. Horiuchi Y: Natural cell mediated cyto toxicity in systemic lupus erythematosus. Arthritis Rheum 1980; 23:1274-1281. 7 Miyasaka N. Seaman W. Bakashi A. et al: Natural killing activity in Sjogren’s syndrome. Arthritis Rheum 1983;26: 954-960. 8 Balch CM . Dougherty PA. Cloud GA. et al: Prostaglandin F.2 -mediated suppression of cellular immunity in colon cancer patients. Surgery 1984:95:71 77. 9 Pislarasu M, Taranu AOD. Herberman RB. ct al: Modulation of natural killer cell activity by scrum from cancer patients: Preliminary results of a study of patients with colorectal adeno carcinoma or other types of cancer. Cancer Res 1988:48: 2596-2603. 10 Sulica A, Gherman M. Galatiuc C, et al: Inhibition of human natural killer cell activity by cytophilic immunoglobulin G. J Immunol 1982;128:1031-1036. II Whitehead JS, Kim YS: An inhibitor of lymphocyte prolifera tion produced by a human colonic adenocarcinoma cell line in culture. Cancer Res 1980:40:29-35. 12 Roth JA, Osborne BA, Ames RS. et al: Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochcmical characterization of a human sar coma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin related determinants. J Immunol 1983; 130:303 308. 13 Hersey P, Bindon C, Czerniecki M. et al: Inhibition of inter leukin 2 production by factors released from tumor cells. J Immunol 1983:131:2837 2842. 14 Guillou P. Scdman PC. Ramsden CW, et al: Inhibition of lymphokine-activated killer cell generation by cultured tumor cell lines in vitro. Cancer Immunol Immunother 1989;28:43-53. 15 Welsh RM. Karrc K, Hansson M, et al: Interferon-mediated protection of normal and tumor target cells against lysis by mouse natural killer cells. J Immunol 1981:126:219-225. 16 Uchida A. Vanky F. Klein E: Natural cytotoxicity of human blood lymphocytes and monocytes and their cytotoxic factors: Effect of interferon on target cell susceptibility. J Natl Cancer Inst 1985:75:849-857. 17 De Fries RU, Golub SH: Interferon-gamma reduces the sen sitivity of cultured and fresh human tumor cells to lysis by lymphokine-activated killer cells. Nat Immun Cell Growth Regul 1988:7:65-76. 18 Groenberg A. Ferm M. Tsai Li. et al: Interferon able to reduce tumor cell susceptibility to human lymphokine-activated killer (LAK) cells. Cell Immunol 1989:118:10 21.
Dr. Hirobumi Kumazawa Division of Surgical Oncology Department of Otorhinolaryngology Kansai Medical University Fumizonocho-1, Moriguchi city Osaka 570 (Japan)
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It is, however, not easy to simply evaluate our results, comparing it with their [10] results, since the experimental systems used were considerably dif ferent. In their studies, lymphocytes alone were in cubated with serum, whereas in our study the serum was exposed to both tumor cells and lymphocytes to see the influence of serum on NK cell activity before 2 weeks of cultivation in Petri dishes. It is known that extracts or supernatants from human tumors have inhibitory effects on cellular functions including NK cells and lymphokine-activated killer cells [11-14], In addition, recent studies have suggested that pretreat ment of tumor cells with some cytokines or soluble factors leads to a change in the susceptibility of target cells of effector cells in vitro [15-18], In this respect, the experimental system used in this study seems to be better to obtain the information of serum, which may influence not only effector cells but also tumor cells during 2 weeks of cultivation. In conclusion, we observed that the culture con dition containing serum from cancer patients with head and neck cancer affected the results as to NK cell activity. These findings may provide the argument that some soluble factors capable of inhibiting NK cell activity should be considered in analyzing host resis tance to tumor growth.
