fnt. J. Cancer: 15, 308-320 (1975)

CELL-MEDIATED CYTOTOXICITY TO HUMAN PULMONARY NEOPLASMS

B. M. VOSE, M . MOOREa n d G. D. JACK’ Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M2O 9BX; and I Deparfmenf of Surgery, Wythenshawe Hospital, Manchester, England

Peripheral blood leukocytes from patients with confirmed pulmonary neoplasia were tested for cytotoxicity against cultured cells derived from lung tumours of various histological types, foetal and normal adult lung tissue and tumours arising in organs other than the lung. Leukocytes jrorn 73 % of patients were cytotoxic jor lung-turnour derived cells compared with age- and sex-matched normal donors, while the frequencies of reactivity against normal adult lung-derived cells and cells from unrelated tumours (e.g. bladder, colon, breast) were 42% and 18% respectively. Leukocytes jrorn lung cancer patients were also cytotoxic for cells derived from foetal lung but susceptibility to cytolysis was variable, cells from 13- and 14-week embryos revealing greatest reactivity (88 %) . Leukocytes from patients with a variety ojtumours ofnon-pulmonary origin or with non-malignant conditions (including respiratory disorders) were also reactive with lung-tumour-derived target cells but with a lower overall frequency (35%) than those from lung-cancer patients. The significance of these cytotoxicity data for the existence of turnour-specific host immunorcactivity in lung neoplasia is discussed,

The demonstration of cell-mediated immune reactions against different human neoplasms using a variety of in vivo and in vitro techniques has been claimed (reviewed by Herberman, 1973). In vitro cytotoxicity studies have in general purported to show that patients with malignancy have peripheral blood leukocytes which are reactive not only with tumour cells derived from the autochthonous host but also with allogeneic cells cultured from tumours arising at the same anatomical site (Bubenik et al., 1970; Fossati et al., 1972; Hellstrom et al., 1971 ; Baldwin rt al., 1973). However, in several of these studies it could not be established whether the reactivity of patients’ leukocytes with cultured tumour cells was tumour-specific rather than organ-specific since cultures from the relevant normal tissues

Received: August 15, 1974.

308

were not available for comparison. Furthermore, Takasugi et al. (1 973) have recently reported that lymphocytes from normal individuals possess a degree of cytotoxicity for cultured tumour cells equivalent to, o r greater than, those of tumourbearing patients, thereby raising doubts about earlier claims for tumour-specific immune reactivity against human neoplasms. Carcinoma of the lung, the most prevalent malignancy in men, has received comparatively little attention in studies of this type. The availability of these tumours a n d their normal tissue counterpart provided a convenient system in which to determine whether specific tumourrelated reactivity was detectable in leukocytes of lung cancer patients.

CELLULAR CYTOTOXICITY IN

HUMAN LUNG NEOPLASIA

TABLE I ORIGIN OF HUMAN TUMOUR CULTURES Cell line

Patient

Sex

Age

Histological diagnosis

Adenoma ” i.e. an argentaffin carcinoma of bronchus Poorly-differentiated squamous-cell carcinoma of bronchus Large-cell carcinoma of bronchus Squamous-cell carcinoma of bronchus Squamous-cell carcinoma of bronchus Moderately-differentiated squamous-cell carcinoma of bronchus Well-differentiated squamous-cell carcinoma of bronchus Undifferentiated carcinoma, large-cell type, bronchus Oat-cell carcinoma of bronchus Well-differentiated squamous-cell carcinoma of bronchus Well-differentiated squamous-cell carcinoma of bronchus Well-differentiated adenocarcinoma of colon Well-differentiated adenocarcinoma of colon Well-differentiated adenocarcinoma of colon Squamous-cell carcinoma of oesophagus Osteosarcoma of distal femur Transitional cell carcinoma of urinary bladder

L1

E.J.

Female

65

L2

W.J.S.

Male

63

L3 L5 L6 L7

A.B. J.D.J. F.L. E.0”.

Male Male Male Male

51 52 64 53

L17

H.B.

Male

64

L19

W.H.

Male

62

L22 L27

B.W. T.S.

Female Male

64 66

L30

B.H.

Female

51

C06 C015 C027 OESI os2 T24

K.R. A.W. T.B. H.R. I.M.G. C.M.

Female Female Male Female Female Female

77

59 12 82

“

Bubenik et al., 1973.

MATERIAL AND METHODS

Tumours Biopsies of tumours were obtained from patients undergoing surgery a n d transported t o the laboratory for the initiation of tissue cultures within 2 h of removal. Clinical data relating t o patients from whom tumour-cell cultures were successfully prepared are summarized in Table I. Cultures were also initiated from macroscopically normal areas of lung tissue removed during block dissection of lung neoplasms L2, L3 a n d L7 a n d from lung tissue removed during thoracic surgery for a nonmalignant condition. These cultures were designated NL2, NL3, NL7 a n d NL respectively.

Embryo lung Foetal tissues were derived from patients undergoing hysterotomy for the therapeutic

termination of pregnancy. The periods of gestation from fertilization were calculated from foetal size a n d appearance (Table 11) as described by Moore (1973). TABLE I1 ORIGIN OF HUMAN EMBRYO LUNG CULTURES Culture

E3 E4 E6 El 1 El2 El6 El7 El8 E26 E27

Crown-rump length (mm)

Foot length (mm)

Estimated age (weeks)

102 15 13 Triplets-age estimated from last menstrual period 14 weeks 127 23 14-15 151 29 17 90 16 12 98 17 13 107 17 13 122 25 14 90 16 12 6 8 43

309

VOSE ET A t .

Preparation of tissue cultures

Monolayer cell cultures were prepared from tissue fragments or trypsinized cells. Tumours were dissected free of necrotic areas and as much surrounding stroma as possible, finely minced and washed in Hanks’ balanced salt solution (HBSS). Tissue fragments (1-2 mm3) were allowed to adhere to the surface of 25 cm2 Falcon flasks (Falcon Plastics, Oxnard, Calif., USA) or alternatively dissociated into single-cell suspensions with 0.25 % trypsin (Cambrian Chemicals) in the presence of DNase (Sigma, St. Louis, Mo. USA). Cells were harvested by centrifugation (30 ~ g and ) washed in HBSS, then 5 x 1 0 - 5 x 10“ were seeded into flasks. Cultures were maintained, by serial subculture, in Waymouth’s medium supplemented with 20% foetal calf serum and antibiotics and used as targets for leukocytotoxicity tests between the first and 14th passages.

by Hellstrom et al. (1971). Briefly, 100-200 target cells were seeded into the wells of Microtest plates No. 3034 or Microtest I1 plates No. 3040 (Falcon Plastics) in Eagle’s MEM buffered with HEPES and supplemented with 10% foetal calf serum. After 4-24 h incubation at 37” C to allow cell attachment the medium was replaced by leukocyte suspension (2 x 1 O5 cells/well in Microtest I1 and sufficient to give an effector:targetcell ratio of 75:l in Microtest plates). After a further 2 days’ incubation the plates were washed with saline to remove non-adherent cells, fixed with methanol and stained with crystal violet. The number of cells surviving in each well was counted and the percentage reduction of cell survival in those wells treated with patients’ lymphocytes compared with those treated with control lymphocytes calculated. The significance of the percentage reduction was determined by Student’s t-test at the 95 % level.

Leukocytes

Heparinised blood samples were taken from patients 7-21 days after surgery for pulmonary neoplasia, from patients attending this hospital for local radiotherapy for various malignant conditions including lung cancer, and from patients with non-malignant disorders. Age and sex-matched control specimens were obtained from the National Blood Transfusion Service. All samples from patients receiving radiation treatment were taken before or 1 day after the first exposure (maximum skin dose 250-450 R) of a series of fractionated dosages. Leukocytes were prepared by centrifugation of blood samples on gradients of Ficoll (Pharmacia, Uppsala, Sweden) and Triosil (Nyeggard and Co., Oslo, Norway) by the method of Boyum (1968). This resulted in a preparation containing a uniformly high proportion of lymphocytes with minimal erythrocyte contamination but occasionally variable contamination with granulocytes, thrombocytes and monocytes. Leukocytes were washed three times in HBSS before resuspension for counting in Eagle’s MEM buffered with 40 mM HEPES supplemented with 10% foetal calf serum. Microtoxicity tests

The cytotoxicity for cultured target cells of peripheral blood leukocytes from patients was assessed by the microassay method of Takasugi and Klein (1970) or the modification described

310

RESULTS

Cytotoxicity against lung-tumour-derived cells

Leukocytes from patients with confirmed pulmonary malignancies were regularly cytotoxic for cultured cells derived from lung neoplasms compared with those from age- and sex-matched controls (Tables 111, IV). Significant reductions of cell survival (p