Int. J. Cancer: 20, 512-519 (1977)

LYMPHOCYTE CYTOTOXICITY AGAINST AUTOLOGOUS TUMOUR BIOPSY CELLS IN HUMANS B. M . VOSE1* 3, F. VANKYl , and Eva KLEIN Department of Tumour Biology, Karolinska Institutet, S-I04 01 Stockholm 60; and Karolinska Hospital S- 104 01 Stockholm 60, Sweden

By the application of separation techniques i n a stepwise manner t o mechanically prepared cell suspensions from human tumour biopsies it has been possible t o isolate tumour cells having high viability and low contamination w i t h host cells. These tumour cells have been used as targets in 5'Cr release cytotoxicity assays using autologous lymphocytes f r o m blood o r lymph node as effectors. It has been possible t o investigate this reactivity in 30 patients w i t h malignant and four patients w i t h benign tumours. Significant autologous cytotoxicity i n the blood lymphocytes was detectable i n t w o of 12 lung tumours, nine of 18 other carcinomas and sarcomas but in none of the benign cases. Cytotoxicity was not limited by the presence of obvious residual disease at the t i m e o f operation although reactivity was less frequently detectable i n patients with secondary involvement. Reactivity in the tumourdraining lymph nodes largely paralleled that i n the blood in the limited number of patients examined. The technical problems encountered during this study and the role o f natural cytotoxicity i n the interpretation o f these data are discussed.

Cytotoxic activity of blood lymphocytes from cancer patients for cultured cells derived from human tumours has been considered to be indicative of the presence of a tumour-associated host response (Hellstrom et a/., 1971; Baldwin et a/., 1973). However, since it has been observed that in certain situations lymphocytes from healthy donors can show the same and sometimes higher cytotoxic activity against cultured cells (Takasugi et a/., 1973) these results have to be reinterpreted. A number of cell lines have been shown to be highly sensitive to the killing effect of certain categories of blood lymphocytes, a phenomenon which may not be totally due to their cultivation in medium containing foetal calf serum (Sulit ef a/., 1976). Cell culture can also lead to the loss of cell surface antigen (Konigsberg, 1963). Of critical importance in attempts to define the presence of tumour-associated antigens on human tumours by this type of methodology may be the report in a number of animal model systems that T-cell-mediated damage is strictly limited by the histocompatibility relationship of effector and target cells (Zinkernagel and Doherty, 1974; Shearer ef a/., 1975). If this is a general rule and applied to the control of human T-cell-mediated killing as some recent evidence suggests (Goulmy et a/., I977), studies performed in allogeneic combi-

Radiurnhemmet,

nation may be unlikely to yield information concerning the antigenicity of the cells from malignant neoplasms. With these facts in mind we initiated studies to investigate the possibility of using cells prepared from human turnour biopsies in autologous lymphocyte cytotoxicity (ALC) tests. Target cells were obtained by the application of separation techniques consisting of several steps, and the assays were performed without involvement of prolonged culture. MATERIAL AND METHODS

Lymphocytes Heparinized blood samples (50 ml) were obtained from untreated patients on the morning of operation prior to medication. The blood was allowed to sediment in the upright syringe at room temperature for 1 h and the leukocyte-rich plasma expelled into 10-ml conical centrifuge tubes in 3- to 4-ml aliquots. These were underlayered by 3 ml of a Ficoll-Isopaque (FI) solution corresponding to 75% of the stock solution (dilution with balanced salt solution-BSS), then 3 ml of stock solution were injected below this. Stock FI solution was prepared by admixture of 104 m19 % Ficoll400 (Pharmacia Uppsala, Sweden), 45 ml distilled water and 25 ml Isopaque (Nyegaard and Co., Oslo, Norway), and had a density of 1.077. Following centrifugation (10 min at 900 g) the lymphocyte-enriched layer at the 100% (stock solution) interface was collected and washed twice. Cells adherent to plastic (principally monocytes) were removed by incubation in 75 cm2 culture flasks (Falcon Plastics No. 3024) for 30 min at 37" C in RPMI medium containing 10% heatinactivated AB, Rh+ serum from healthy donors (NHS). Non-adherent leukocytes were collected and washed once, then further purification was effected by passage of the cells through columns of nylon fibre. Received: April 25, 1977 and in revised form July 26, 1977.

Present address and correspondence : Department of Immunology, Paterson Laboratories, Christie Hospital and Holt Radium lnstitute, Manchester, M20 9BX England.

LYMPHOCYTOTOXICITY AGAINST TUMOUR CELLS

Nylon column separation

Separation was performed according to Julius et a/. (1973). A 5-ml disposable syringe was packed with 0.4 g scrubbed nylon fibre (FT 242 Fenwal Laboratories, Chicago, Ill., USA) and a 23 G needle attached. The column was washed with RPMI+10% NHS and leukocytes ( 3 - 5 ~ 1 0in~ 0.5 ml RPMI+lO% NHS) were run in, incubated for 1 h at 37" C before elution of non-adherent cells by addition of medium with collection of the eluate at a rate of approximately 1 ml/min. Eluted cells consisted of 85-92 % T lymphocytes as assessed by their capacity to form rosettes with SRBC, 1-4 % cells forming rosettes with antibodycoated ox erythrocytes (EA rosettes) and less than 1 % contamination with cells of non-lymphocyte morphology. The lymphocytes were stored overnight in 25-cm2 culture flasks (Falcon Plastics No. 3013) at 37" C in RPMI-1640+10% NHS prior to use in cytotoxicity assays. Lymph-node cells ( LN C )

Lymph nodes were finely minced with scissors and pressed through a stainless steel grid with a spatula into medium. Cells were washed twice in RPMI-1640 without serum and cells adherent to plastic removed as described above. Non-adherent leukocytes were separated on FI gradient and the lymphocyte-enriched layer washed once with RPMI1640 10 % NHS and applied to nylon columns as previously described. Eluted lymphocytes were stored overnight in 25-cm2 culture flasks prior to use as effectors in cytotoxicity assays and had a similar composition to the peripheral blood lymphocyte preparations.

+

Tumour

Tumours were examined on removal and assessed for their suitability for use in subsequent assays. Those with a hard consistency, such as welldifferentiated fibrosarcoma or chondrosarcoma, were avoided since experience showed that the cell yield from these tumours was low and viability poor. Suitable material was transported to the laboratory in RPMI-1640 without serum and immediately processed. Tumour tissue was minced, connective tissue and remaining necrotic areas removed. Tumour cell suspensions

No single method of tumourcell separation can be given since different tumours even of the same type varied in their characteristics with regard to cell separation. The procedures used and the order in which they were applied were decided step by step, depending on the composition of the fractions as judged by morphology and trypan blue staining.

513

Selection was then largely a matter of experience although the separations outlined below were found particularly useful. The objective of the separation procedures was to remove recognizably non-malignant elements such as macrophages, erythrocytes, lymphocytes and dead cells from the preparations. Undoubtedly the final preparations also contained a proportion of cells of non-malignant origin with isolation characteristics similar to those of the tumour cells. Preparalion of cell suspension

Attempts to prepare suspensions containing viable tumour cells were made in two ways. The finely minced tumour tissue was shaken vigorously thrze or four times with changes of medium. This regularly resulted in suspensions containing large numbers of red cells, live and dead tumour cells, and lymphocytes. The residual material was then transferred to a stainless steel mesh and forced through with a spatula into BSS. The resulting suspensions were examined to determine if further separation procedures were justified. Separation procedures

The initial step consisted of centrifugation at low speed (60 g, 5 min) followed by more rapid centrifugation of the supernatant (500 g, 5 min). From the sediments red cells, dead cells and some of the macrophages were removed on FicollIsopaque gradients similar to those employed for the separation of blood lymphocytes. The two interfaces and the bottom fractions were examined for enrichment of tumour cells. In early experiments separation by velocity sedimentation at unit gravity o n bovine serum gradients was used. Although this procedure was found to be useful it was recognized that it presented a possible hazard to the interpretation of the final assays in view of the fact that tumour cells can adsorb components from bovine serum (Sulit et a/., 1976). It was therefore abandoned in favour of a similar separation at unit gravity into a discontinuous gradient of Ficoll (generally 25 %, 50% and 75 % of the stock solution). In some preparations it was found that overnight attachment to plastic at 37" C in 75 cm2 culture flask provided a useful separation of tumour cells while in others a shorter period of attachment (30 min) could be used to deplete macrophages (whose presence could be recognized by ingestion of carbon particles). When necessary during the procedure, dead cells could be eliminated by treatment of the preparation with 0.20% trypsin for 1 min at room temperature in the presence of 10 Kunitz unitsjml DNase (Sigma, St. Louis, Mo., USA) to prevent cell clumping. If separation was judged successful, tumour cells were incubated

514

VOSE ET AL.

overnight in Falcon culture flasks at 37" C prior to the cytotoxicity assays. This overnight incubation of tumour cells was found to be advantageous in the autologous lymphocyte stimulation (ATS) assay in which this simple manipulation increased the number of positive tests detected (Vanky et a!., in preparation) and was therefore included in this procedure. A final examination was made before W r labelling to determine the suitability of the cells for testing. If necessary a further attempt to eliminate dead cells was performed (FI gradient). Only cell preparations exceeding 80 % viability were used. From approximately 60% of tumours in which separation procedures were attempted a usable tumour cell preparation was obtained. The autologous lymphocytotoxicity assay (ALC)

The cytotoxicity of peripheral blood lymphocytes for autologous tumour cells was determined in a 51Cr release assay. Tumour cells (lo6 in 0.5 ml RPMI-1640 10 % NHS) were labelled by dropwise addition of 75 pCi sodium [61Cr] chromate (Radiochemical Centre, Amersham, Bucks., UK) and incubation with occasional agitation at 37" C for 2 h. They were then washed four times in BSS and resuspended in RPMI 10% NHS. Cell suspensions were adjusted to 1 x lo5 cells/ml and 0.1 ml aliquots dispersed into 1.5 ml Eppendorf capped reaction tubes (No. 3810). Lymphocytes (0.2 ml of a suspension of 2.5 x 106/ml in RPM1+10% NHS) were added to give an effector: target cell ratio of 5O:l in all tests. The final volume was adjusted to 0.6 ml with medium. Following centrifugation to compact the reactants, tubes were incubated at 37" C for 4 h before 0.2 ml of the supernatant was removed. Supernatants and pellets were counted in a gamma counter. Spontaneous release of 61Cr from target cells was measured in tubes containing these alone and maximum release by lysis with I : 50 dilution of detergent (Triton X-100 Sigma). All tests were performed in triplicate. Percentage 51Cr release for each tube was then calculated from the formula: 3 x supernatant counts x loo. Percent 51Cr release = supernatant and pellet counts

K-562 (Lozzio and Lozzio, 1973) derived from a patient with chronic myeloid leukaemia in blast crisis was included in all tests. This cell line has been shown to be extremely sensitive to the killing effect of normal human blood lymphocytes: spontaneous cytotoxicity (Jondal and Pross, 1975). Tests in which the spontaneous release from the tumour cell preparations exceeded 50% were not considered. This entailed discarding seven of the 43 tests performed where initially the tumour cells had been considered adequate. In two additional tests tumour cell preparation showed extremely low labelling with W r and these too were not considered in the final assessment reported here. Significance was calculated by Student's t-test and in all cases it could be considered positive if cytotoxicity higher than 20% was recorded (pt0.05). Scatter in the replicates was small and did not exceed 6% cytotoxicity.

+

+

Cytotoxicity was then derived:

% 61Crrelease -spontaneous Cytotoxicity

=

51 Cr release x 100. maximum "Cr release-spontaneous 61Cr release

If possible a second preparation of tumour cells from a different individual was included as a specificity control. Additionally the long-term cell line

RESULTS

Separation procedures

The ease of separation and yield of tumour cells varied enormously from different biopsies when they were subjected to the separation procedures described above. Not surprisingly the yield obtained depended primarily on the amount of tumour received and the viability of the initial, mechanically prepared cell suspension. Examples of the separation protocol for three tumours are presented here for reference. The procedures used were chosen on the basis of the cell composition of the suspension obtained at the previous step. They indicate some of the problems encountered and the methods employed to overcome them. Tu 1269 low differentiated squamous cell carcinoma. This tumour biopsy measured approximately 2 x 2 cm without necrosis. Mechanical separation of the tumour followed by low-speed centrifugation yielded a cell suspension consisting primarily of dead cells, erythrocytes, live tumour cells and lymphocytes. In order to remove the majority of the dead cells whose presence could hamper subsequent separation procedures, the suspension was first treated with 0.2% trypsin and DNase, centrifuged and resuspended in RPMI+10% NHS. The cell suspension was then applied to a discontinuous gradient of Ficoll-Isopaque corresponding to 25 % and 50% of the stock solution and incubated for 2 h at 37" C. The supernatant then consisted of erythrocytes, lymphocytes and some tumour cells and the material entering the gradient showed good enrichment for tumour cells but with contaminating erythrocytes and lymphocytes. This included material washed with BSS, resuspended in RPMI+10%

515

LYMPHOCYTOTOXICITY AGAINST TUMOUK CELLS

NHS and applied to a discontinuous Ficoll-Isopaque gradient, corresponding to 75% and 100% of the stock solution. Following centrifugation (10 min, 900 g) the two interfaces and bottom fraction were examined. The bottom showed erythrocytes, some dead cells and some lymphocytes. The 100% interface contained predominantly lymphocytes with a small number of live tumour cells and erythrocytes and the top fraction contained live tumour cells, with a small number of lymphocytes also present. This top fraction was incubated overnight in a Falcon culture flask and the adherent fraction which showed less than 2% macrophages and no lymphocytes, was used as the source of target cells in cytotoxicity assays. Further tumour cells could be obtained by subjecting the non-adherent fraction to a further centrifugation of Ficoll-Isopaque. The tumour cell yield from this biopsy was 7 x lo8 cells and viability was 93 %. Fibrosarcoma. Following mechanical separation and centrifugation, examination of the suspension showed that 90% of the cells were dead as assessed by trypan blue staining. Treatment with trypsin was effective in removing the dead cells but the yield of cells was too low t o make further separation justified and the material was therefore discarded. Tu 764 brain metastasis of meianoma. A large, soft tumour approximately 4 cm x 4 cm without necrosis was mechanically separated and centrifuged, then the suspension was examined. Almost all the cells were live and had melanin granules. Some dead cells and erythrocytes but an extremely low proportion of lymphocytes were also present. The material was centrifuged on a 75 %/loo% FicollIsopaque gradient. The 75 % interface consisted of debris with few intact tumour cells present, the bottom fraction contained red cells, dead cells and some live tumour cells and the 100% interface live tumour cells. No further separation was performed, the material being incubated overnight and then used as a source of targets in cytotoxicity assays. Cell yield was 13 x lo7 cells and viability 87%. Cytotoxicity The cytotoxicity of peripheral blood lymphocytes from 30 cancer patients and four patients with benign tumours of connective tissue origin were tested for cytotoxicity against autologous tumour cells. Clinical details of the patients are presented in Table I. Particular attention was paid to the presence of metastasis at the time of operation. Cytotoxic reactivity was obtained in 11 cases of malignant and in none of the patients with benign tumours. An example of the results of a cytotoxicity test is presented in Table 11. In this assay it was possible to test lymphocytes from blood and lymph node from a patient with nasopharangeal carcinoma.

Lung malignancies Twelve patients with pulmonary neoplasia have been examined and significant reactivity determined in two cases (17%) (Fig. 1) (one adenocarcinoma and one squamous cell carcinoma). The blood TABLE I HUMAN TUMOURS EXAMINED No.

Diagnosis

Sex

~~

~

Lung tumours 1250 M Low differentiated adenocarcinoma 1253 F Low differentiated adenocarcinoma 1259 M Low differentiated adenocarcinoma 1262 M Undifferentiated adenocarcinoma 1265 M Adenoid carcinoma 1267 M Undifferentiated adenocarcinoma 1269 M Low differentiated squamous carcinoma 1277 M Low differentiated sqiiamous carcinoma 1281 M Undifferentiated carcinoma 1282 M Undifferentiated carcinoma 1283 M Oat-cell carcinoma 1284 M Squamous carcinoma Sarcomas 1002 F Osteoblastic osteosarcoma 1003 I; Osteogenic osteosarcoma 1007 M Polymorphocellular fibrosarcoma 1009 M Fibroliposarcoma 1005 M Osteoblastic osteosarcoma metastasis in lung 1270 M Fibrosarcoma metastasis in lung

Benign connective tissue disorders 1006 F Giant cell tumour 1007 F Giant cell turnour 1008 M Synoviomu pigmentmu 1001 M Mass on femur. Pathologically non-malignant Glioma 757 M Glioma 762 F Astrocytoma grade 111 763 M Glioma Secondary tumours 1279 F Hypernephroma in lung 1280 M Melanoma in lung 504 Hypernephroma in lung 1285 M Rectal adenocarcinoma in lung 764 F Melanoma in brain 765 F Melanoma in brain Miscellaneous 1272 M Thymoma 1004 F Chordoma 7 M Nasopharyngcal carcinoma (from Nairobi)

t

+

-

i-

i-

+-

+i

++i ++ + T

-

516

VOSE ET AL. TABLE I1 CYTOTOXICITY TEST WITH LYMPHOCYTES FROM PERIPHERAL BLOOD A N D LYMPH NODE FROM PNC AGAINST AUTOLOGOUS CELLS, K562 A N D MELANOMA Target cells

%7 SICr release

Lymphocytes

Spontaneous Maximuni

PNC 7

Spontaneous Maximum PBL LNC

Spontaneous Maximum PBL LNC

764 (melanoma)

'

-

34.4 (3 1, 34, 36) 94.2 (93, 94, 95) 61.2 (59, 60, 63) 52.8 (49, 52, 55) 27.3 (25, 21, 29) 99.6 (99, 100, 100) 33.2 (33, 33, 33) 26.5 (25, 26, 28)

PBL LNC K562

% cytotoxicity

44.8 30.8 .-

8.2 0

14.2 (12, 15, 15) 100.0 (100, 100, 100) 15.0 (14, 15, 15) 19.0 (18, 19, 20)

0.9 5.5

Numbers in parentheses indicate the cytotoxicity value calculated from individual replicates.

lymphocytes were more frequently cytotoxic for the K562 cell line (6/10 positive) although significant cytotoxic potential against these cells was not found in the two autologous positive cases. Sarconias Significant cytotoxic reactivity against autologous cells was noted in 4/6 (67%) cases of sarcoma (Fig. 2). It is of interest that both tumours (1005 and 1270) in which target cells were prepared from lung metastasis were positive. Lack of autologous reactivity was found against an osteogenic sarcoma with proven recurrent disease and against a fibroliposarcoma. Cytotoxicity against K562 was detected in 4 cases. Reactivity against autologous tumour and K562 coincided in 2 cases. Studies against 4 histologically benign tumours of connective tissue origin failed to reveal autologous reactivity. K562 cells were damaged in one case.

.. r.. .... ., .::. - . ...-.. .. .. .... .'.. ::.* .. ..

8 2 0.

OA

....'. ..

9 7

3

m

v SARCOMATA

BENIGN

F I G ~ J R2E Cytotoxicity of peripheral blood lymphocytes from patients with tumours of connective tissue origin for autologous tumour cells (open columns) and K562 cell line (dotted columns).

Other tumours Lymphocyte cytotoxicity was detectable against autologous cells from 2/3 gliomas, 1 thymoma, 1 nasopharyngeal carcinoma and in 1 of 2 metastases of hypernephroma to lung. No significant reactivity was found in 3 melanomas (1 lung metastasis and 2 brain metastases) in 1 lung metastasis of colon carcinoma or in I chordoma. Reactivity against K562 was found in 7/11 of these patients (Fig. 3). 1277

1265

I

1281

1262 LUNG

1253 1267

I259 1282

1283

1269

1284

CARCINOMATA

FIGURE1 Cytotoxicity of peripheral blood lymphocytes from lung cancer patients against autologous tumour cells (open columns) and K562 cell line (dotted columns).

Relationship to the stage of disease The presence of cytotoxicity against either autologous tumour cells or K562 was not limited by the presence of obvious residual disease at the time of operation. Of 16 patients who were apparently

51 7

LYMPHOCYTOTOXICITY AGAINST TUMOUR CELLS

Specificity

In 14 cases it was possible to test the specificity of the observed reactions using allogeneic biopsy cells prepared on the same day (Table 111). In 6 cases (Table 11) significant reactivity against autologous cells was noted in the absence of reactivity against allogeneic biopsy cells. In no case were the autologous and allogeneic tumours of similar anatomical derivation. Three cases showed reactivity against allogeneic but not autologous cells.

; - no -

0

60 I-

c 0 > 40

c 20

0

757

762 763 GLIOMA

504 1279

764 1280

SECONDARY

1285 765

1004

Lymph-node cell reactivity

1272

TUMORS

FIGURE 3 Cytotoxicity of peripheral blood lymphocytes from patients with glioma, metastases to lung and brain and with malignancies at different sites against autologous tumour cells (open columns) and K562 cell line (dotted columns).

disease-free, 7 (44 %) showed autologous reactivity while 4/14 (29%) patients with metastases were positive (Table I). Generally tests against cells prepared from secondary tumours showed a low frequency of autologous reactivity with only 2 sarcomas and 1 hypernephroma metastasis to lung being positive from 8 tested. Nine out of 13 (69%) patients with metastases and 8/15 (53 %) without secondary spread showed cytotoxic potential against K562. Differences are not statistically significant.

In a limited number of cases it was possible to carry out similar studies with lymph-node cells (Table IV). These samples were mainly from patients with tumours in the lung (2 lung tumours and 3 metastases of fibrosarcoma, melanoma and hypernephroma) and from 1 osteosarcoma and 1 nasopharyngeal carcinoma. Five of 7 cases showed autologous reactivity. In none of these was cytotoxicity demonstrable against either K562 or allogeneic biopsy cells. In these cases lymphocytes froin peripheral blood were tested in parallel (Fig. 4). In 4/5 cases in which significant reactivity was found in the lymph-node preparation, blood lymphocytes were also cytotoxic and there is a good correlation between the level of cytotoxicity in the two effector populations. Patient 1280 showed reaction in neither effector population while in patient 1279 peripheral blood lymphocytes but not the lymph-node cells were cytotoxic for autologous cells. In 1262 lymph-node cells but not peripheral blood lymphocytes had ATC.

TABLE 111 SPECIFICITY TESTS WITH TUMOUR BIOPSY CELLS-PERIPHERAL

BLOOD LYMPHOCYTE EFFECTORS

Cytotoxic activity against Patient

Diagnosis Autologous tumour

1262 1002 1270 1279 1280 1281 7 1004 1265 1272 762 763 I283 1284 765

Lung Ca Osteosarcoma Fibrosarcoma Hypernephroma Melanoma Lung Ca PNC Chordoma Lung Ca Thymoma Glioma Glioma Lung Ca Lung Ca Melanoma

' Numbers in parentheses indicate spontaneous

8 (43) 27 (37) 76 (16) 25 (50) 3 (33) 24 (46) 45 (34) 0 (22) 17 (18) 43 (45) 6 (49) 33 (30) 0 (36) 0 (29) 3 (20) "Cr release from the target cells.

KS62

Allogeneic tumour (type)

10 (0)

41 (37) sarcoma 14 (43) lung 2 (27) lung 0 (49) glioma I (26) sarcoma 1 (14) lung 37 (18) lung 27 (22) sarcoma 0 (38) melanoma 4 (33) lung 2 (46) lung 0 (20) melanoma 0 (20) melanoma 4 (29) lung

32 (9) 0 (34) 6 (8) 21 (14) 10 (14) 8 (27) 57 (19) 48 (17) 6 (14) 16 (14) 27 (23) -

_-

-

518

VOSE ET AL. TABLE IV SPECIFICITY TESTS WITH TUMOUR BIOPSY CELLS-LYMPH-NODE

Patient

Cytotoxic activity against

Diagnosis Autologous tumour

Lung Ca Osteosarcoma Fibrosarcoma Hypernephroma Melanoma Lung Ca PNC

1262 1002 1270 1279 1280 1281 7

CELL-EFFECTORS

20 (43) 28 (37) 59 (16) 0 (50) 0 (33) 40 (46) 56 (34)

K562

0 (9)

-

0 (34)

3 (8) 31 (14) 9 (14) 2 (27)

Allogeneic tumour (type)

8 (37) sarcoma 7 (43) lung -

0 (49) lung 0 (26) glioma 19 (14) sarcoma 0 (14) lung

' Numbers in parentheses indicate spontaneous S'Cr release from the target cells.

> '0°1 c 80

1270

1002

7

1279

1281

1262

1280

FIGURE4 Cytotoxicity of peripheral blood lymphocytes (dotted columns) and lymph-node cells (open columns) against autologous tumour cells.

DISCUSSION

The greatest technical difficulty encountered in this study has been the preparation and characterization of target cell suspensions with high viability and low host cell contamination. Principal contaminants in the original cell suspensions were dead cells, erythrocytes, lymphocytes and macrophages. By stepwise application of separation procedures it was possible to eliminate these cells in several but not all cases. Among the target cells at least some cells were of nonmalignant derivation since separation was performed only on the basis of the physical characteristics of the different cell types, e.g. a small number of live ciliated epithelial cells

were seen in some lung tumour cell preparations. However, we believe that many of target cells were malignant by the observation of (1) secretion of mucin by isolated adenocarcinoma cells, (2) presence of melanin granules in melanoma cells, (3) multinucleated cells of giant-cell tumour preparations and (4)the establishment of wholly epithelial type cultures from the carcinomas in each case when attempted. The proportion of contaminating cells of a nonmalignant nature in the preparations cannot, however, be accurately assessed and represents the principal disadvantage of the methodology. In the targets can be considered to be of malignant origin the problem of interpretation of these data then resolves into two questions: can the cytotoxicity be attributed to (1) specific reactivity against tumourassociated antigens or (2) a susceptibility of the biopsy cells to lysis by naturally cytotoxic cells as in the case of many cultured cells? It is known that in viva harvested cells are regularly less sensitive to the killing effect of such cells. Nevertheless a line (K562) known to have extremely high sensitivity to lysis by the naturally cytotoxic cells was included in the majority of the assays. Cytotoxicity against biopsy cells with insignificant reactivity against K562 occurred in 6/11 cases and this would offer strong support for a relatively specific type of cytotoxicity against biopsy cells. Lymph-node cells were uniformly cytotoxic for biopsy cells in the absence of reactivity against K562 and most frequently cytotoxicity in peripheral blood and lymph-node cells against tumour cells coincided. Even in those cases in which cytotoxicity was present against both biopsy cells and K562 there is no reason to suppose that the two arise by the same mechanism. The naturally cytotoxic cell active against K562 has been well characterized and represents a minority non-T-lymphocyte popu-

LYMPHOCYTOTOXICITY AGAINST TUMOUR CELLS

lation carrying the Fc receptor but no surface immunoglobulin on complement receptor. While the effector populations used in this present study have a variable proportion of cells with these properties, cytotoxicity against biopsy cells may arise as a result of activity in a different lymphocyte population. The characterization of those effectors would yield valuable information about the nature of the reaction. Obviously the number of tumours tested must be increased but this paper indicates the possibility of using autologous, freshly prepared tumour cell preparations as targets in W r release cytotoxicity assays.

519

ACKNOWLEDGEMENTS

The authors are grateful to Mrs. Lena Virving and Miss Marianne Isaksson for skilled technical assistance. This work was performed persuant to Contract N01-CB-64023 with the Division of Cancer Biology and Diagnosis National Cancer Institute, US Department of Health, Education and Welfare and was also supported by the Swedish Cancer Society. One of us (BMW) was supported by grants from the Cancer Research Campaign of Great Britain and F.V. by the Stanley Thomas Johnson Foundation, Bern, Switzerland.

LYMPHOCYTOTOXICITE CONTRE DES CELLULES DE BIOPSIE DE TUMEUR AUTOLOGUE CHEZ L’HOMME L’application de techniques de separation progressive B des suspensions de cellules de biopsies de tumeurs humaines preparees mtcaniquement a permis d’isoler des cellules tumorales dont la viabilite etait &leveeet qui etaient peu contaminees par les cellules de 1’hBte. Ces cellules tumorales ont servi de cibles dans des tests de relargage du Cr6’avec comme effecteurs des lymphocytes autologues provenant du sang ou des ganglions lymphatiques. La reactivitt a 6t6 testee dans 30 cas de tumeurs malignes et 4 cas de tumeurs bknignes. Avec les lymphocytes du sang, on a detecte une cytotoxicite autologue significativedans 2 cas de tumeurs pulmonaires sur 12 et dans 9/18 autres carcinomes et sarcomes, mais pas dans les cas de tumeurs benignes. La cytotoxicite n’etait pas limitbe par la presence d’une maladie residuelle Bvidente au moment de l’operation, bien que la r6activitC soit moins souvent decelable chez les sujets souffrant d’un envahissement secondaire. Chez les quelques malades examines, la rCactivitC observee avec les cellules des ganglions lymphatiques drainant la tumeur 6tait dans l’ensemble analogue B celle que l’on constatait avec les lymphocytes du sang. Les problhmes techniques rencontrds au cours de cette etude et le rBle de la cytotoxicite naturelle dans I’interprktation de ces donnees sont discutb.

REFERENCES

BALDWIN, R. W., EMBLETON, M. J., JONES,J. S. P. I., and LANGMAN, M. J. S., Cell-mediated and humoral immune reactions to human tumours. Znt. J . Cancer, 12, 73-83 (1973). GOULMY, E., TERMIJTELEN, A., BRADLEY,B. A., and VAN ROOD,J. J., Y-antigen killing by T cells of women is restricted by HLA. Nature (Lond.), 266, 544-545 (1977). HELLSTROM, I., HELLSTROM, T. E., SJOGREN,H. O., and G. E., Dmonstration of cell-mediated immunity WARNER, to human neoplasms of various histological types. Int. J. Cancer, 7, 1-6 (1971). JONDAL, M., and PROSS,H., Surface markers on human B and T lymphocytes. VI. Cytotoxicity against cell lines as a functional marker for lymphocyte subpopulations. Znt. J. Cancer, 15, 596-605 (1975). JULIUS,M. H., SIMPSON, E., and HERZENBERG, L. A., A rapid method for the isolation of functional thymus-derived lymphocytes. Europ. J. Immunol., 3, 645-649 (1973). KONIGSBERG, I. R., Clonal analysis of myogenesis. Science, 140, 1273-1384 (1963).

Lozzio, C. B., and LOZZIO,B. B., Cytotoxicity of a factor isolated from human spleen. J . nut. Cancer Znst., 50, 535-538 (1973).

SHEARER, G. M., REHN,T. G., and GARBARINO, C. A., Cellmediated lympholysis of trinitrophenyl-modified autologous lymphocytes. J . exp. Med., 141, 1348-1364 (1975). SULIT,H. L., GOLUB,S. H., IRIE, R. F., GUPTA,R. K., GROOMS, G. A. and MORTON,D. L., Human tumor cells grown in fetal calf serum: influences on the tests for lymphocyte cytotoxicity, blocking and serum arming effects. Int. J. Cancer, 17, 461-468 (1976).

TAKASUGI, M., MIKEY,M. R., and TERASAKI, P. I., Reactivity of normal lymphocytes from normal persons on cultured tumor cells. Cancer Res., 33, 2898-2902 (1973). ZINKERNAGEL, R. M.,and DOHERTY, P. C., Restriction of in vitro T cell-mediated cytotoxicity in lymphocytic choriomeningitis within a syngeneic or semi-allogeneic system. Nature (Lond.), 248, 701-702 (1974).

Lymphocyte cytotoxicity against autologous tumour biopsy cells in humans.

Int. J. Cancer: 20, 512-519 (1977) LYMPHOCYTE CYTOTOXICITY AGAINST AUTOLOGOUS TUMOUR BIOPSY CELLS IN HUMANS B. M . VOSE1* 3, F. VANKYl , and Eva KLEI...
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