cell-mediated cytotoxicity in carcinoma of the human urinary bladder

Cancer Immunol. Immunother. 2, 233-243 (1977) ancer mmunolggyand mmunotherapy

© by Springer-Verlag 1977

Cell-Mediated Cytotoxicity in Carcinoma of the Human Urinary Bladder

M. Moore and Nicola Robinson

Paterson Laboratories, Christie Hospital and Holt Radium Institute, Manchester M20 9BX, England

Summary. Lymphoeytes from patients with tumors of the bladder or other unrelated tissues or with non-malignant genitourinary (GU) conditions and from normal subjects were tested in a mierocytotoxicity assay against long- and short-term cultures (T24 and BT) derived from bladder eareinoma and several other target cell types, to determine the validity of the hypothesis that cell-mediated eytotoxieity (CMC) in bladder caneer patients is a specific disease-related phenomenon. At the effector cell level, lymphoeytes from bladder eaneer patients displayed uniformly greater cytotoxieity for T24 and BT cells than those from normal donors. This pro- elivity was shared by the lymphocytes of patients with non-GU cancers but not of those with non-malignant GU disorders. At the target cell level, CMC was observed less frequently against non-bladder tumor targets than against T24 and B T cells and the CMC of bladder cancer patients did not differ signifieantly from that of the other groups. The pattern of CMC observed against the bladder tumor-derived targets was thus one of target cell sensitivity rather than tumor-speeifieity and disease- related only to the extent that the CMC of patients with cancer was greater overall than of healthy subjects. A brogation of CMC by passage of lymphoeytes through immunoglobulin-eoated columns indicated that the ef-

fector cells were principally of non-T type, bearing a superficial resemblance to those in normal individuals which induce non-disease related CMC.

Introduction

Comparison of cell-mediated cytotoxicity (CMC) of peripheral blood lymphocytes of cancer patients and normal individuals against cultured allogeneic human tumors has frequently been used to ascertain the specificity of cellular immune responses to a wide spectrum of cancers of different histological type and anatomical derivation. In the extensive literature relevant to this

Reprint requests should be addressed to: M. Moore

field a basic dichotomy exists, the results of which fall broadly into two categories: those which purport to demonstrate specific disease-related activity (directed against putative organ-related tumor-specific antigens) (e.g., Hellstr6m et al., 1971) and those which describe patterns of CMC against both disease-related and non- disease-related target cells (e.g. Oldham et al., 1975).

A critical development in the interpretation of in vitro cytotoxicity data was the observation of Takasugi et al. (1973) that lymphocytes from normal subjects as well as cancer patients were frequently cytotoxic for allogeneic targets, irrespective of their origin, thereby implying that CMC reactions from testing of patients on cultured cells derived from the same tumor type are not limited to tumor type-specific antigens.

In studies of CMC in human neoplasia, carcinoma of the urinary bladder has featured prominently (Bube- nik et al., 1970a and b; O'Toole et al., 1972; Bloom et al., 1974; Bean et al., 1975; Oldham et al., 1975) partly on account of the availability of established cell lines (e.g., T24) the use of which permits a limited degree of standardization among different laboratories. In the present study, this system has been adopted to determine whether the susceptibility of targets derived from bladder cancer was restricted to the lymphocytes of patients with bladder carcinoma, and thereby to determine the validity of the hypothesis that these cells express tumor-type specific antigens to which bladder cancer patients are sensitized. For this purpose, CMC of lymphocytes from patients with bladder cancer, non-malignant GU conditions or unrelated neoplasms has been assessed against bladder tumor-derived targets as well as against various other cultures originating from a diversity of human tissues.

Materials and Methods

Target Cells. All target cells were tissue culture cells derived from either normal adult, malignant, or embryonic human tissue (Table 1).

234

Table 1. Human tissue culture cells used as targets in the microcytotoxicity assay

M. Moore and Nicola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer

Tissue origin Cell culture Source designation in text

Bladder cancer T24 Dr. C. O'Toole, b (long-term)

Bladder cancer BT Dr. S. Kumar (short-term)

Lung cancer Lu (L6, L27, L49, L53) a, c

Normal lung NLu a

Teratoma Ter Dr. S. Kumar

Embryo Em (Era37, Era38) a, d

Kidney Hyp a

Liver Ch (Chang) e (long-term)

Colon Co (Co22, Co27, Co32) a, f

Cervix CCU a

Skin SCC a

a = Miscellaneous short-term cultures initiated de novo in this labo- ratory from solid tumor specimens b = Bubenik et al. (1973) c = Vose et al. (1975a) d = Vose and Moore (1976a) e = Obtained commercially f = Vose et al. (1975b)

Those described as short-term had been carried in vitro for up to 3 months while those designated long-term had been in continuous culture for 6 months or longer when used in cytotoxicity tests. T-24, a long-term epithelioid cell line, was employed because of previous reports that these cells express bladder tumor-associated antigens demonstrable by microcytotoxicity (O'Toole et al., 1972; Bubenik et al., 1973); and for comparison, a short-term culture (BT) of epithelial cells derived from an early bladder papilloma (Ib-IIa) of a 63-year- old female patient. The other cultures were selected from a spectrum of different human tissues so as to control the specificity of cytotoxicity at the target cell level. All cultures could be satisfactorily maintained in Waymouth's medium supplemented with 10% fetal bovine serum and antibiotics (ampicillin, 200 ~g/ml; streptomycin, 100 ~g/ml and amphotericin B, 2.5 ~g/ml) and were serially pas- saged once or twice weekly depending on the rate of prolifera- tion.

Effector Cells. Twenty ml specimens of defibrinated peripheral blood were allowed to sediment in a 3% (w/v) solution of gelatin at 37 ° C for 30 min (Coulson and Chalmers, 1964). The leucocyte-rich buffy coat was carefully aspirated and transferred to a warm (37 ° C) nylon column (0.6 g sterile nylon in a 2 ml plastic syringe barrel) for 30 min, according to the procedure of Greenwalt et ah (1962). Nonad- herent cells were then eluted with warm Hanks balanced salt solution (HBSS), remaining erythrocytes lysed with Tris ammonium chloride and washed a further 3 times (Boyle, 1968). The cell recovery by this method (30-50%) was lower than that for other separation procedures (e.g., Ficoll-Hypaque).

Lymphocyte populations were further purified on human IgG- anti IgG coated Degalan columns using the technique described by Wigzell et al. (1972) with slight modifications (Peter et al., 1975).

Briefly, HC1 and phosphate buffered saline (PBS) washed Degalan beads (Degussa AG, West Germany) were incubated with an equal volume of IgG (7 ml of solution + 7 ml beads), at a concentration of 2.5 mg protein/ml for 45 rain at 45 ° C, followed by overnight incubation at 4 ° C. The beads were then poured into several 5 ml plastic syringes and washed with 100 ml of PBS and incubated for at least 2 h at 25 ° C with Coombs reagent (supplied by the National Blood Transfusion Service), diluted 1 : 6. After this period, the columns were rinsed again with 100 ml of PBS and charged with 107 lymphocytes in MEM + HEPES which had previously been separated by gelatin sedimentation followed by a nylon column incubation. Cells not retained by the column were eluted at a flow rate of approximately 1 ml/min, washed once, and resuspended to the requisite concentration for the microassay. Recovery of lymphocytes with high viability from Degalan beads was accomplished by shaking in MEM + HEPES, centrifugaton, washing, and appropriate concentration adjustment.

Surface marker analysis of effector cell preparations consisted of enumeration of E-rosette forming cells and those bearing surface immunoglobulin (Ig) using standard procedures (Potter and Moore, 1975).

Cytotoxicity Assay. The cytotoxicity of peripheral blood lymphocytes from patients and healthy donors for cultured target cells was evaluated by the conventional microassay technique of Takasugi and Klein (1970). Monolayers of short-term or established tissue cultures from normal, neoplastic, and embryonic human tissues were treated with trypsin (0.1%) to produce a single cell suspension. The celIs were washed thoroughly in tissue culture medium (Eagles MEM supplemented with 10% calf serum) and the suspension adjusted to con- tain 100-150 cells per 10 ~1 medium. Ten-~d aliquots were then dispensed into the wells of sterile disposable microtest plates (Falcon; 3034) using a 500 ~1 capacity Hamilton syringe. Mierotest plates were incubated overnight (18 h) at 37 ° C in a moist atmosphere of air and 5% CO2. After incubation the medium was shaken out of the wells and the latter were washed gently to remove non-adherent target cells and debris. Effeetor cells were then added so as to give an effector cell : target cell (E : T) ratio of approximately 100 : 1 in most tests. The plates were thereafter incubated under similar conditions for a further 48 h. Finally they were washed to remove effector cells, fixed in methanol for 30 rain, stained with crystal violet for 15 min, washed and counted to determine the number of residual adherent cells (RAC). Ten replicates for each effector cell preparation were included.

The "reactivity" of all effector cell preparations was evaluated by comparison of the numbers of RAC with those cultivated in the absence of added lymphocytes (i.e., medium alone); in addition, the "reactivity" of all lymphocyte preparations from patients (with both malignant and benign disorders) was calculated with reference to the numbers of RAC surviving exposure to normal lymphocytes. The results of tests on each lymphocyte preparation are thus presented both as a percentage (cytotoxic index, CI) of the number of cells exposed to medium alone, and of the number of cells exposed to lymphocytes from normal donos.

Statistical Methods. The significance of the percentage reduction for individual lymphocyte preparations was determined by the Student's t-test at the 95% level. Since the data were not normally distributed, non-parametric techniques were also used: the Mann Whitney U test for comparison of reactivity between patient groups and controls and the Wilcoxon matched pairs signed ranks test for comparison of effector cell reactivity before and after passage through Degalan columns (Siegel, 1956).

Experimental Design. The majority of the experiments were designed so as to permit simultaneous tests of effector cell preparations from

M. Moore and Nicola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer 235

normal donors and patients with various malignant and non-malignant disorders against both bladder cancer and unrelated target cells. Thus, in practice effector cell preparations from patients with bladder carcinoma, 2 or more with unrelated cancers (or benign GU disorders), and 1 (or occasionally 2) unselected normal donor(s) were tested simultaneously against the T24 cell line or the BT cell or both, plus at least 1 other unrelated target cell

Clinical Material. Blood specimens were obtained during the morn i ing of the experiment and effector cells isolated and used the same day. No patient with carcinoma of the bladder or any other neo- plasm was currently receiving or had previously received chemother- apy or radiotherapy. Samples from patients with malignancy were from new case presentations with the exception of a small number of patients with recurrent breast carcinoma. Patients with carcinoma of the urinary bladder, in whom interest was centred, were classified according to the UICC (1963) staging system viz: tumor with infiltration of subepithelial connective tissue - T1 (9 patients); tumor with infiltration of superficial muscle - T2 (38); tumor with infiltration of deep muscle - T3 (37); and tumor fixed or invading adjoining organs - T4 (14). Patients with other neoplasms were a heteroge- neous group with respect to both tumor type and clinical staging and comprised the following: tumors of the head and neck (12 patients); esophagus (2); lung (10); breast (6); cervix (5); uterus (3); ovary (2); vagina (1); kidney (1) and sarcoma (1). Non-malignant GU conditions were represented by those of more common type, viz., calculi, stricture, benign prostatic hypertrophy, primary kidney conditions such as glomerular nephritis and pyelonephritis. Most of the patients in this group were undergoing treatment at the time when blood was taken. Most normal donors were either hospital medicaI or technical staff, and no attempt was made to identify and select donors with minimal reactivity against cultured targets. Controls were matched to patients with respect to sex but there was some unavoidable disparity in age. The mean age (± S.E.) of the controls was 28 ± 1 years; while those of bladder cancer patients and patients with unrelated cancers and non-malignant genitourinary conditions were 68 ± 1, 66 ± 2, and 53 ± 3 years, respectively.

Results

phonuclear leucocytes and monocytes of preparat ions

from controls, and various cl inicopathological catego-

ries are shown in Table 2. The da ta reveal that the nylon column purification procedure in general yielded preparations of uniformly high lymphocyte content with some polymorphonuclear leucocyte contaminat ion but virtual absence of monocytes . Differences in the relative pro- por t ion of any of the three major cell types amog the various patient groups were not significantly different from those of normal subjects.

I t was further shown that the principal component of the purified effector cell populat ion was the T lymphocyte which accounted for 79 + 1% and 81 _+ 1% of the cells present in the preparat ions from normal donors and bladder cancer patients respectively. Ig-bearing cells which may have included small numbers of non-lym- phocyt ic elements accounted for 22 _+ 2% and 19 + 1% of the same preparat ions (Table 3).

CMC of Normal Allogeneic Lymphoeytes Evaluated with Reference to Medium Alone

Twenty of 63 (32%) preparat ions were significantly cytotoxic for T24 cells, although the range of activities was wide (range, - 3 7 to 59%; mean CI, 11 +_ 3% S.E.) (Fig. 1). "Feeder effects", in which the number of targets in the presence of added lymphocytes exceeded those in medium alone, were observed for a minori ty (4/63 or 6%) of preparat ions. Comparab le results were obtained for BT cells, against which 9/22 (41%) preparat ions were cytotoxic (range, - 4 0 to 59%, mean CI, 14 + 5%) (Fig. 2). Reduct ion of a further 9 unrelated targets was significant in 32/69 (46%) tests (Fig. 3).

Composition of Effeetor Ceil Preparations

Differential counts were made on the major i ty of the effector cell preparat ions utilized in microcytotoxici ty assays and mean values for the lymphocytes , polymor-

CMC of Lymphocytes from Bladder Cancer Patients Evaluated with Reference to Medium Alone

Sixty-six of 81 (82%) preparat ions were cytotoxic for T24 (range, - 1 2 to 86%, mean CI 42 _+ 2%) (Fig. 1);

Table 2, Gross cellular composition of effector cell populations a

Donors No. examined

Percentage cell Types (± S.E.)

Lymphocytes Polymorphs Monocytes

Normal subjects 65 93.4 _+ 0.5 5.6 _+ 0.4 0.8 ± 0.2 Bladder cancer (T1) 5 93.4 ± 1.9 5.4 ± 1.5 1.0 ± 0.4 Bladder cancer (T2) 34 91.7 +_ 1.2 7.1 ± 1.0 0.9 ± 0.5 Bladder cancer (T3) 22 90.0 _+ 1.0 8.9 ± 1.0 0.9 ± 0.2 Bladder cancer (T4) 10 86.0 ± 2.7 12.2 ± 2.5 1.5 ± 0.5 Other cancers 35 89.7 ± 1.1 9.3 _+ 1.1 1.1 ± 0.2 Non-malignant GU 24 90.2 + 1.2 8.5 ± 1.2 1.2 ± 0.4

a Prepared by gelatin sedimentation and nylon column-incubation

236 M. Moore and Nicola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer

Table 3. Proportions of E-rosette forming and Ig-bearing cells in effector cell preparations from bladder cancer patients and normal donors

Separation procedure Donors (no. examined)

Percentage cell type (± S.E.)

E-rosette forming Ig-bearing cells cells

Gelatin alone Mixed (11)" 55 + 4 ND

Gelatin + Normals (40) 79 ± 1 22 ± 2 nylon column

Gelatin + Bladder cancer 81 _+ 1 19 + 1 nylon column patients (29)

Gelatin + nylon column followed by Degalan (Ig-anti Ig) column

Degalan (Ig-anti Ig) column b

Normals (29) 91 _+ 1 11 _+ 2

Bladder cancer 93 + I 14 ± 2 patients (37)

Bladder cancer 34 _+ 3 b 78 ± 1 b patients (13) b

ND, not determined a Comprised preparations from 7 patients with bladder cancer and 4 healthy controls b Illustrates composition of population retained and subsequently recovered from Dega- lan beads See "Materials and Methods"

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Fig. I. Cytotoxicity of peripheral blood lymphocytes from patients with bladder cancer (all stages), unrelated cancers, non-malignant GU conditions, and normal donors for the long-term bladder cancer cell line, T24. Per cent cytotoxicity is calculated with reference to a medium control and unselected normal donors. Open and closed circles denote reactions of statistical significance (P (0 .05) and insignificance respectively in comparison with the appropriate control


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Fig. 2. Cytotoxicity of peripheral blood lymphocytes from patients with bladder cancer (all stages), unrelated cancers, non-malignant GU conditions, and normal donors for the short-term bladder cancer cell culture, BT. Per cent cytotoxicity is calculated with reference to a medium control and unselected normal donors. Open and closed circles denote reactions of statistical significance (P < 0.05) and insignificance respectively in comparison with the appropriate control

and 37/40 (93%) were cytotoxic for BT cells (range, 0 to 99%, mean CI 51 + 4%) (Fig. 2). Against the unrelated targets from 9 different tissues, cytotoxicity was observed in 41/95 (43%) tests (Fig. 3). The difference in cytotoxic activity between bladder cancer patients (n = 81) as a group and normal subjects (n = 63), against T24 (U=4135, Z=6 .8596 , P(<0.001) was highly significant and a similar distinction in reactivity (where n = 40 and 22 for bladder cancer patients and normals respectively) was observed against BT cells (U-- 724, Z = 4.1792, P << 0.001). However there were no significant differences in reactivity between patients classified according to tumor stage (Table 4).

CMC of Lymphocytes from Patients with Non GU Cancers Evaluated with Reference to Medium Alone

In common with normal donors and bladder cancer patients, lymphocytes from patients with non-GU cancers were cytotoxic for both T24 and BT cells. Against the former culture 24/30 (80%) preparations were cytotoxic

(range, - 4 8 to 94%; mean 37 + 5%) (Fig. 1) and against the latter, 21/23 (91%) preparations were cytotoxic (range, 11 to 93%; mean 51 + 5%) (Fig. 2). The frequency of positive tests against the unrelated targets was 38/70 (54%) (Fig. 3). There was no significant difference between the cytotoxicity of bladder cancer patients (n = 81) and those with non-GU cancers (n = 30) against T24 ( U = 1300, Z---0.5644; P > 0 . 5 ) nor against BT cells (where n = 40 and 23 for the respective groups of patients) ( U = 465, Z = 0.0714, P > 0.94).

CMC of Lymphocytes from Patients with Non-Malignant GU Conditions Evaluated with Reference to Medium Alone

Twelve of 24 (50%) preparations were significantly cytotoxic for T24 (range --3 to 88%, mean 23 + 5%) (Fig. 1), but the reactivity of the group as a whole did not differ significantly from that of normal donors (n = 63) ( U = 908, Z = 1.72, P > 0.08). Tests on BT cells and the unrelated targets were limited to small numbers (Figs. 2 and 3).


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Fig. 3. Cytotoxicity of peripheral blood lymphocytes from patients with bladder cancer (all stages) (O), unrelated cancers ([3), non-malignant GU conditions (V), and normal donors (&) for target cells derived from several normal, neoplastic and embryonic tissues (see Table 1 for details). Per cent cytotoxicity is calculated with reference to a medium control and unselected normal donors. Open and closed symbols denote reactions of statistical significance (P < 0.05) and insignificance respectively, in comparison with the appropriate control

Table 4. Cell-mediated cytotoxicity (CMC) of bladder cancer patients' lymphocytes as a function of tumor stage

Clinical No. staging patients

tested

Mean eytotoxicity index (CI)

With respect to With respect to medium alone a normal donors b

T1 9 53 + 7 42 + 6 T2 38 43 + 4 38 _+ 3 T3 37 39 + 4 38 4- 5 T4 14 36 _+ 5 33 _+ 5

Evaluated against T24 and BT ceils a Statistical analyses by Mann-Whitney U test:

T1 vsT2, U = 2 0 7 , Z = 0 . 9 7 3 , P > 0.3 T1 vsT3, U = 2 1 7 , Z = 1.398, P > 0.15 T1 vsT4, U = 79, Z = 1.369, P > 0.17

b T1 vsT2, U = 192.5, Z = 0.581, P > 0.5 T1 vsT3, U = 197; Z = 0 . 8 4 5 , P > 0.4 T1 vs T4, U = 69.5, Z = 0.735, P > 0.4

CMC of Cancer Patients' Lymphocytes Evaluated with Reference to Normal Donors

Sixty-nine of 81 (85%) preparations from bladder cancer patients and 25/30 (83%) preparations from patients with unrelated cancers were cytotoxic for T24 cells (mean CIs 37 _+ 2% and 31 + 5%, respectively) (Fig. I). There was thus no difference between these groups in respect of reactivity toward T24 ( U = 1389, Z = 1.1554, P>0.02) . Similarly, 35/39 (90%) and 17/23 (74%) preparations from the respective groups of patients were cytotoxic for BT cells (mean CIs 40 + 4% and 32 + 6%) (Fig. 2) and these differences were not significant ( U = 539, Z = 1.3264, P > 0 :18 ) .

Subdivision of the bladder cancer patients according to tumor stage failed to disclose significant differences in CMC (Table 4).

M. Moore and Nicola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer

Sixty-two of the 81 lymphocyte preparations from bladder cancer patients and those from all of the patients with unrelated cancers were simultaneously tested against at least one other unrelated target. In all tests, the control lymphocytes originated from the same donors as those in the parallel tests against T24 cells and/or BT cells. The numbers of tests in which these preparations were cytotoxic among bladder cancer patients and those with unrelated tumors were comparable, viz., 11/92 (12%) and 12/73 (16%), respectively, and revealed that these targets were markedly less susceptible to CMC than the bladder tumor-derived cultures.

CMC of Lymphocytes from Patients with Non-Malignant GU Conditions Evaluated with Reference to Normal Donors

These patients comprised the only group in which reactivity was exceeded by that of the normal donors with which they were matched. Thus 4/24 (17%) of preparations were significantly cytotoxic for T24, whereas 5/24 (21%) enhanced target cell survival (Fig. 1). The difference between the reactivity of these patients and those with bladder cancer (n = 81) as groups was thus highly significant ( U = 1700, Z = 5.5563, P<(0.001).

Effect of Passage of Effector Cells through Degalan Coated (IgG-anti IgG) Columns

239

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Fig. 4. Comparative cytotoxicity of peripheral blood lymphoeytes initially prepared by gelatin sedimentation and nylon column incubation, before and after passage through IgG-anti IgG coated Degalan beads. Pre- and post-Degalan lymphocyte populations were tested against 3 target cells: T24 ((3); BT (A) and Hyp (O). Per cent cytotoxicity is calculated with reference to unseleeted normal donors. Open and closed symbols denote reactions of statistical significance (P < 0.05) and insignificance respectively in comparison with normal donor reactivity. For details of the lymphocyte separation procedures see "Materials and Methods"

The cytotoxic potential of nylon column purified lymphocytes from a total of 33 bladder cancer patients was evaluated against T24, BT and Hyp cells before and after passage through Degalan coated (IgG-anti IgG) columns. From both cancer patients and normal donors these effluents contained a uniformly higher proportion of E-rosette forming cells (93 _+ 1 and 91 + 1% respectively) and correspondingly fewer Ig-bearing cells (14 + 2 and 11 + 2% respectively) than the nylon column eluates (Table 3). The results of cytotoxicity tests, calculated with respect to the reactivity of normal donors the lymphocytes of whom were subjected to identi- cal isolation procedures, are presented in Figure 4. Pas- sage of effector cells through coated Degalan columns effectively abrogated the cytotoxicity of bladder cancer patients' lymphocytes for both T24 and BT cells. Thus, against T24 cells, the frequency of positive reactions and mean CIs respectively for patients' lymphocytes were 22/33 (67%) and 33 + 4% pre-Degalan; and 1/32

O (3%) and - 7 + 3 ~ post-Degalan. These differences in reactivity were highly significant ( n = 3 2 , T = 2 ,

Z = 4 . 9 , P<<0.001). Similar results were obtained against BT cells where the corresponding figures were 17/18 (95%) and 46 + 7% pre-Degalan; and 2/18 (11%) and 2 + 5% post-Degalan ( n = 18, T = 4 , Z = 3.46, P < 0.001). Against the unrelated and less susceptible target, Hyp, 2/9 (22%) lymphocyte preparations from bladder cancer patients were cytotoxic pre- Degalan (mean CI, 8 _+ 7) and 1/9 (11%) post-Degalan (mean CI, - 2 + 4). This difference was not significant (n -- 9, T = 10, Z = 1.48, P > 0.13).

On 13 occasions, lymphocytes retained by coated Degalan columns were recovered and tested against T24 cells under the standard conditions of the microtest. Eleven of these preparations were cytotoxic for T24 although the mean CI of this group (26 + 3%) was lower than that of the lymphocytes prior to passage through the column (33 + 4%, based on 33 patients). The proportion of Ig + cells in the recovered population was 78 + 1% and of E-rosette forming cells, 34 _+ 3% (Table 3).

240

Discussion

The cytotoxicity of purified lymphocyte preparations from the peripheral blood of normal subjects and patients with bladder cancer, tumors arising in other organs and non-malignant GU conditions against in vitro cultivated cells derived from two bladder carcinomas and a variety of other human tissues, has been compared. The conventional microplate technique based on visual enumeration of surviving target cells following exposure to a single concentration of effector cells was adopted as the assay procedure, and any statistically significant reduction in the number of target cells in- duced by a given effector population compared with appropriate controls was presumed to be indicative of CMC. Results were expressed with reference to medium alone and to the reactivity of unselected normal donors. In this way any inherent reactivity in normal subjects could be monitored and simultaneous unbiased comparison made with preparations from patients.

The first main feature of this investigation was the finding that coincubation of healthy donor lymphocytes with several targets resulted in diminished survival compared with medium alone. Supportive effects of added normal lymphocytes on target cells were also observed, but less frequently than inhibitory effects. The probabil- ity that reduction in survival represented a cytotoxic event and was not simply due to exhaustion of nutrients was indicated by the fact that only a proportion of lymphocytes from normal subjects possessed significant cytotoxic activity and in quantitative terms this varied markedly between individuals. In this respect these data agree closely with several reports from our own (Vose et al, 1975b; Vose and Moore, 1976a and b) and other laboratories (Takasugi et al, 1973; Pavie-Fischer et al., 1975; Oldham et al., 1976., Bloom and Seeger, 1976), imputing spontaneous cytotoxicity against various targets to lymphocytes from individuals with no apparent pathological abnormality, (non disease-related CMC) which was not disclosed in many early CMC studies. In concordance with some investigators (Oldham et al, 1975) but by contrast with others (Bean et al., 1973; 1974) this reactivity was not wholly non-specific. Thus, in those instances where sufficient targets were simultaneously included for evaluation, normal reactivity was limited to certain targets rather than non-specific in the sense that all targets were equally susceptible to cytolysis by a given lymphocyte preparation.

The second major feature of this study was the de- monstration that CMC against a diversity of targets encountered among normal controls is also common among cancer patients irrespective of tumor type. This was particularly evident in tests utilizing T24 cells where the reactivity of the bladder cancer patients as a group

M. Moore and Nieola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer

was indistinguishable from that of patients with unrelated tumors.

In common with all studies of this type the wide spectrum of eytotoxic activity encountered among normal subjects and patients with neoplastic and non-neoplastic diseases makes it difficult to impose strict criteria for the estimation of the frequency, magnitude, or specificity of CMC reactions. On the basis of the variable activity of normals, some investigators (e.g., Oldham et al., 1975; Pierce and DeVald, 1975) have argued that the least active normal (LAN), determined by comparison of the reactivity of several normal donors, is the superior control; whereas others (e.g., Bloom and See- ger, 1976) on the basis of their observed reactivity among nonmalignant disease controls have maintained that medium alone should comprise the reference standard. Although neither control is without disadvantages, we have presented our data with reference both to medium alone and to normal donor activity. However, in the interpretation of our data, there are other limitations which must also be recognized: first, the percentage reduction of target cells calculated from tests utilizing a single concentration of lymphocytes is not proportional to their eytotoxic activity and, second, the possibilty that differences in activity falling outside the range estimated at a particular concentration might be missed (Hakala et al., 1976). Even so, the majority of patients' lymphocytes tested against the bladder tumor-derived targets revealed quantitatively greater reactivity than those from unselected normal subjects. The manner of effector cell separation and testing was such that this consistent increase was not an artefact of sample collec- tion or of the assay system, and it seems unlikely that the disparity in age between patients and controls could account for the difference (Oldham et al., 1975). Fur- thermore, the degree of reactivity among the cancer patients, with reference to normal donors, against the bladder tumor-derived cells was greater than that against the unrelated targets. In essence, therefore, the pattern of CMC observed against bladder tumor targets in this study was one of target cell sensitivity, rather than tumor specificity, and disease-related only in the broad sense that the lymphocytes of patients with malignancy had a greater overall capacity for CMC than those of normal subjects. Takasugi et al. (1973), in a study in- volving more than 2000 tests on 7 established cell lines and 12 short-term cultures, demonstrated greater cytotoxicity by lymphocytes from bladder cancer patients over those from normal subjects against a short-term culture of bladder tumor derivation (designated 458). However, no data on the susceptibility of culture 458 to effector cells from patients with other cancers are avail- able for comparison.

Confining the discussion to studies which have con- centrated on cellular immunity in urinary bladder carci-

M. Moore and Nieola Robinson: Cell-Mediated Cytotoxicity in Bladder Cancer 241

noma, our data have points of resemblance with those of several other investigators, but with no single study is there precise overlap. This was not unexpected in view of the many variables in methodology and patient selection entailed in CMC testing (Herberman and Oldham, 1975; Bean et al., 1975). Thus, our data differ from those of Bubenik et al. (1970a and b) and O'Toole et al. (1972; 1973a) in respect of tumor specificity while ap- parently concurring with the nature of the cytotoxic cells involved (discussed below). Similarly, there are several features in common with the results of Bloom et al. (1974), who demonstrated uniformly strong CMC against T24 cells by bladder cancer patients but differences in regard to the distribution of reactivity among bladder cancer patients classified according to tumor stage and among patients with non-malignant GU disorders or carcinoma. Although there are technical differences between the two studies the anomaly might be partially attributable to patient selection. In the majority of T4 stage bladder cancer patients, any metastases beyond the pelvis were occult at the time of CMC testing by contrast with the advanced cases studied by Bloom et al. (1974), where disease was widely dissemi- nated. Furthermore, in the present study those patients with non-malignant GU diseases comprised the most he- terogeneous group and the only one in which patients were undergoing, or had recently undergone, treatment; while the majority of the cancer patients tested had max- illo-facial or pulmonary neoplasms of variable body burden and thus differed from those with renal and prostatic carcinoma studied by Bloom et al. (1974).

The lack of concordance with other studies in which CMC against bladder cancer has been a dominant theme appears to be primarily a reflection of methodo- logical differences, rather than of patient selection. For instance, Oldham et al. (1975) failed to distinguish significant differences in reactivity against T24 (and RT4) cells between bladder cancer patients and selected (i.e., least active) normal controls. A notable difference in these tests was the adoption of the proline-labelling modification (Beat et al., 1973), which differs in some respects from the visual microcytotoxicity assay and the use of higher E : T ratios.

The observed pattern of target cell sensitivity, as dis- tinct from tumor specificity, raises obvious questions about the nature and relevance of the various targets. Differences in the susceptibility of T24 and BT cells to cytolysis were marginal. This finding contrasts with experience in other systems (De Vries et al., 1975), where stronger non-disease related CMC was observed against long-term cell lines compared with short-term cultures. An important feature of both bladder tumor-derived cultures was that they were uniformly epithelial and possessed a high plating efficiency and short doubling time in the microplate wells. T24 cells, in particular, gave rise

to palpable tumors (anaplastic carcinomas) on subcuta- neous injection into athymic "nude" mice (Pimm, M. V. and Moore, M., unpublished observation). Although the possibility was not formally examined, it is conceivable that the relative sensitivity of T24 and BT cells to cytolysis compared with unrelated targets, most of which were short-term cultures and not all tumor-derived, may be a reflection of factors such as cellular heterogeneity, lower plating efficiency and longer doubling time. Pierce and DeVald (1975) similarly stressed the importance of the "growth status" of target cells used in CMC tests, based on the differential sensitivity of tumor cells and adult fibroblasts to cytotoxicity by lymphocytes from cancer patients and normal subjects. Diminished sensitivity of normal tissue cell lines to CMC was also em- phasized by Skurzak et al. (1973).

Any likelihood that tumor specificity of CMC in the present study is obscured by nonselective effects arising from the method of effector cell preparation is remote. The method used was chosen so as to circumvent Ficoll- Hypaque which reportedly non-specifically activates lymphoid cells (Oldham et al., 1973), although the pattern of CMC observed for T24 cells did not differ significantly from that previously reported from this labora- tory in another context (Vose and Moore, 1976a), using the latter separation technique. The degree of non-lymphoid cell contamination to which some nonspecific cytotoxic effects (particularly by polymorphs) can un- doubtedly be attributed (Takasugi et al., 1975; Vose and Moore, 1976b) was much lower than in many of the earlier studies purporting to demonstrate tumor-specificity ofCMC (Bubenik et al., 1970a and b). However, it is significant that by avoidance of the use of Ficoll-Hypa- que we have been able to derive no more definitive evidence for organ-related tumor-associated antigens on urinary bladder carcinoma than we have by adopting the procedure in other tumor-host systems (Vose et al., 1975a and b). In this respect at least our cumulative experience concurs with that of several other laboratories (Oldham et al., 1975; Bloom and Seeger, 1976).

Abrogation of CMC of bladder cancer patients lymphocytes for T24 cells by passage through Degalan coated (IgG-anti IgG) columns confirmed that the principal effector cells were of non-T type (O'Toole et al., 1973; 1974). Although in this respect they bear a superficial similarity to the cells known to be responsible for non-disease related CMC in normal individuals (Kiuchi and Takasugi, 1976), our data do not distinguish whether the reactivity against sensitive cells exhibited by patients with other malignant and non-malignant disorders differs from that of normals in kind or degree. In the absence of demonstrable tumor type-specific reactivity we are inclined to the view that the differences between healthy subjects and patients with malignant or non- malignant diseases are primarily a reflection of differ-


ences in the numbers or unit activity of cytotoxic non-T cells in peripheral blood. Conceivably the effect of various "pathological processes" could be to augment these, though not necessarily to the exclusion of patterns of CMC which might be vested in other populations.

Acknowledgements: This study was supported by grants from the Medical Research Council and the Cancer Research Campaign. We are grateful to the consultants of the University Hospital of South Manchester for access to the patients under their care, in particular Dr. R. C. S. Pointon, Consultant Radiotherapist, and Dr. R. Hunter for assistance in the collation of clinical material. In addition, we thank Ian Kimber and Roger Ferguson for their skilled technical assistance and Mr. R. Swindell for undertaking the statistical analyses.

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Received October 12, 1976/Accepted November 11. 1976

cell-mediated cytotoxicity in carcinoma of the human urinary bladder

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