characterization of cytolytic effector cells in … · 2013-12-18 · ii. cytotoxicity to...

THE JOURNAL OF IMMUNOLOGY Vol. 118, No. 6, June 1977 Copyright © 1977 by The Williams & Wilkins Co. Printed in U.S.A.

CHARACTERIZATION OF CYTOLYTIC EFFECTOR CELLS IN PERIPHERAL BLOOD OF HEALTHY INDIVIDUALS AND CANCER

PATIENTS

II. Cytotoxicity to Allogeneic or Autochthonous Tumor Cells in Tissue Culture

GERD R. PAPE, 2 MARITA TROYE, AND PETER PERLMANN

From the Department of Immunology, University of Stockholm, Fack, S-10691 Stockholm 1, Sweden

Peripheral blood lymphocytes from patients with transitional cell carcinoma of the urinary bladder, from control tumor patients, and from healthy individuals were fractionated on columns retaining 1) cells with Fc-receptors for IgG, as defined by rosette formation with IgG-coated erythrocytes, 2) cells with surface-bound immunoglobulin, or 3) cells with both types of surface markers. The spontaneous cytotoxicity of these lymphocytes against allogeneic or autochthonous tumor cells of bladder carcinoma or unrelated origin was studied in a ~lCr-release assay. The cytotoxicity to allogeneic target cells of lymphocyte fractions depleted of B cells (Slg + cells) was only slightly reduced, unchanged, or, occasionally, augmented when compared to that of unfractionated lymphocytes. Fractions containing the majority of the B cells present initially but depleted of most of the Fc-receptor-bearing lymphocytes (EA ÷ cells) displayed a strongly reduced cytotoxicity against the same tumor cells. These effector cell fractions were also depleted o f a majority of the complement receptor-bearing lymphocytes (EAC ÷ cells) and of a minor but significant fraction of T cells (E + cells) and of a minor but significant fraction of T cells (E + cells). Removal of both SIg +- and Fc-receptor-bearing cells gave the same results. Thus, in most instances, the spontaneous cytotoxicity against allogeneic tumor cells o f fractionated lymphocytes was similar to their K cell activity against chicken erythrocytes (Ec) in the presence of rabbit anti-Ec antibodies (preceding paper). In conjunction with other data on the immunoglobulin dependence of this spontaneous cytotoxicity these results suggest that a significant fraction of this reactivity, displayed by both tumor patients' and normal individuals' lymphocytes, may be antibody dependent. The antibodies may either be carried over cytophil- ically during lymphocyte isolation or may be formed and released by some antibody-producing cells during the in vitro incubation. In two cases, in which bladder cancer patients' lymphocytes were tested with both autochthonous and allogeneic target cells, the effector cells killing the autochthonous target cells were neither B cells nor Fc-recep- tar-bearing cells. These results suggest that different effector mechanisms were involved in these cases.

Submitted for publication December 15, 1976. 1 This work was supported by Grants 365-B76-08XB from the

Swedish Cancer Society and B 2032-047 from the Swedish Natural Science Research Council.

2 G. R. Pape is a holder of a fellowship from Deutsche Forschungs- gemeinschaft, F.R.G. Permanent address: Medizinische Klinik (II), Universit~t Tiibingen, F.R.G.

Purified peripheral blood lymphocytes of cancer patients display cytotoxicity when tested in various in vitro assays against a variety of allogeneic and autologous tumor target cells (1-5). Lymphocytes of healthy controls may also display cytotoxic reactivity in vitro against various cell types. This phenomenon has been reported for both human (6-10) and animal (11-15) tumor target cells. However, in a large number of test systems the nature of the cytotoxic effector cell(s) is either unknown or not precisely defined. Antibody dependent as well as antibody-independent cell-mediated cytotoxicity may both operate in tumor-bearing individuals as described for Moloney sarcoma-bearing mice (16-18). In humans, cytotoxicity mediated by sheep erythrocyte binding (E-rosetting) cells (thymus-derived lymphocytes) has been reported for malignant melanoma (19) and for colon carcinoma (20). Other groups have shown that "nonthymus-derived" effector cells may be cytotoxic in malignant melanoma (6, 21) or in urinary bladder carcinoma (22, 23). Several different types of effector cells have also been reported to be responsible for "spontaneous" cytotoxicity in man. These comprise Fc-receptor-bearing cells (6), activated T cells (24), lymphocytes with both Fc- receptors, and complement receptors (25), E--EAC ÷3 lymphocytes (21), or ~'nonselective cytotoxic cells" which are SIg--E-- EAC- (26). Cytotexicity due to soluble factors has also been discussed (27, 28). For the mouse, a natural killer cell of lymphoid type has been described, which lacks all these markers as well as monocyte-macrophage characteristics (12, 13, 15). It is not established to what extent this "spontaneous" cytotoxicity reflects on immune response to target cell antigens. These seemingly contradictory results imply a heteroge- neity of effector mechanisms to be operative in tumor-associated immunity and in "spontaneous" cytotexicity. Precise characterization of the nature of the effector cells is essential for a valid interpretation of the results obtained in various in vitro assay systems. We have approached this problem by fractionating lymphocytes from bladder cancer patients and

3 Abbreviations used in this paper: CTL, antibody-independent cytolytic T cells; E + cells, cells forming spontaneous rosettes with sheep erythrocytes; EA ÷ cells, cells forming rosettes with IgG-coated bovine erythrocytes; EAC + cells, cells forming rosettes with IgM and complement-coated sheep erythrocytes; Ec, chicken erythrocytes; F(ab')janti-F(ab')~ column, column charged with human F(ab')~ fragments and F(ab')2 fragments of rabbit IgG antibodies to human F(ab')~; IgG/anti-IgG column, column charged with human IgG and intact antibodies of rabbit anti-human IgG; OAtanti-OA column, column charged with ovalbumin and rabbit antibodies to ovalbtumin; SIg + cells, surface immunoglobulin-positive cells; HD, healthy donors.

1925

1926 GERD R. PAPE, MARITA TROYE, AND PETER PERLMANN [voL. 118

healthy individuals on three different types of columns retaining 1) cells with Fc-receptors for IgG, 2) cells with surface bound immunoglobulin, or 3) cells with both types of surface markers. The results of the fractionation procedures, the surface marker profiles, and K cell activity in a model system of the different cell fractions were reported in the preceding paper (29). In this paper, the cytotoxicity of the different lymphocyte fractions from either cancer patients or healthy individuals to allogeneic or autochthonous tumor cells was studied.

M A T E R I A L S A N D METHODS

Patients and healthy controls. Patients and healthy donors (HD) in the present study represent a selected group. Only those individuals were chosen whose lymphocytes display cytotoxic reactivity in the ~lCr-release assay against tumor target cells. Most of the patients and controls were identical with those in the preceding paper (29). We tested lymphocytes from 13 patients with transitional carcinoma of the urinary bladder, nine males and four females; their ages ranged from 51 to 76 years (mean 64 years). Nine patients were untreated in regard to their cancer whereas two had received radiotherapy 9 and 12 years earlier. In two patients the tumor was removed surgically several months before the blood was drawn. All five patients with cancer prostatae were untreated. Their ages ranged from 52 to 77, mean 64 years. Since no attempts were made in this study to correlate the in vitro results with the clinical status of the patients the clinical data are not further detailed. Nine healthy donors were included in our study, six were males and three were females. Seven of the HD were between 25 and 35 years old, 2 between 50 and 60 years.

Purification and fractionation of lymphocytes. This procedure has been described in the preceding paper (29). Surface immunoglobulin positive (SIg +) lymphocytes were removed by fractionation on glass bead columns charged with F(ab')2 fragments of human IgG and F(ab')2 fragments of rabbit IgG antibodies to human F(ab')2 (F(ab')Janti-F(ab')2). Fc-receptor-positive cells were removed on columns charged with ovalbumin and rabbit anti-ovalbumin (OA/anti-OA). Cells with Fc-receptors and/or SIg were removed on columns charged with human IgG and rabbit anti-human IgG (IgG/anti-IgG).

Characterization of the lymphocytes. Surface marker tests by rosette formation for sheep erythrocytes (E+), complement receptors (EAC+), and Fc-receptors (EA+), and by direct or indirect immunofluorescence for SIg were done as described in (29). Antibedy-dependent cell-mediated cytotoxicity (K cell activity) was assayed with 51Cr-labeled chicken erythrocytes (E~) as target cells and rabbit anti-Ec as inducing antibody (30).

Target cells. Several established cells lines were used as target cells in the cytotoxicity assay: T 24 (31) derived from transitional cell carcinoma of the urinary bladder (TCC), HCV 29 possibly nonmalignant specimen of urinary bladder epithelium (established by J. Fogh, Sloan Kettering Institute, New York), HT 29, a CEA-producing cell line, derived from a colon carcinoma (32), and Mel-1 from a metastatic malignant melanoma (established by O'Toole, 1972). In some experiments we also used cells from two other TCC lines, J 82 (established by O'Tocle, 1973) and MANO (established by Troye, 1974), and cells from a primary culture (patient E.J.) in its fifth passage. The MANO-line and the E.J. primary culture were tested with both autelogous and allogeneic lymphocytes. All cultures were passaged once a week and were free of mycoplasm. The tissue culture medium was Parker 199 (Biocult, Labs., Ltd., Glas-

gow, Scotland) supplemented with 2 mM glutamine, penicillin (100 IU/ml), streptomycin (100 tLg/ml), and 10% fetal bovine serum (FBS).

~lCr-release assay. The isotope release assay was performed as described elsewhere. 4 Twenty-four hours before the cytotoxic tests a short-term monolayer culture of the target cells was established in 35-mm tissue culture Petri dishes (Falcon 3001, Oxnard, Calif.). The target cells were removed from the Petri dishes by trypsinization (0.25% in PBS with 0.2% EDTA), washed, and then labeled by incubation for I hr with Na2~lCrO4 (specific activity 0.5 to 1 mCi/ml, 3 to 20 tLg Cr/ml, Radiochemical Centre, Amersham, England). One-tenth milliliter 5~Cr/0.7 × 106 target cells was used, total volume 0.6 ml. The cells were washed, counted in trypan blue, and used for the test. The cytotoxic test was performed in conical plastic tubes (0.7 × 11 mm) containing 5 × 103 target cells in 0.25 ml TCM + 10% FBS, 1.5 or 3 × 105 lymphocytes (ratio L/T 30:1 or 60:1) in 0.25 mt TCM + 10% FBS, and 0.25 TCM + 10% FBS. After incubation for 18 hr at 37°C in humidified air containing 5% CO2 the cells were centrifuged for 10 min at 1200 to 1500 rpm (300 × G). Half a milliliter of the supernatant was with- drawn and counted. Spontaneous release of 51Cr in the majority of experiments was between 10 and 20%. Cells showing a spontaneous release >25% were not used. The percentage of specific lysis was calculated according to the formula:

cpm in of tube supernatant experimental % Lysis = [ Total cpm.

cpm in supernant of control tube~ - J × 100

All tests were performed in duplicates. The error between duplicates was -<2.5%.

R E S U L T S

As already stated, all lymphocyte donors included in this study were selected on the basis of showing some cytotoxicity above background against one or several of the target cells used. The question of the disease-related specificity of the bladder cancer patients' lymphocytes against bladder tumor cells has been dealt with elsewhere. 4 The experiments described below were performed to elucidate the nature of the effector cells operative in either patients or controls.

Cytotoxicity of bladder patients' lymphocytes to allogeneic target cells of different types. The cytotoxicity of the patients' lymphocytes before and after fractionation through F(ab')J anti-F(ab')2 columns for different target cell types is shown in Figure 1. As seen, removal of the SIg + cells by this procedure caused in most instances a slight reduction of cytotoxicity. The mean reduction for T24 or HCV, tested at the ratio 60:1 with lymphocytes from eight or seven donors, respectively, was 3.4 -+ 5.0 or 4.2 _+ 3.8, respectively. The difference between unfractionated and fractionated lymphocytes at the ratio 60:1 was statistically not significant for T24, but well for HCV (p -< 0.05). At the ratio 30:1 there was no statistical significance in any case. The same trend was seen with the other target cells but here the number of experiments was smaller. It will be noted that in some patients fractionation of the lymphocytes increased this cytotoxicity. This was particularly evident at the lower lymphocyte to target cell ratio (30:1).

4Troye, M., P. Perlmann, /k. Larsson, H. Blomgren, and B. Johansson. Cellular cytotoxicity in vitro in transitional cell carcinoma of the human urinary bladder: 51Cr-release assay. Int. J. Cancer (submitted for publication).

25"

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HUMAN LYMPHOCYTES CYTOLYTIC FOR TUMOR CELLS IN VITRO 1927

? ,

m io-

5 ,

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T 24 t HCV 29 HT 29 l ~ e ! - 1

Figure i. Effect of F(ab')Janti-F(ab')2 column fractionation of lymphocytes from individual bladder cancer patients (n = 8) on s'Cr- release from allogeneic target cells. T24, transitional cell carcinoma of the urinary bladder; HCV29, possibly nonmalignant specimen of urinary bladder epithelium; HT29, colon carcinoma; Mel-1, malignant melanoma; U, unfractionated lymphocytes; F, fractionated lymphocytes. Ordinate: ~'Cr-release, corrected for background release after 18 hr of incubation; solid lines: ratio of lymphocytes: target cells 60:1; dotted lines ratio 30:1.

25 2o "•.. .

. . . . . . . . . . . .

Figure 2. Effect of OA/anti-OA column fractionation of lympho. cytes from individual bladder cancer patients (n = 9) on 5~Cr-release from allogeneic target cells. For details see legend to Figure 1.

I t has been reported ear l ier t ha t fractionation of bladder cancer pat ients ' lymphocytes on IgG/ant i - IgG columns reduces the i r cytotoxicity when tested ei ther in the microcytotoxicity assay or by ~lCr-release (22). A few exper iments performed in the present series also indicated tha t this fract ionat ion procedure resulted in a s t rong or complete reduction of cytotoxicity aga ins t al l allogeneic ta rge t cells l ines invest igated.

The cytotoxicity of pat ients ' lymphocytes before and after fractionation through OA/ant i -OA columns is shown in F igure 2. The lymphocytes from nine pat ients tested a t a ra t io of 60:1

aga ins t T24 had a mean cytotoxicity (% corrected ~lCr-release) of 11.5 _+ 4 before and 2.1 _+ 1.2 after fractionation, respec- t ively. This difference was s ta t i s t ica l ly h ighly signif icant (p -< 0.001). In al l instances, cytotoxicity was e i ther completely abolished or very s t rongly reduced. S imi la r resul ts were ob- ta ined with the other t a rge t cells. I t will also be noted tha t tests a t rat io 30:1 gave the same results. The occasional increase of cytotoxicity appear ing after F(ab ' ) Jant i -F(ab ' )2 frac- t ionat ion was never seen here.

Cytotoxicity of bladder cancer patients' lymphocytes to autochthonous target cells. For two pat ients , t a rge t cells ob- ta ined from the pa t ien ts ' own tumors were available. Table I shows the resul ts of two exper iments in which lymphocyte cytotoxicity agains t e i ther autochthonous or allogeneic ta rge t cells was invest igated. I t will be seen tha t pa t ien t S.F. had the h ighest cytotoxicity aga ins t the autochthonous ta rge t cells and this reaction was not a t al l affected by fract ionation through e i ther F(ab')2/anti-F(ab')~ columns or through OA/ ant i -OA columns. This was in contrast to the reductions seen with the allogeneic t a rge t cells• The unfract ionated lymnpho- cytes of pa t ien t E.J. were only weakly cytotoxic aga ins t all t a rge t cells. However, fractionation through OA/ant i -OA increased cytotoxicity in the autochthonous combinat ion but not in any of the allogeneic combinations. I t should be ment ioned tha t both MANO- and EJ - ta rge t cells behaved in the same way at T24 and the other ta rge t cells when tested with unfract ionated or f ract ionated allogeneic lymphocytes (footnote 4 and unpublished)

Cytotoxicity of lymphocytes from patients with cancer prostatae or from healthy donors against allogeneic target cells. Figure 3 shows the resul ts of fractionation on the cytotoxicity of lymphocytes from five patients . OA/ant i -OA fractionation very s t rongly reduced cytotoxicity in al l instances. F(ab')2/ anti-F(ab')2 fractionation was only performed in one experi- ment, resul t ing in weak reduction of cytotoxicity aga ins t HCV and a s l ight increase of cytotoxicity aga ins t melanoma. The effects of OA/ant i -OA fract ionation on the cytotoxicity of lymphocytes from hea l thy donors a re shown in F igure 4. The lymphocytes from eight donors tested a t a ra t io of 60:1 aga ins t T24 or HCV had a mean cytotoxicity (% corrected ~lCr-release) of 10.5 +- 5 or 9.5 +- 5.5 before and of 1 _+ 2.5 or 1.5 -+ 1.5 after fractionation, respectively. These differences were s tat is t i - cally highly significant (p < 0•001). The same resul ts were obtained af ter fract ionat ion on IgG/ant i - IgG columns (five experiments): % corrected 5~Cr-release a t a ra t io of 60:1 aga ins t T24 or HCV before fract ionation were 12 -+ 5 or 11 +_ 7, and after fractionation 1 -+ 3.6 or 1 +_ 1, respectively. Two heal thy

TABLE I 5'Cr-release assay with autologous and allogeneic target cells from

two bladder cancer patients

Patient Lymphocytes Autolo- % 5'Cr-Release (Corr,) ~

gous Tumor Cetlsa T24 c HCV29 c HT29 ~"

S .F . Unfractionated 22 7 - 2 10 F(ab')Janti-F(ab')~ 22 3 0 7 OA/anti-OA 19 2 - 1 5

E . J . Unfractionated 4 2 5 3 OA/anti-OA 8 2 1 2

a MANO for patient S. F., E. J. for patient E. J. b ~lCr.releas e corrected by subtracting background release

(<20%). Incubation time 18 hr. Lymphocyte target cell ratio 60:1. For cell lines see legend to Figure 1.

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GERD R. PAPE, MARITA TROYE, AND PETER PERLMANN [voL. 118

\ e

o°

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T Z4 SCV 29 HT29 MeL - I

Figure 3. Effect of OA/anti-OA column fractionation of lymphocytes from individual patients with cancer prostatae (n = 5) on 51Cr- release from allogeneic target cells. For details see legend to Figure 1.

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Figure 4. Effect of OA/anti-OA column fractionation of lymphocytes from selected healthy donors (n = 9) on ~lCr-retease from allogeneic target cells. For details see legend to Figure 1.

donors' tymphocytes were also tested after fractionation on F(ab')2/anti-F(ab')2 columns. A weak reduction of cytotoxicity was seen in these experiments (unpublished).

D I S C U S S I O N

Most donors displayed selective cytotoxicity (33) in that their lymphocytes were cytotoxic to some but not all target cells included in this study. In a few instances cytotoxicity was nonselective (33), i.e., the lymphocytes killed most or all of the target cell types under investigation. In the material used herein, no disease-related cytotoxicity of bladder cancer patients' lymphocytes against bladder carcinoma target cells can be discerned by studying individual cases. However, the blad-

der cancer patients as a group had a higher mean cytotoxicity toward T24 (and other bladder carcinoma target cells, unpublished) than against the other target cells on the panel. This was not the case with the cytotoxicity displayed by the control donors' lymphocytes. In a detailed investigation on a larger material published elsewhere 4 it has been shown that this disease-related cytotoxicity of lymphocytes from untreated bladder cancer patients is highly significant. That the differences between the different groups of donors were small was to be expected since control donors were selected for cytotoxicity above background to one or more of the target cells on the panel.

The occurrence of a %pontaneous" cytotoxicity of human blood lymphocytes for human cells in tissue culture greatly hampers the usefulness of the assay for monitoring disease- related reactions in cancer patients. Elucidation of both tumor-associated and %pontaneous" cytotoxicity requires: 1) im- munochemical studies of the various antigens on the target cell membranes, and 2) detailed analysis of different effector mechanisms involved. This study deals exclusively with the second question. Previous studies have indicated that the cytotoxicity of bladder cancer patients' lymphocytes against allogeneic bladder carcinoma target cells was not mediated by antibody-independent cytotoxic T cells (CTL). Rather, it re- quired the presence of cells bearing Fc-receptors for IgG (22, 23). The previous results have been confirmed and extended in the present study by using OA/anti-OA-coated columns. In contrast to the IgG/anti-IgG columns used previously (22), the OA/anti-OA columns remove the majority of the Fc-receptor- positive cells (EA +) but leave most of the B cells in the passaged lymphocyte preparation (29). This procedure also abolished or strongly reduced cytotoxicity of both bladder tumor patients' and control donors' lymphocytes against all allogeneic target cells under investigation. This indicates that similar effector mechanisms may be responsible for cytotoxicity in all these instances.

The results further indicate that B cells are not directly involved in these cytotoxic reactions. Direct evidence for this was obtained by using F(ab')Janti-F(ab')2 columns which removed B cells (SIg +) but left the majority of the EA ÷ and EAC + cells in the effector cell preparation. This fractionation procedure did not significantly decrease cytotoxicity of either bladder cancer patients or controls. On the contrary, in several instances it was noted that removal of B cells enhanced cytotoxicity particularly when assayed at lower lymphocyte to target cell ratios. Similar observations have recently been made in an antibody-dependent model system (34). This en- hancement may reflect a suppressor effect of B cells but the mechanism of this phenomenon remains to be elucidated.

In the preceding paper (29) we have shown that fractionation on OA/anti-OA columns but not that on F(ab')Janti- F (ab')5 columns strongly reduced antibody-dependent lymphocyte-mediated cytotoxicity (K cell activity) in a model system. This does not prove that the cytotoxicity of the patients and control donors' lymphocytes against allogeneic tumor cells also is antibody mediated. However, good evidence that this may indeed be the case has been obtained in inhibition experiments, in which Fab-fragments to human IgG (or its Fc- or F(ab')~-fragments) were added to the lymphocyte-target cell

'~ Troye, M., P. Perlmann, G. R. Pape, H. L. Spiegelberg, I. N~islund and A. GidlSv. The use of Fab-fragments of rabbit anti-human immunoglobulin as analytic tools for establishing the involvement of immunoglobulin in the spontaneous cytotoxicity of tymphocytes from patients with bladder carcinoma and from health) ~lonors. J. Immunol. (submitted for publication).

1977] HUMAN LYMPHOCYTES CYTOLYTIC FOR TUMOR CELLS IN VITRO 1929

mixtures. 5 The cytotoxicity of the majority of patients and control donors, investigated so far, was inhibited, suggesting antibody involvement in both disease related and ~spontaneous" cytotoxicity. Inhibi t ion was dose dependent and either complete or partial (60 to 70% inhibition). The na ture of the ant igens recognized by these antibodies remains to be elucidated. It remains also to be seen whether or not incomplete inhibit ion or lack of inhibi t ion observed in some cases reflects the occurrence of an antibody-independent cytotoxicity caused by a '~natural killer cell" (NK-cell) as seen in mice (12, 13) or soluble factors as described by others (27, 28).

If the na tura l cellular cytotoxicity of the patients ' and controls' lymphocytes against allogeneic tumor target cells is an antibody-dependent phenomenon, it has to be established where the inducing antibodies come from. Two al ternative explanations may be considered. 1) Antibody, perhaps in the form of immune complexes, may have been picked up by K cells in vivo and may then be utilized for induction of cytotoxicity in the in vitro assay (35-38). Since it is likely that some of these antibody-carrying K cells may be retained on the F(ab')Janti-F(ab')2 columns this would explain the weak reduction of cytotoxicity noted after this fractionation in some experiments. 2) Antibody may be released from antibody- producing cells and utilized by K cells during the cytotoxicity assay. Since removal of B cells by F(ab ' ) jant i -F(ab ' )2 columns does not significantly reduce cytotoxicity, such antibody-producing cells would not be the B cells with a high concentration of SIg. However, more mature immunoglobulin-producing B cells and plasma cells have less or no SIg (39, 40). When fractionated at room temperature such cells are expected to pass through the F (ab ' )Jant i -F (ab')2 columns and could easily release enough antibody in vitro to induce cytotoxicity which does not require high antibody concentrations to become mea- surable (35). By May Gr/inwald-Giemsa s ta in ing and immunofluorescence for cytoplasmic immunoglobulin, plasma cells were frequently detected in the lymphocyte preparations passaged through F(ab')2/anti-F(ab')2 columns (unpublished ob- servation).

Although the mechanisms described above may account for the cytotoxicity observed in many human tumor systems in which allogeneic target cells are used, other effector mechanisms may occur as well, and may be predominant in some individual cases. Furthermore, our results suggest that other cytotoxic mechanisms (as well as other antigenic specifici- ties) may be involved when lymphocytes and target cells are tested in autologous combinations. In the few instances tested so far, the effector cells from bladder tumor pat ients reacting with autochthonous tumor cells were nei ther B cells nor Fc- receptor-bearing lymphocytes. It remains to be seen whether these effector cells are of the antibody-independent CTL-type displaying a syngenically restricted cytotoxicity as seen in animal tumor systems (41).

Acknowledgment. Blood from cancer patients was obtained through the courtesy of Dr. K. A. Backman, Urology Dept., St. Erik's Hospital, Stockholm, Drs. A. GidlSv and B. Johans- son, Urology Dept., Karol inska Hospital, Stockholm, Drs. N. Hul tengren and T. Svenberg, Danderyd's Hospital, Danderyd, and Dr. I. N~islund, Radiumhemmet, Karol inska Hospital, Stockholm.

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