VoL 113,
THE JOURNAL OF UROLOGY
Copyright © 1975 by The Williams & Wilkins Co.
Printed in U.8.A
LYMPHOCYTE ANTIBODY INTERACTION IN CYTOTOXICITY AGAINST HUJ'\!IAN CELL CARCINOMA THOMAS R. HAKALA, PAUL H. LANGE, ANTHONY E. CASTRO, ARTHUR Y. ELLIOTT AND ELWIN E. FRALEY
From the Division of Urology, Department of Surgery, Veterans Administration Hospital and Department of Urologic Surgery, University of Minnesota Health Sciences Center, Minneapolis, Minnesota
ABSTRACT
The interaction of antibodies and lymphocytes in their immune reaction human transitional cell carcinomas was studied using the in vitro assay. A non-complement dependent, IgG antibody was detected in the serum of against occasional transitional cell carcinoma patients, which induced~,,,-~,.~=, transitional cell carcinoma target cells by lymphocytes from donors with and without transitional. cell carcinoma. The observation that lymphocytes from transitional cell carcinoma donors were more sensitive to activation this anti-transitional cell lymphocyte dependent is with the hypothesis that the surface of lymphocytes from some transitional cell carcinoma donors is coated in vivo with an anti-transitional cell carcinoma lymphocyte dependent antibody and that this antibody may be a significant factor in immunity to transitional cell carcinomas of the urinary tract. Complement dependent, antibody mediated immunity as well as lymphocyte mediated immunity to transitional cell carcinoma (TCC) has been described by investigators using the microcytotoxicity assay. •- 3 Since in vivo cell mediated immunity occurs in the presence of antibodies, the results of microcytotoxicity assays against TCC using lymphocytes plus antibodies may be expected to complement information gained by studies of TCC cytotoxicity caused lymphocytes alone. In the studies reported herein we attempted to detect tumor associated, lymphocyte mediated immunity against human TCCs and to investigate the relation of antibody to such u~,~,h~.n,d·n mediated immunity. METHODS
Lymphocyte preparation. Mononuclear cells (lymphocytes) were isolated from heparinized or defibrinated venous blood samples 1 of 3 methods. In the first mononuclear cells were concentrated at the interface of a Ficoll-hypaque barrier during centrifugation.' In the second erythrocytes were sedimented in the presence of plasmaAccepted for publication July 19, 1974. Read at annual meeting of American Urological Association, St. Louis, Missouri, May 19-23, 1974. Supported in part by Research Funds of Veterans Administration, The American Cancer Society Institutional Grant, Minnesota Medical Foundation, United States Public Health Service Training Grant AM 05514-08, National Cancer Institute Grant CA 13095-02 and National Bladder Cancer Project Grant CA 1551-01.
gel* and the leukocyte-rich supernatant was bated on nylon fiber to remove neutrophils and monocytes. 5 • 6 The were washed from the nylon fiber and blood cells were lysed using tris-buffered ammo-· nium chloride. 7 In the third method, used to obtain lymphocyte preparations free of monocyte contamination, the mononuclear cell suspension obtained after centrifugation over Ficoil. hypaque was incubated with lysine coated carbonyl iron particles. Leukocytes iron (monocytes/macrophages) were removed netic trapping. 0 Serum preparation. Serum. was obtained venous blood samples, stored in the vapor liquid nitrogen (less than lOOC) and the ment activity was removed heating at 56C minutes before use. Target cells. TC Cs of the renal ureter bladder, renal cell carcinoma as well as nornial. bladder epithelium, kidney and testis were ob· tained from patients at operation and were propagated in tissue culture as described ouslv. 1 , 9 • 10 Human embryonic skin and musde, and "human embryonic kidney cell strains were tained from HEM, Inc., Rockville, Maryland. T24, an established cell line derived from TCC of the bladder was provided by Dr. Jorgen Fogh the Sloan-Kettering Institute in New York. Target cell strains obtained from non-mesenchymal tissues were epithelial in morphology. All target cell strains used in these assays were free of evidence of *H.I.T. Corporation, Buffalo, New York. 663
664
HAKALA AND ASSOCIATES
Mycoplasma when examined by culture* and elec- and incubation was resumed for 1 hour. After this tron microscopic techniques. lymphocytes and fetal calf serum were added as in Microcytotoxicity assay. A modification of the the lymphocyte mediated cytotoxicity assay previmicrocytotoxicity assay described by Hellstrom ously described and incubation was continued for a and associates was used to detect lymphocyte further 48 hours. Target cell survival was determediated cytotoxicity and has been described mined as in the lymphocyte mediated cytotoxicity elsewhere. 11 In brief, a uniform number of target assay and the effect of serum on lymphocyte cells (averaging 50 to 100) was inoculated into each cytotoxicity was expressed as a cytotoxic index of the 96 wells of the micro-test II plate.t These (CIAoccl which was calculated as follows: target cells were propagated in Waymouth medium supplemented with 10 per cent tryptose x 100 - CI, ADCC, phosphate broth, 20 per cent fetal calf serum, TCc+ NS non-essential amino acids, glutamine, 100 units per ml. penicillin and 100 mcg. per ml. streptomy- Where TCuNs equaled the mean number of target cin at 37C in a humidified atmosphere of 5 per cent cells surviving exposure to lymphocytes from a carbon dioxide in air. The plates were incubated given donor plus normal serum the TCuTs equaled overnight to allow attachment of the target cells. the mean number of target cells surviving exposure Medium was then removed by shaking the inverted to lymphocytes obtained from the same donor plus plate and lymphocytes (2 by 10 5 lymphocytes in test serum. The cytotoxic index derived from this 0.2 ml. Waymouth medium without fetal calf se- formula thus controls for intrinsic cytotoxicity rum) from each donor were added to each of 6 to 8 demonstrated by the lymphocytes themselves and wells. After incubation for 1 hour 0.05 ml. medium reflects only the effect of serum on lymphocyte containing 50 per cent fetal calf serum (heat inac- cytotoxicity. The difference in the number of tivated at 56C for 30 minutes) was added and in- target cells surviving exposure to lymphocytes plus cubation was continued on a rocking platform:j: test serum and exposure to lymphocytes plus for 2 days. Lymphocytes and non-adherent target control serum was considered significant if the p cells were then removed by washing with phos- value resulting from Student's t test was less than phate buffered saline, pH 7.4. The remaining ad- 0.05. herent target cells were fixed in 95 per cent RESULTS ethanol, stained with crystal violet (0.1 per cent) and counted visually using a microscope. Results of a representative experiment designed The mean number of target cells surviving in the to detect the presence of cell mediated cytotoxicity wells which contained test patient lymphocytes against TCC are presented in table 1. Lymphowas compared to the mean number surviving in cytes from all 4 patients with TCC showed signifiwells which contained control lymphocytes. The cant cytotoxicity against the TCC target cells percentage reduction in target cell survival caused when compared to TCC target cell survival followby the test patient lymphocytes was expressed as ing exposure to lymphocytes obtained from the a cytotoxic index (CILMc) and was calculated as normal donor (Cl). Lymphocytes from 3 (Tl, T2 follows: and T3) of the 4 TCC patients were not cytotoxic against the control target cells (derived from norTCcL - TC'Jl, - - - - - x 100 - CILMC mal testis) tested simultaneously. These results TCcL suggest that the cytotoxicity of lymphocytes deWhere TCcL equaled the mean number of target rived from the TCC patients Tl, T2 and T3 is cells surviving exposure to control lymphocytes directed preferentially against TCC target cells and TCTL equaled the mean number of target cells and is compatible with a lymphocyte mediated, surviving exposure to test lymphocytes. The differ- TCC associated immunity. In contrast, the cytoence in the number of target cells surviving expo- toxicity of lymphocytes from patient T4 was disure to test and to control lymphocytes was consid- rected against target cells derived from TCC and ered significant if the p value resulting from normal tissue. Under these assay condition,s it Student's t test was less than 0.05. cannot be concluded that lymphocytes of patient To estimate the effect of serum upon lymphocyte T4 had TCC specific immunity. Patient T4 had mediated cytotoxicity (in this case an antibody received multiple whole blood transfusions in the dependent, cell mediated cytotoxicity) the afore- past and had a pelvic abscess at the time of lymmentioned microcytotoxicity assay was modi- phocyte testing. Whether the strong non-specific fied. After the overnight incubation to allow at- cytotoxicity demonstrated here resulted from 1) tachment of the target cells, plating medium was sensitization to histocompatability antigens presremoved, serum (diluted in phosphate buffered ent in the transfusions, 2) non-specific immunosaline) from TCC and non-TCC donors was added stimulation secondary to the pelvic abscess or 3) other unrecognized factors is unknown. Lymphocytes from 48 patients with TCC and 45 *Flow Laboratories, Rockville, Maryland. patients without malignancy (control patients) tFalcon Plastics, Los Angeles, California. :j:Bellco Glass Co., New York, New York. were tested in this fashion against target cells
665
LYMPHOCYTE ANTIBODY INTERACTION TABLE
1. Target cell survival after exposure to lymphocytes from TCC and control donors Target Cell Origin Normal Testis
TCC Bladder
Lymphocyte Donor (diagnosis)
Surviving Target Cells*
Surviving Target Cells* Cl (Normal) Tl (TCC bladder) T2 (TCC bladder) T3 (TCC bladder) T4 (TCC bladder)
83.6 (6) 26.0 (4.4) 44.8 (7 .3) 56.0 (7.8) 0.0
68 p 46 p 33 p 100 p
< < <
THE JOURNAL OF UROLOGY
Copyright © 1975 by The Williams & Wilkins Co.
Printed in U.8.A
LYMPHOCYTE ANTIBODY INTERACTION IN CYTOTOXICITY AGAINST HUJ'\!IAN CELL CARCINOMA THOMAS R. HAKALA, PAUL H. LANGE, ANTHONY E. CASTRO, ARTHUR Y. ELLIOTT AND ELWIN E. FRALEY
From the Division of Urology, Department of Surgery, Veterans Administration Hospital and Department of Urologic Surgery, University of Minnesota Health Sciences Center, Minneapolis, Minnesota
ABSTRACT
The interaction of antibodies and lymphocytes in their immune reaction human transitional cell carcinomas was studied using the in vitro assay. A non-complement dependent, IgG antibody was detected in the serum of against occasional transitional cell carcinoma patients, which induced~,,,-~,.~=, transitional cell carcinoma target cells by lymphocytes from donors with and without transitional. cell carcinoma. The observation that lymphocytes from transitional cell carcinoma donors were more sensitive to activation this anti-transitional cell lymphocyte dependent is with the hypothesis that the surface of lymphocytes from some transitional cell carcinoma donors is coated in vivo with an anti-transitional cell carcinoma lymphocyte dependent antibody and that this antibody may be a significant factor in immunity to transitional cell carcinomas of the urinary tract. Complement dependent, antibody mediated immunity as well as lymphocyte mediated immunity to transitional cell carcinoma (TCC) has been described by investigators using the microcytotoxicity assay. •- 3 Since in vivo cell mediated immunity occurs in the presence of antibodies, the results of microcytotoxicity assays against TCC using lymphocytes plus antibodies may be expected to complement information gained by studies of TCC cytotoxicity caused lymphocytes alone. In the studies reported herein we attempted to detect tumor associated, lymphocyte mediated immunity against human TCCs and to investigate the relation of antibody to such u~,~,h~.n,d·n mediated immunity. METHODS
Lymphocyte preparation. Mononuclear cells (lymphocytes) were isolated from heparinized or defibrinated venous blood samples 1 of 3 methods. In the first mononuclear cells were concentrated at the interface of a Ficoll-hypaque barrier during centrifugation.' In the second erythrocytes were sedimented in the presence of plasmaAccepted for publication July 19, 1974. Read at annual meeting of American Urological Association, St. Louis, Missouri, May 19-23, 1974. Supported in part by Research Funds of Veterans Administration, The American Cancer Society Institutional Grant, Minnesota Medical Foundation, United States Public Health Service Training Grant AM 05514-08, National Cancer Institute Grant CA 13095-02 and National Bladder Cancer Project Grant CA 1551-01.
gel* and the leukocyte-rich supernatant was bated on nylon fiber to remove neutrophils and monocytes. 5 • 6 The were washed from the nylon fiber and blood cells were lysed using tris-buffered ammo-· nium chloride. 7 In the third method, used to obtain lymphocyte preparations free of monocyte contamination, the mononuclear cell suspension obtained after centrifugation over Ficoil. hypaque was incubated with lysine coated carbonyl iron particles. Leukocytes iron (monocytes/macrophages) were removed netic trapping. 0 Serum preparation. Serum. was obtained venous blood samples, stored in the vapor liquid nitrogen (less than lOOC) and the ment activity was removed heating at 56C minutes before use. Target cells. TC Cs of the renal ureter bladder, renal cell carcinoma as well as nornial. bladder epithelium, kidney and testis were ob· tained from patients at operation and were propagated in tissue culture as described ouslv. 1 , 9 • 10 Human embryonic skin and musde, and "human embryonic kidney cell strains were tained from HEM, Inc., Rockville, Maryland. T24, an established cell line derived from TCC of the bladder was provided by Dr. Jorgen Fogh the Sloan-Kettering Institute in New York. Target cell strains obtained from non-mesenchymal tissues were epithelial in morphology. All target cell strains used in these assays were free of evidence of *H.I.T. Corporation, Buffalo, New York. 663
664
HAKALA AND ASSOCIATES
Mycoplasma when examined by culture* and elec- and incubation was resumed for 1 hour. After this tron microscopic techniques. lymphocytes and fetal calf serum were added as in Microcytotoxicity assay. A modification of the the lymphocyte mediated cytotoxicity assay previmicrocytotoxicity assay described by Hellstrom ously described and incubation was continued for a and associates was used to detect lymphocyte further 48 hours. Target cell survival was determediated cytotoxicity and has been described mined as in the lymphocyte mediated cytotoxicity elsewhere. 11 In brief, a uniform number of target assay and the effect of serum on lymphocyte cells (averaging 50 to 100) was inoculated into each cytotoxicity was expressed as a cytotoxic index of the 96 wells of the micro-test II plate.t These (CIAoccl which was calculated as follows: target cells were propagated in Waymouth medium supplemented with 10 per cent tryptose x 100 - CI, ADCC, phosphate broth, 20 per cent fetal calf serum, TCc+ NS non-essential amino acids, glutamine, 100 units per ml. penicillin and 100 mcg. per ml. streptomy- Where TCuNs equaled the mean number of target cin at 37C in a humidified atmosphere of 5 per cent cells surviving exposure to lymphocytes from a carbon dioxide in air. The plates were incubated given donor plus normal serum the TCuTs equaled overnight to allow attachment of the target cells. the mean number of target cells surviving exposure Medium was then removed by shaking the inverted to lymphocytes obtained from the same donor plus plate and lymphocytes (2 by 10 5 lymphocytes in test serum. The cytotoxic index derived from this 0.2 ml. Waymouth medium without fetal calf se- formula thus controls for intrinsic cytotoxicity rum) from each donor were added to each of 6 to 8 demonstrated by the lymphocytes themselves and wells. After incubation for 1 hour 0.05 ml. medium reflects only the effect of serum on lymphocyte containing 50 per cent fetal calf serum (heat inac- cytotoxicity. The difference in the number of tivated at 56C for 30 minutes) was added and in- target cells surviving exposure to lymphocytes plus cubation was continued on a rocking platform:j: test serum and exposure to lymphocytes plus for 2 days. Lymphocytes and non-adherent target control serum was considered significant if the p cells were then removed by washing with phos- value resulting from Student's t test was less than phate buffered saline, pH 7.4. The remaining ad- 0.05. herent target cells were fixed in 95 per cent RESULTS ethanol, stained with crystal violet (0.1 per cent) and counted visually using a microscope. Results of a representative experiment designed The mean number of target cells surviving in the to detect the presence of cell mediated cytotoxicity wells which contained test patient lymphocytes against TCC are presented in table 1. Lymphowas compared to the mean number surviving in cytes from all 4 patients with TCC showed signifiwells which contained control lymphocytes. The cant cytotoxicity against the TCC target cells percentage reduction in target cell survival caused when compared to TCC target cell survival followby the test patient lymphocytes was expressed as ing exposure to lymphocytes obtained from the a cytotoxic index (CILMc) and was calculated as normal donor (Cl). Lymphocytes from 3 (Tl, T2 follows: and T3) of the 4 TCC patients were not cytotoxic against the control target cells (derived from norTCcL - TC'Jl, - - - - - x 100 - CILMC mal testis) tested simultaneously. These results TCcL suggest that the cytotoxicity of lymphocytes deWhere TCcL equaled the mean number of target rived from the TCC patients Tl, T2 and T3 is cells surviving exposure to control lymphocytes directed preferentially against TCC target cells and TCTL equaled the mean number of target cells and is compatible with a lymphocyte mediated, surviving exposure to test lymphocytes. The differ- TCC associated immunity. In contrast, the cytoence in the number of target cells surviving expo- toxicity of lymphocytes from patient T4 was disure to test and to control lymphocytes was consid- rected against target cells derived from TCC and ered significant if the p value resulting from normal tissue. Under these assay condition,s it Student's t test was less than 0.05. cannot be concluded that lymphocytes of patient To estimate the effect of serum upon lymphocyte T4 had TCC specific immunity. Patient T4 had mediated cytotoxicity (in this case an antibody received multiple whole blood transfusions in the dependent, cell mediated cytotoxicity) the afore- past and had a pelvic abscess at the time of lymmentioned microcytotoxicity assay was modi- phocyte testing. Whether the strong non-specific fied. After the overnight incubation to allow at- cytotoxicity demonstrated here resulted from 1) tachment of the target cells, plating medium was sensitization to histocompatability antigens presremoved, serum (diluted in phosphate buffered ent in the transfusions, 2) non-specific immunosaline) from TCC and non-TCC donors was added stimulation secondary to the pelvic abscess or 3) other unrecognized factors is unknown. Lymphocytes from 48 patients with TCC and 45 *Flow Laboratories, Rockville, Maryland. patients without malignancy (control patients) tFalcon Plastics, Los Angeles, California. :j:Bellco Glass Co., New York, New York. were tested in this fashion against target cells
665
LYMPHOCYTE ANTIBODY INTERACTION TABLE
1. Target cell survival after exposure to lymphocytes from TCC and control donors Target Cell Origin Normal Testis
TCC Bladder
Lymphocyte Donor (diagnosis)
Surviving Target Cells*
Surviving Target Cells* Cl (Normal) Tl (TCC bladder) T2 (TCC bladder) T3 (TCC bladder) T4 (TCC bladder)
83.6 (6) 26.0 (4.4) 44.8 (7 .3) 56.0 (7.8) 0.0
68 p 46 p 33 p 100 p
< < <
