Cytotoxicity Inhibition Assay: Cryopreservation and Standardization: Brief Communication 1 R. K. Oldham, 2.3.4 J. R. Ortaldo," H. T. Holden," and R. B. Herberman
The central problem with the use of in vitro assays for assessment of ceIl-mediated immunity has been the standardization and reproducibility of these assays. With the widespread use of a variety of methods to assess immunologie competence in ceIl-mediated immunity to tumor-associated antigens in vitro (1, 2), various technical problems associated with such assays have become obvious. Not only is the day-to-day variation of the assay of considerable concern to those working in the field, but also the effector cell donor and target cell function in assays of ceIl-mediated immunity can fluctuate with time as can the antigenie content and lysability of target cells taken from the same source over time. In addition to the chronologie variation within each animal, considerable variation occurs among animals (even when age- and sex-matched inbred strains of animals are used and when all animals are immunized at the same point with the same immunogen) when testing is done as part of a systematic program to standardize the assays of ceIl-mediated immunity. We have reported the use of cryopreserved cells to standardize the CRA in the mouse (3), rat (4), and man (5). In addition, we have investigated the use of cryopreserved cells in various other in vitro assays of cellular immunity in man [inc1uding lymphocyte transformation, leukocyte migration inhibition, and rosette formation (5)] . Competitive inhibition of cell-rnediated cytotoxicity, as measured by the CRA, has been utilized in our laboratory as an investigative tool in specificity testing (6). This assay is based on the finding that the addition of unlabeled cells carrying the same antigen as the labeled cells will competitively inhibit 51Cr release from labeled target cells. With the demonstration that cytotoxicity in the CRA is cryopreservable, utilizing cryopreserved attacker cells and cryopreserved target cells (3-5), we reasoned that all the elements in the cytotoxicity inhibition assay might be cryopreservable as weIl and that this method would help reduce the variability in this assay. This paper reports on the use of cryopreserved cells as attacker, target, and inhibitor cells in this assay. This system for specificity testing represents a powerful tool in the standardization of in vitro testing of cellular cytotoxicity. VOL. 59. NO. 4. OCTOBER 1977
1321
MATERIALS AND METHODS Tumors and animals.- The MuSV tumor system has been detailed (3, 7,8). C57BL/6N (B6) mice were obtained from the Rodent Production Section, Division of Research Services, National Institutes of Health. Two in vivo-maintained ascites tumor cell lines were utilized in this work: RBL-5, a B6 Rauscher virus-induced lymphoma (7), and EL 4 (G-), a benzo[a]pyrene-induced lymphoma in B6 mice (3). Each was passaged ip weekly in syngeneic mice. Immunization was performed by means of the im inoculation of the Moloney strain of MuSV into the thigh of 8- to 12-week-old B6 mice (3). Tumors appeared by 7-9 days after injection, reached a maximum size by day 14, and regressed by days 21-25. Spleen cells were harvested 12-17 days after MuSV inoculation during the peak of primary cytotoxic activity. The Gross virus-induced tumor system (C58NT)D has also been described in detail (9). Inbred WF rats were obtained from Microbiological Associates, Inc., Walkersville, Maryland. The (C58NT)D rat lymphoma was induced by Gross leukemia virus and maintained in syngeneic WF rats by serial ip transplantation of 1x10 7 cells weekly. The 13762 ascites line (MAT), an ascites-adapted line derived from the 13762 breast adenocarcinoma tumor induced by 7,12-dimethylbenz[a]anthracene in syngeneic F344 rats (10), was passaged in a manner similar to that of the (C58NT)D cells. Spleen cells were harvested approximately 10 days after sc inoculation of (C58NT)D when the peak cytotoxic reactivity of spleen ce1ls had been previously demonstrated to occur (9). Cryopreservation. - Cells were cryopreserved as described (3-5). Cells in complete media (RPMI-1640 containing,20% fetal calf serum) were suspended in appropriate concentrations and frozen in 7.5 or 10% dimethyl sulfoxide in a controlled-rate freezer (Model R201; G. V. Planar, Ltd., Middlesex, England). We had previously determined that -1 0 C/minute was the optimal freezing rate for these cells. Cells were stored in vapor-phase liquid nitrogen below -150 0 C. Cryopreserved cells were recovered by rapid thawing in a
ABBREVIATIONS USED: CRA = 51Cr·release cytotoxicity assay; MuSV murine sarcoma virus; cpm = counts per minute.
=
Received December 20,1976; accepted April 20, 1977. Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Present address: Division of Oncology, Department of Medicine, Vanderbilt Vniversity, Nashville, Tenn. 37232. • Address reprint requests to Dr. Oldham, Division of Oncology, Department ofMedicine, Vanderbilt Vniversity. 1
2
J
NA TL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
ABSTRACT-The cytotoxicity inhibition assay is an extremely useful tool for examination of the specificity of celt-medlated cytotoxicity. With this demonstration of cryopreservation of the aUacker, target, and inhibitor cells, a powerful new tool is available for the standardization of this assay to beUer examlne the speclficity of cytotoxicity assays. Not only can cryopreserva· tion be used to standardize the assay wlthln a single laboratory, but also reagents may now be exchanged between laboratories to beUer understand the dlfferences In cell·medlated cytotoxiclty as observed by different Investlgators.-J Natl Cancer Inst 59: 1321-1323,1977.
2
1322
OLDHAM, ORTALDO, HOLDEN, AND HERBERMAN
Percent cytotoxicity =
cpm SlCr released into supernatant - cpm counter background total cpm incorporated - cpm counter background
x 100.
The specific release was ascertained by the following formula: Percent experimental cytotoxieity = percent cytotoxicity in experimental group - percent release by autologous control.
For the autologous control, unlabeled "target cells" were added in place of and in the same concentration as the effector cells. These same autologous cells were used as the positive control in the inhibition assays. Percent inhibition was calculated by the following formula: Percent inhibition
= 1-
percent speeific release with inhibitor percent speeific release without inhibitor
FROZ~FROZEN TARGET. ATIACKER
L
FROZ~FRESH
I
TARGET. ATIACKER
X
0 ~ 0 ~
>u
75
NTD ~ F~SH
u.
0
z
50
0
~FRESH --'-'-Q
AIDZE~ NTD
NTD FROZEN NTD
-
u
~
80
60
Q
~ 40
~ ~
o
20
~~EC~~ FROZE~
FRESH RBL-5
RBL-5
FROZEN RBL-5
FRESH EL 4
5/1
~~10/1
20/1
5/1
10/1
FRESH EL4
2Q/1
ATIACKER/INHIBITOR RATIO
TEXT-FIGURE 2.-Cytotoxicity inhibition for the MuSV system: The mean and standard error of five experiments are shown. Both the effector and target cells were cryopreserved in (A), whereas the effector cell was fresh in (B). VOL. 59, NO. 4, OCTOBER 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
37 0 C water bath until the last crystal of ice was present. The cell suspension was then transferred to a 50-mI tube. A volume of complete media of one-fiftieth of the cell suspension was added to the tube by drops and mixed for 5 minutes. The volume of medium added each time was doubled until the final 1: 10 dilution of the original cell suspension had been achieved. The cells were then centrifuged at 250xg for 10 minutes and resuspended in complete media for testing. When cryopreserved, the attacker, target, and inhibitor cells were handled in a like manner with regard to freezing and recovery. Cytotoxicity inhibition assay.- The cytotoxicity inhibition assay was performed as described previously (6, 11). Unlabeled inhibitor cell suspensions were added at three concentrations (2X10 5 , lXIOs, and 5X10', respectively). This resulted in ratios of attacker.inhibitor cells of 5: 1, 10: 1, and 20: 1, respectively. The inhibition test was considered "positive" when: 1) Lysis was significantly reduced; 2) a dear dose-response relationship with an increasing number of inhibitor cells was observed; and 3) the inhibitor curve with test cells was similar to that produced by the positive control (the positive control was always the same cell population as that being used as the target). Experimental results were expressed by calculation of the percent cytotoxicity:
CYTOTOXICITY INHIBITION: CRYOPRESERVATION
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
REFERENCES (1) HERBERMAN RB: Cell-mediated immunity to tumor cells. Adv Cancer Res 19:207-263,1974 (2) HERBERMAN RB, OLDHAM RK: Problems associated with study of cellmedia ted immunity to human turnors by microcytotoxicity assays. J Natl Cancer lust 55:749-753,1975 (3) HOLDEN HT, OLDHAM RK, ORTALDOJR, et af: Standardization ofthe chromium-51 release, cell-rnediated cytotoxicity assay: Cryopreser-
VOL. 59, NO. 4, OCTOBER 1977
(12)
(13)
vation of mouse effector and target cells. J Natl Cancer Inst 58:611-622,1977 ORTALDO JR, OLDHAM RK, HOLDEN HT, et al: Immune response to Gross virus-induced lymphoma: Cryopreservation of functional activity of rat lymphocytes and tumor cells. Cell Immunol 25:60-73, 1976 OLDHAM RK, DEAN JH, CANNON GB, et al: Cryopreservation of human lymphocyte function as measured by in vitro assays. Int J Cancer 18:145-155, 1976 ORTIZ DE LANDAZURI M, HERBERMAN RB: Speeificity of cellular immune reactivity to virus-induced tumours, Nature [New Biol] 238:18-19,1972 LAVRIN DH, HERBERMAN RB, NUNN M, et al: In vitro cytotoxicity studies of murine sarcoma virus-induced immunity in mice. J Natl Cancer Inst 51: 1497 -1508, 1973 HERBERMAN RB, HOLDEN HT, TING CC, et al: Cell-mediated immunity to leukemia virus and tumor-associated antigens in mice. Cancer Res 36:615-621, 1976 OREN ME, HERBERMAN RB, CANTY TG: Immune response to Gross virus-induced lymphoma. 11. Kinetics of the cellular immune response. J Natl Cancer lust 46:621-628, 1971 CAMPBELL DA, MANDERS EK, OEHLERJR, et al: Inhibition of in vitro lymphoproliferative response by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infective agent. Cell Immunol. In press HERBERMAN RB, NUNN ME, HOLDEN HT: Cytotoxicity inhibition assay for analysis of specificity of cell-mediated 5ICr release cytotoxicity. In In Vitro Methods of Cell-Mediated Tumor Immunity (Bloom BR, DavidJR, eds). New York: Academic Press. In press HERBERMAN RB, AOKI T, NUNN ME, et al: Specificity of SlCr-release cytotoxicity by lymphocytes immune to murine sarcoma virus. J NatlCancer Inst 53:1103-1111, 1974 OLDHAM RK, SIMMLER MC: The use of cryopreserved lymphocytes and lymphoblasts in SlCr lymphocyte cytotoxicity. In The Cryopreservation of Normal and Neoplastic Cells (Weiner RS, Oldham RK, Schwarzenberg L, eds). Paris: INSERM, 1973, pp 161-169
J NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
ty of testing and retesting on multiple oeeasions to deerease the variability inherent in eytotoxieity assays. Partieularly, with the inereased use of immunotherapy, areal need exists to reproducibly assess immunologie responsiveness. Therefore, the ability to send eells for testing from one laboratory to another to eonfirm results and standardize assays is eritieally important to the progress in immunology in this area. The demonstration that attaeker and target eells ean be eryopreserved represented a major step forward in this area. This paper demonstrates that all the elements in the eytotoxicity inhibition assay, a powerful tool for the examination of the specifieity of cell-mediated eytotoxicity, are eryopreservable. With sueh a three-eell system as a standard in the laboratory, various inhibitor eells, both fresh and frozen, ean be tested on multiple oeeasions to determine if similar antigens are on these eell surfaees. This ability allows one to disseet out the specificity of eellular responses to a degree previously available only in serologie testing. The applieation of this method in eell-mediated eytotoxicity will make the specificity testing mueh more praetieal and reproducible.
1323
The central problem with the use of in vitro assays for assessment of ceIl-mediated immunity has been the standardization and reproducibility of these assays. With the widespread use of a variety of methods to assess immunologie competence in ceIl-mediated immunity to tumor-associated antigens in vitro (1, 2), various technical problems associated with such assays have become obvious. Not only is the day-to-day variation of the assay of considerable concern to those working in the field, but also the effector cell donor and target cell function in assays of ceIl-mediated immunity can fluctuate with time as can the antigenie content and lysability of target cells taken from the same source over time. In addition to the chronologie variation within each animal, considerable variation occurs among animals (even when age- and sex-matched inbred strains of animals are used and when all animals are immunized at the same point with the same immunogen) when testing is done as part of a systematic program to standardize the assays of ceIl-mediated immunity. We have reported the use of cryopreserved cells to standardize the CRA in the mouse (3), rat (4), and man (5). In addition, we have investigated the use of cryopreserved cells in various other in vitro assays of cellular immunity in man [inc1uding lymphocyte transformation, leukocyte migration inhibition, and rosette formation (5)] . Competitive inhibition of cell-rnediated cytotoxicity, as measured by the CRA, has been utilized in our laboratory as an investigative tool in specificity testing (6). This assay is based on the finding that the addition of unlabeled cells carrying the same antigen as the labeled cells will competitively inhibit 51Cr release from labeled target cells. With the demonstration that cytotoxicity in the CRA is cryopreservable, utilizing cryopreserved attacker cells and cryopreserved target cells (3-5), we reasoned that all the elements in the cytotoxicity inhibition assay might be cryopreservable as weIl and that this method would help reduce the variability in this assay. This paper reports on the use of cryopreserved cells as attacker, target, and inhibitor cells in this assay. This system for specificity testing represents a powerful tool in the standardization of in vitro testing of cellular cytotoxicity. VOL. 59. NO. 4. OCTOBER 1977
1321
MATERIALS AND METHODS Tumors and animals.- The MuSV tumor system has been detailed (3, 7,8). C57BL/6N (B6) mice were obtained from the Rodent Production Section, Division of Research Services, National Institutes of Health. Two in vivo-maintained ascites tumor cell lines were utilized in this work: RBL-5, a B6 Rauscher virus-induced lymphoma (7), and EL 4 (G-), a benzo[a]pyrene-induced lymphoma in B6 mice (3). Each was passaged ip weekly in syngeneic mice. Immunization was performed by means of the im inoculation of the Moloney strain of MuSV into the thigh of 8- to 12-week-old B6 mice (3). Tumors appeared by 7-9 days after injection, reached a maximum size by day 14, and regressed by days 21-25. Spleen cells were harvested 12-17 days after MuSV inoculation during the peak of primary cytotoxic activity. The Gross virus-induced tumor system (C58NT)D has also been described in detail (9). Inbred WF rats were obtained from Microbiological Associates, Inc., Walkersville, Maryland. The (C58NT)D rat lymphoma was induced by Gross leukemia virus and maintained in syngeneic WF rats by serial ip transplantation of 1x10 7 cells weekly. The 13762 ascites line (MAT), an ascites-adapted line derived from the 13762 breast adenocarcinoma tumor induced by 7,12-dimethylbenz[a]anthracene in syngeneic F344 rats (10), was passaged in a manner similar to that of the (C58NT)D cells. Spleen cells were harvested approximately 10 days after sc inoculation of (C58NT)D when the peak cytotoxic reactivity of spleen ce1ls had been previously demonstrated to occur (9). Cryopreservation. - Cells were cryopreserved as described (3-5). Cells in complete media (RPMI-1640 containing,20% fetal calf serum) were suspended in appropriate concentrations and frozen in 7.5 or 10% dimethyl sulfoxide in a controlled-rate freezer (Model R201; G. V. Planar, Ltd., Middlesex, England). We had previously determined that -1 0 C/minute was the optimal freezing rate for these cells. Cells were stored in vapor-phase liquid nitrogen below -150 0 C. Cryopreserved cells were recovered by rapid thawing in a
ABBREVIATIONS USED: CRA = 51Cr·release cytotoxicity assay; MuSV murine sarcoma virus; cpm = counts per minute.
=
Received December 20,1976; accepted April 20, 1977. Laboratory of Immunodiagnosis, National Cancer Institute, National Institutes of Health, Public Health Service, V.S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Present address: Division of Oncology, Department of Medicine, Vanderbilt Vniversity, Nashville, Tenn. 37232. • Address reprint requests to Dr. Oldham, Division of Oncology, Department ofMedicine, Vanderbilt Vniversity. 1
2
J
NA TL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
ABSTRACT-The cytotoxicity inhibition assay is an extremely useful tool for examination of the specificity of celt-medlated cytotoxicity. With this demonstration of cryopreservation of the aUacker, target, and inhibitor cells, a powerful new tool is available for the standardization of this assay to beUer examlne the speclficity of cytotoxicity assays. Not only can cryopreserva· tion be used to standardize the assay wlthln a single laboratory, but also reagents may now be exchanged between laboratories to beUer understand the dlfferences In cell·medlated cytotoxiclty as observed by different Investlgators.-J Natl Cancer Inst 59: 1321-1323,1977.
2
1322
OLDHAM, ORTALDO, HOLDEN, AND HERBERMAN
Percent cytotoxicity =
cpm SlCr released into supernatant - cpm counter background total cpm incorporated - cpm counter background
x 100.
The specific release was ascertained by the following formula: Percent experimental cytotoxieity = percent cytotoxicity in experimental group - percent release by autologous control.
For the autologous control, unlabeled "target cells" were added in place of and in the same concentration as the effector cells. These same autologous cells were used as the positive control in the inhibition assays. Percent inhibition was calculated by the following formula: Percent inhibition
= 1-
percent speeific release with inhibitor percent speeific release without inhibitor
FROZ~FROZEN TARGET. ATIACKER
L
FROZ~FRESH
I
TARGET. ATIACKER
X
0 ~ 0 ~
>u
75
NTD ~ F~SH
u.
0
z
50
0
~FRESH --'-'-Q
AIDZE~ NTD
NTD FROZEN NTD
-
u
~
80
60
Q
~ 40
~ ~
o
20
~~EC~~ FROZE~
FRESH RBL-5
RBL-5
FROZEN RBL-5
FRESH EL 4
5/1
~~10/1
20/1
5/1
10/1
FRESH EL4
2Q/1
ATIACKER/INHIBITOR RATIO
TEXT-FIGURE 2.-Cytotoxicity inhibition for the MuSV system: The mean and standard error of five experiments are shown. Both the effector and target cells were cryopreserved in (A), whereas the effector cell was fresh in (B). VOL. 59, NO. 4, OCTOBER 1977
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
37 0 C water bath until the last crystal of ice was present. The cell suspension was then transferred to a 50-mI tube. A volume of complete media of one-fiftieth of the cell suspension was added to the tube by drops and mixed for 5 minutes. The volume of medium added each time was doubled until the final 1: 10 dilution of the original cell suspension had been achieved. The cells were then centrifuged at 250xg for 10 minutes and resuspended in complete media for testing. When cryopreserved, the attacker, target, and inhibitor cells were handled in a like manner with regard to freezing and recovery. Cytotoxicity inhibition assay.- The cytotoxicity inhibition assay was performed as described previously (6, 11). Unlabeled inhibitor cell suspensions were added at three concentrations (2X10 5 , lXIOs, and 5X10', respectively). This resulted in ratios of attacker.inhibitor cells of 5: 1, 10: 1, and 20: 1, respectively. The inhibition test was considered "positive" when: 1) Lysis was significantly reduced; 2) a dear dose-response relationship with an increasing number of inhibitor cells was observed; and 3) the inhibitor curve with test cells was similar to that produced by the positive control (the positive control was always the same cell population as that being used as the target). Experimental results were expressed by calculation of the percent cytotoxicity:
CYTOTOXICITY INHIBITION: CRYOPRESERVATION
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
REFERENCES (1) HERBERMAN RB: Cell-mediated immunity to tumor cells. Adv Cancer Res 19:207-263,1974 (2) HERBERMAN RB, OLDHAM RK: Problems associated with study of cellmedia ted immunity to human turnors by microcytotoxicity assays. J Natl Cancer lust 55:749-753,1975 (3) HOLDEN HT, OLDHAM RK, ORTALDOJR, et af: Standardization ofthe chromium-51 release, cell-rnediated cytotoxicity assay: Cryopreser-
VOL. 59, NO. 4, OCTOBER 1977
(12)
(13)
vation of mouse effector and target cells. J Natl Cancer Inst 58:611-622,1977 ORTALDO JR, OLDHAM RK, HOLDEN HT, et al: Immune response to Gross virus-induced lymphoma: Cryopreservation of functional activity of rat lymphocytes and tumor cells. Cell Immunol 25:60-73, 1976 OLDHAM RK, DEAN JH, CANNON GB, et al: Cryopreservation of human lymphocyte function as measured by in vitro assays. Int J Cancer 18:145-155, 1976 ORTIZ DE LANDAZURI M, HERBERMAN RB: Speeificity of cellular immune reactivity to virus-induced tumours, Nature [New Biol] 238:18-19,1972 LAVRIN DH, HERBERMAN RB, NUNN M, et al: In vitro cytotoxicity studies of murine sarcoma virus-induced immunity in mice. J Natl Cancer Inst 51: 1497 -1508, 1973 HERBERMAN RB, HOLDEN HT, TING CC, et al: Cell-mediated immunity to leukemia virus and tumor-associated antigens in mice. Cancer Res 36:615-621, 1976 OREN ME, HERBERMAN RB, CANTY TG: Immune response to Gross virus-induced lymphoma. 11. Kinetics of the cellular immune response. J Natl Cancer lust 46:621-628, 1971 CAMPBELL DA, MANDERS EK, OEHLERJR, et al: Inhibition of in vitro lymphoproliferative response by in vivo passaged rat 13762 mammary adenocarcinoma cells. I. Characteristics of inhibition and evidence for an infective agent. Cell Immunol. In press HERBERMAN RB, NUNN ME, HOLDEN HT: Cytotoxicity inhibition assay for analysis of specificity of cell-mediated 5ICr release cytotoxicity. In In Vitro Methods of Cell-Mediated Tumor Immunity (Bloom BR, DavidJR, eds). New York: Academic Press. In press HERBERMAN RB, AOKI T, NUNN ME, et al: Specificity of SlCr-release cytotoxicity by lymphocytes immune to murine sarcoma virus. J NatlCancer Inst 53:1103-1111, 1974 OLDHAM RK, SIMMLER MC: The use of cryopreserved lymphocytes and lymphoblasts in SlCr lymphocyte cytotoxicity. In The Cryopreservation of Normal and Neoplastic Cells (Weiner RS, Oldham RK, Schwarzenberg L, eds). Paris: INSERM, 1973, pp 161-169
J NATL CANCER INST
Downloaded from http://jnci.oxfordjournals.org/ at University of Lethbridge on October 19, 2015
ty of testing and retesting on multiple oeeasions to deerease the variability inherent in eytotoxieity assays. Partieularly, with the inereased use of immunotherapy, areal need exists to reproducibly assess immunologie responsiveness. Therefore, the ability to send eells for testing from one laboratory to another to eonfirm results and standardize assays is eritieally important to the progress in immunology in this area. The demonstration that attaeker and target eells ean be eryopreserved represented a major step forward in this area. This paper demonstrates that all the elements in the eytotoxicity inhibition assay, a powerful tool for the examination of the specifieity of cell-mediated eytotoxicity, are eryopreservable. With sueh a three-eell system as a standard in the laboratory, various inhibitor eells, both fresh and frozen, ean be tested on multiple oeeasions to determine if similar antigens are on these eell surfaees. This ability allows one to disseet out the specificity of eellular responses to a degree previously available only in serologie testing. The applieation of this method in eell-mediated eytotoxicity will make the specificity testing mueh more praetieal and reproducible.
1323
