Secondary Cytotoxic Response In Vitro Against Moloney Lymphoma Cells Antigenically Altered by Drug Treatment In Vivo 1 Angela Santoni, 2 Yvonne Kinney, 2 and Abraham Goldin 2,3
ABSTRACT-Cell-mediated cytotoxic response in vitro to a Moloney strain 01 murine leukemia virus-induced tumor (LSTRA) altered by 5-(3,3-dimethyl-1-triazeno)i midazole·4-carboxamide (DTIC) was investigated with the use 01 mixed-Ieukocyte tumor cell cultures (ML TC). As assessed by means 01 a short-term 51 Crrelease assay, minimal or no cytolytic activity was generated in primary MLTC. In contrast, high specific cytotoxic response against the altered tumor was obtained in secondary MLTC. Partial cross-reactivity was tound between the LSTRA and LSTRA/DTIC tumor Iines.-J Natl Cancer Inst 60: 109-112, 1978.
Novel tumor-associated antigenic determinants were found in murine lymphoma cells after treatment of tumor-bearing hosts with DTIC (1-5). The new antigenic specificity (specificities) mediated by DTIC treatment in vivo has been tentatively called DMTA. Specific cell-rnediated immunity against DMTA was found in mice sensitized in vivo with DTIC-treated tumor sublines (3, 4). The recently developed techniques of immune response in vitro (6-8) offer the opportunity for analysis of several aspects of generation of cytotoxic lymphocytes against tumor-associated antigens for both primary and secondary response. Nevertheless, no studies on cytotoxic immune response in vitro against DMTA have been conducted. In our laboratory, DMTA was evidenced in a MMuLV-induced lymphoma (LSTRA) after treatment with DTIC in vivo (5). The present experiments were performed with this DTIC-treated lymphoma subline to determine the possibility of obtaining primary or secondary cytotoxic responses in vitro. Our study indicates that highly cytotoxic lymphocytes were produced in the secondary response in vitro. However, little or no primary response was obtained.
MATERIALS AND METHODS Mice.-Inbred BALB/cCr and C57BL/6Cr male mice , 2-3 months old, were obtained from the Mammalian Genetics and Animal Production Section, National Cancer Institute. Tumors.-Ascites LSTRA of BALB/cCr origin and RBL-5 of C57BL/6Cr origin were maintained in syngeneic mice by weekly ip injections of 106 and 107 cells, respectively, suspended in 0.2 ml of medium 199. The DTIC-treated LSTRA lymphoma subline, hereafter referred to as LSTRA/DTIC, was obtained after several transplant generations in BALB/cCr mice treated ip with 100 mg DTIC/kg daily from day 1 through day 7 VOL. 60, NO. 1, JANUARY 1978
109
after challenge with 106 tumor cells (5). The line is maintained routinely in BALB/cCr animals treated with DTIC. Sensitization in vitro (mixed lymphocyte - tumor cell cultures) and cytotoxicity test. -Spleen lymphocytes (RSC) (2.Bx 106/ml, 2.5 ml/well) were cocultured in vitro with X-irradiated [5,000 rads for tumor cells or 2,000 rads for spleen cells by means of a Quandrocondex Westinghouse machine (Westinghouse Electric Corp., Pittsburgh, Pa.) with 0.25-mm copper and 0.55-mm aluminum filters at 200 kV, 15 m A, and 661 rads/min] stimulator cells (0.1 ml/well at the desired concentration) in 16-mm wells (tissue-culture cluster dishes; Costar, Data Packaging Corp., Cambridge, Mass.). The cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum and supplemented with glutamine, sodium pyruvate, nonessential amino acids, 2-mercaptoethanol, penicillin, and streptomycin, as described by Mishell and Dutton (9). After 1-7 days ofincubation at 37° C in a 7% CO 2 incubator, lymphoid cells were harvested, washed, resuspended in RPMI-1640 medium containing 10% fetal calf serum, and used as attacker cells in a 51Cr-release microcytotoxicity test. Attacker cells at various concentrations in 0.1 ml were mixed in 96-well plastic round-bottom microtiter plates with 2.5X104 51Cr-Iabeled target cells in 0.1 ml, centrifuged at 35 Xg for 4 minutes , and incubated for 4 hours at 37° C in a 7% CO 2 incubator. Cell labeling was performed as described by Canty and Wunderlich (10). After incubation, the plates were centrifuged at BOOxg for 10 minutes, and 0.1 ml of the supernatant was read in a gamma-scintillation counter (Packard Model #3002; Packard Instrument Co .; Inc., Downers Grove, 111.). The percentage of lysis was calculated as: cpm 51Cr released from cells Percent lysis= X100. cpm 51Cr released from cells by being frozen and thawed four times ABBREVIATIONS USED: DTIC = 5-(3,3-dimethyl-l-triazeno)imidazole4-carboxamide; DMTA = drug-mediated tumor antigen(s); M-MuLV = Moloney strain of murine leukemia virus; RSC = responder spleen cell(s); cpm = counts per minute; AC = autologous control(s). 1 Received ]uly 16, 1976; revised August 8, 1977; accepted August 15,1977. 2 Laboratory of Experimental Chernotherapy, Drug Research and Development Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Public Health Service, U .S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Address reprint requests to Dr. Goldin.
J
NATL CANCER INST
110
SANTONI, KINNEY, AND GOLDIN 100
Experimental results were expressed as: Percent cytotoxicity = percen t lysis in experimental wells - percen t lysis in AC, in which labeled target cells were incubated with cold target cells at the same concentration but in place of attacker cells.
90 LSTRA/DTIC TARGET
80
u
70
«
RESULTS
w
60 6 co
The primary and secondary in vitro response of BALB/cCr spleen cells against LSTRA/DTI C is illustrated in table 1. The primary stimulation in vitro (groups 7-9) by LSTRA/DTIC did not generate cytotoxic lymphocytes against either the parental LSTRA, LSTRA/DTIC, or the unrelated RBL-5 lines. No cytotoxicity was found when coculture times varied from 1 to 9 days or different RSC:stimulator cell ratios or attacker:target cell ratios (data not reported) were tested. Nevertheless, when C57BL/6 (H-2 fJ) allogeneic spleen cells were used as stimulators (groups 4-6), high cytotoxic response was obtained against H-2 b RBL-5 lymphoma cells (group 6). Strong cytotoxic activity against LSTRA/DTIC cells was obtained when spleen cells collected from mice presensitized with LS TRA/ DTIC cells were cocultured with the same lymphoma line (secondary response, group 14). On the contrary, low 51Cr-release values were found when the parental LSTRA line was used as target tumor (group 13). Moderate cytotoxic activity was detected against the unrelated RBL-5 line (group 15). When in vivo-presensitized lymphocytes were cultured in vitro for 5 days without stimulator cells (groups 10-12), only minimal cytotoxic activity was observed. To find the optimal RSC:stimulator cell ratio in the secondary response, we cocultured 7x 106 spleen cells from LSTRA/DTIC-sensitized mice with graded numbers of LSTRA/DTIC stimulator cells. The data reported in text-figure 1 indicate optimal response at 20: 1-80: 1 RSC:stimulator cell ratios. As in the previous experiment (table 1), spleen cells immune against LSTRA/DTIC were more cytotoxic for the DTIC tumor line than for the parental LSTRA (text-fig. 1). In further experiments, the secondary response in vitro was obtained with the use of parental as weIl as LSTRA/ DTIC cells as stimulators in coculture with spleen cells of LSTRA/DTIC-sensitized donor inbred BALB/c mice (table 2). At optimal RSC:stimulator cell ratios, lymphocytes cocultured with LSTRA/DTIC (groups 9-14) were
TABLE
«
~50
« w --l
~ 40 LSTRA TARGET
Cl:
u "'
cf.
30 20
I
1:1
5:1
"
10:1
20:1
40:1
80:1
RSC:SC RATID
TEXT-FIGURE 1.-Secondary response in vitro of BALB/cCr spleen cells to LSTRA/DTIC. SC=stimulator cell ,
more cytotoxic against the DTIC tumor line (groups 10, 12, and 14) than against the parental LSTRA lymphoma cells (groups 9,11, and 13). Moreover, LSTRA/ DTIC cells (groups 9-14) were more effective than were LSTRA parental cells (groups 3-8) as stimulators in the induction of cytotoxic lymphocytes against both the drug-altered and the original tumors. The data in tables 1 and 2 and text-figure 1 suggested the possibility that novel antigen(s) eliciting cell-mediated immunity would be on the LSTRA/DTIC. Therefore, studies on the cold-cell inhibition test were conducted (table 3). The 51Cr release by labeled LSTRA/DTIC in the presence of anti-LSTRA/DTIC immune spleen cells (secondary response) was not appreciably affected by the addition of unlabeled (inhibitor) LSTRA cells (groups 1, 3, and 5). When cold inhibitor LSTRA/DTIC cells were added to the attacker-target cell mixture , com petition was evidenced by a decrease in the 51Cr release (groups 2, 4,6, and 8). The differences were statistically significant (P 0 60 co
«
>-
I-
u
.,i-"
r.'
I-
- LSTRA - - LSTRA/DTIC .'-- RBL-5
I-
_.- NC57BL16
X
40
"
0 0
>- 20 u -;{( 0
0 5:1
20:1 80:1 A:I RATIO TEXT-FIGDRE 2.-Secondary response in vit.ro of C57BLj6Cr spleen cells 10 RBL-S lymphorna. Inhibitory effects of unlaheled cells added to attacker-target cell rnixture. Attacker:target cell ratio was 40: 1. A: I ratio=attacker:unlabeled cells ratio ; MSV -eS7 aRBL-5 = C57BLj6Cr rnice sensitized in vivo with Moloney murine sarco ma virus and cocultured in vitro with RBL-5 cells (RSC:stiInulator cell ratio was 20:1); LSTRA, LSTRA/DTIC, and RBL-5=unlabeled t.urnor cells; NC57BL/6=unlabeled normal spleen cells of C57BL/ 6er mice.
sponse was obtained in vitro against LSTRA/DTIC and M-MuLV-induced tumor of BALB/c origin. The presence of an immunodepressive virus (12) associated with LSTRA/D~rIC cells appears to have been ruled out in prelirninary studies where lirnited nu mbers of X-irradiated cells of the same tumor did not prevent primary sensitization in vitro against. normal, H-2-incOlTIpatible allogeneic spleen cells (Santoni A, TaramelliD: Unpublished data). Thus t.he lack of primary response in vitro could be ascribed to Iimited immu nogenicitv of the tumor for syngeneic l yrn phocytes. N evertheless, such an interpretation must be viewed with caution since a vigorous secondary response could be obtained readily in vitro. The data illustrated here seern consistent with previous observations (5), which showed cross-reactivity between LS1'RA and LSTRA/DTIC lines and appearanee of noble antigenicity on LSTRA cells after rreatment with DTIC in vivo. At.tacker splenocytes sensitized in vitro against LSTRA cells are equally cytotoxic against LS'TRA or LS'I'RA/D'I'IC targets (table 2, groups 3-8); this fact suggest.s that LSTRA and LSTRA/I)TIC possess cross-reacting antigen(s) possibly in co m parable amounts , When attacker splenocytes sensitized wit.h LSTRA/DrrIC were used, higher cytotoxic effects we re observed against both LS'-rRA and LS'l'RA/D]'IC lines. Nevertheless, killer effect.s were strikingly higher against LSTRA/DT~IC when corupared with those against the parentalline (table 1, groups 13-14; table 2, groups 9-14; anel text-fig. 1). This finding suggests that LSTRA/I)~rIC expresses additional antigen(s) not present in the paren tal lym phoma cells. Difference bet ween LSTRA and LsrrRA/D~rIC lines in their susceptibility to cell-mediated killing was ruled out by the followiug observations: a) No significant difference (P >0 .05, according to the Student's t-test) could be faund bei ween
J
NATL CANCER
I~ST
112
SANTONI, KINNEY, AND GOLDIN
51Cr release by parental and DTIC tumor lines exposed to anti-LSTRA attacker splenocytes (table 2, groups 38); b) target LSTRA or LSTRA/DTIC cells were equally susceptible to cell killing by allogeneic spleen cells sensitized in vitro against H-2 d alloantigens (Santoni A, Taramelli D: Unpublished observations). Further support for the presence of antigenicity on LSTRA/DTIC cells was provided by the cold-cell inhibition assay reported in table 3. Progressive inhibition of 51Cr release from labeled LSTRA/DTIC cells incubated with specifically sensitized splenocytes was provided by the increasing numbers of cold cells of the LSTRA/DTIC tumor subline. However, minimal or no inhibition was afforded by cold parental LSTRA cells in the same system. The specificity of cold-cell inhibition assay was supported by nonspecific inhibitory effects of the LSTRA/DTIC line being ruled out, as shown in text-figure 2. Both LSTRA and LSTRA/DTIC cells exerted minimal or no inhibitory effects in an in vitro systern of attacker-target cells, known to be unaffected specifically by the addition of cold LSTRA cells (11). Gur data seem to be consistent with the hypothesis that LSTRA/DTIC cells share tumor-associated antigens with LSTRA cells and express additional DMTA that are not detectable with the 51Cr-release test in the parentalline.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9) (10)
(11)
(12)
REFERENCES (1) BONMASSAR E, BONMASSAR A, VADLAMUDI S, et al: Immunclog-
J
NATL CANCER INST
ical alteration of leukemic cells in vivo after treatment with an antitumor drug. Proc Natl Acad Sei USA 66:1089-1095, 1970 - - - : Antigenie changes of Ll210 leukemia in mice treated with 5-(3 ,3-dimethyl-l-triazeno)imidazole-4-carboxamide. Cancer Res 32: 1446-1450, 1972 NICOLIN A, BINI A, CORONETTI E, et al: Cellular immune response to a drug treated L5178Y lymphoma subline. Nature 251;654-655, 1974 NICOLIN A, BINI A, FRANCO P, et al: Cell-mediated response to a mouse leukemic subline antigenically alte red following drug treatment in vivo. Cancer Chemother Rep 58:325-330, 1974 HOUCHENS DP, BONMASSAR E, GASTON MR, et al: Drug-mediated immunogenic changes of virus-induced leukemia. Cancer Res 36:1347-1352,1976 WAGNER H, ROLLINGHOFF M: Cell-rnediated immunity in vitro against syngeneic mouse plasma tumour cells. Nature [New Biol] 241 :53-54, 1973 LUNDAK RL, RAlDT DJ: Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensioris against DBA mastocytoma cells. Cell Immunol 9:60-66, 1973 PLATA F, CEROTTINI JC, BRUNNER RT: Primary and secondary in vitro generation of cytolytic T lymphocytes in the murine sarcoma virus system. Eur J Immunol 5:227-233, 1975 MISHELL TI, DUTTON RW; Immunization of dissociated spleen cell cultures from normal mice. J Exp Med 126:423-442, 1967 CANTY TG, WUNDERLICH JR: Quantitative in vitro assay of cytotoxic cellular immunity. J Natl Cancer Inst 45:761-772, 1970 HERBERMAN RB, AOKI T, NUNN M, et al: Specificity of 51Cr_ release cytotoxicity oflymphocytes immune to murine sarcoma virus. J Natl Cancer Inst 53: 1103-1111, 1974 BONNAItD GD, MANDER EK, CAMPBELL DA, et al: Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. Evidence for minute virus of mice causing the inhibition. J Exp Med 143:187-205, 1976
VOL. 60, NO. 1, JANUARY 1978
ABSTRACT-Cell-mediated cytotoxic response in vitro to a Moloney strain 01 murine leukemia virus-induced tumor (LSTRA) altered by 5-(3,3-dimethyl-1-triazeno)i midazole·4-carboxamide (DTIC) was investigated with the use 01 mixed-Ieukocyte tumor cell cultures (ML TC). As assessed by means 01 a short-term 51 Crrelease assay, minimal or no cytolytic activity was generated in primary MLTC. In contrast, high specific cytotoxic response against the altered tumor was obtained in secondary MLTC. Partial cross-reactivity was tound between the LSTRA and LSTRA/DTIC tumor Iines.-J Natl Cancer Inst 60: 109-112, 1978.
Novel tumor-associated antigenic determinants were found in murine lymphoma cells after treatment of tumor-bearing hosts with DTIC (1-5). The new antigenic specificity (specificities) mediated by DTIC treatment in vivo has been tentatively called DMTA. Specific cell-rnediated immunity against DMTA was found in mice sensitized in vivo with DTIC-treated tumor sublines (3, 4). The recently developed techniques of immune response in vitro (6-8) offer the opportunity for analysis of several aspects of generation of cytotoxic lymphocytes against tumor-associated antigens for both primary and secondary response. Nevertheless, no studies on cytotoxic immune response in vitro against DMTA have been conducted. In our laboratory, DMTA was evidenced in a MMuLV-induced lymphoma (LSTRA) after treatment with DTIC in vivo (5). The present experiments were performed with this DTIC-treated lymphoma subline to determine the possibility of obtaining primary or secondary cytotoxic responses in vitro. Our study indicates that highly cytotoxic lymphocytes were produced in the secondary response in vitro. However, little or no primary response was obtained.
MATERIALS AND METHODS Mice.-Inbred BALB/cCr and C57BL/6Cr male mice , 2-3 months old, were obtained from the Mammalian Genetics and Animal Production Section, National Cancer Institute. Tumors.-Ascites LSTRA of BALB/cCr origin and RBL-5 of C57BL/6Cr origin were maintained in syngeneic mice by weekly ip injections of 106 and 107 cells, respectively, suspended in 0.2 ml of medium 199. The DTIC-treated LSTRA lymphoma subline, hereafter referred to as LSTRA/DTIC, was obtained after several transplant generations in BALB/cCr mice treated ip with 100 mg DTIC/kg daily from day 1 through day 7 VOL. 60, NO. 1, JANUARY 1978
109
after challenge with 106 tumor cells (5). The line is maintained routinely in BALB/cCr animals treated with DTIC. Sensitization in vitro (mixed lymphocyte - tumor cell cultures) and cytotoxicity test. -Spleen lymphocytes (RSC) (2.Bx 106/ml, 2.5 ml/well) were cocultured in vitro with X-irradiated [5,000 rads for tumor cells or 2,000 rads for spleen cells by means of a Quandrocondex Westinghouse machine (Westinghouse Electric Corp., Pittsburgh, Pa.) with 0.25-mm copper and 0.55-mm aluminum filters at 200 kV, 15 m A, and 661 rads/min] stimulator cells (0.1 ml/well at the desired concentration) in 16-mm wells (tissue-culture cluster dishes; Costar, Data Packaging Corp., Cambridge, Mass.). The cells were suspended in RPMI-1640 medium containing 10% fetal bovine serum and supplemented with glutamine, sodium pyruvate, nonessential amino acids, 2-mercaptoethanol, penicillin, and streptomycin, as described by Mishell and Dutton (9). After 1-7 days ofincubation at 37° C in a 7% CO 2 incubator, lymphoid cells were harvested, washed, resuspended in RPMI-1640 medium containing 10% fetal calf serum, and used as attacker cells in a 51Cr-release microcytotoxicity test. Attacker cells at various concentrations in 0.1 ml were mixed in 96-well plastic round-bottom microtiter plates with 2.5X104 51Cr-Iabeled target cells in 0.1 ml, centrifuged at 35 Xg for 4 minutes , and incubated for 4 hours at 37° C in a 7% CO 2 incubator. Cell labeling was performed as described by Canty and Wunderlich (10). After incubation, the plates were centrifuged at BOOxg for 10 minutes, and 0.1 ml of the supernatant was read in a gamma-scintillation counter (Packard Model #3002; Packard Instrument Co .; Inc., Downers Grove, 111.). The percentage of lysis was calculated as: cpm 51Cr released from cells Percent lysis= X100. cpm 51Cr released from cells by being frozen and thawed four times ABBREVIATIONS USED: DTIC = 5-(3,3-dimethyl-l-triazeno)imidazole4-carboxamide; DMTA = drug-mediated tumor antigen(s); M-MuLV = Moloney strain of murine leukemia virus; RSC = responder spleen cell(s); cpm = counts per minute; AC = autologous control(s). 1 Received ]uly 16, 1976; revised August 8, 1977; accepted August 15,1977. 2 Laboratory of Experimental Chernotherapy, Drug Research and Development Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Public Health Service, U .S. Department of Health, Education, and Welfare, Bethesda, Md. 20014. 3 Address reprint requests to Dr. Goldin.
J
NATL CANCER INST
110
SANTONI, KINNEY, AND GOLDIN 100
Experimental results were expressed as: Percent cytotoxicity = percen t lysis in experimental wells - percen t lysis in AC, in which labeled target cells were incubated with cold target cells at the same concentration but in place of attacker cells.
90 LSTRA/DTIC TARGET
80
u
70
«
RESULTS
w
60 6 co
The primary and secondary in vitro response of BALB/cCr spleen cells against LSTRA/DTI C is illustrated in table 1. The primary stimulation in vitro (groups 7-9) by LSTRA/DTIC did not generate cytotoxic lymphocytes against either the parental LSTRA, LSTRA/DTIC, or the unrelated RBL-5 lines. No cytotoxicity was found when coculture times varied from 1 to 9 days or different RSC:stimulator cell ratios or attacker:target cell ratios (data not reported) were tested. Nevertheless, when C57BL/6 (H-2 fJ) allogeneic spleen cells were used as stimulators (groups 4-6), high cytotoxic response was obtained against H-2 b RBL-5 lymphoma cells (group 6). Strong cytotoxic activity against LSTRA/DTIC cells was obtained when spleen cells collected from mice presensitized with LS TRA/ DTIC cells were cocultured with the same lymphoma line (secondary response, group 14). On the contrary, low 51Cr-release values were found when the parental LSTRA line was used as target tumor (group 13). Moderate cytotoxic activity was detected against the unrelated RBL-5 line (group 15). When in vivo-presensitized lymphocytes were cultured in vitro for 5 days without stimulator cells (groups 10-12), only minimal cytotoxic activity was observed. To find the optimal RSC:stimulator cell ratio in the secondary response, we cocultured 7x 106 spleen cells from LSTRA/DTIC-sensitized mice with graded numbers of LSTRA/DTIC stimulator cells. The data reported in text-figure 1 indicate optimal response at 20: 1-80: 1 RSC:stimulator cell ratios. As in the previous experiment (table 1), spleen cells immune against LSTRA/DTIC were more cytotoxic for the DTIC tumor line than for the parental LSTRA (text-fig. 1). In further experiments, the secondary response in vitro was obtained with the use of parental as weIl as LSTRA/ DTIC cells as stimulators in coculture with spleen cells of LSTRA/DTIC-sensitized donor inbred BALB/c mice (table 2). At optimal RSC:stimulator cell ratios, lymphocytes cocultured with LSTRA/DTIC (groups 9-14) were
TABLE
«
~50
« w --l
~ 40 LSTRA TARGET
Cl:
u "'
cf.
30 20
I
1:1
5:1
"
10:1
20:1
40:1
80:1
RSC:SC RATID
TEXT-FIGURE 1.-Secondary response in vitro of BALB/cCr spleen cells to LSTRA/DTIC. SC=stimulator cell ,
more cytotoxic against the DTIC tumor line (groups 10, 12, and 14) than against the parental LSTRA lymphoma cells (groups 9,11, and 13). Moreover, LSTRA/ DTIC cells (groups 9-14) were more effective than were LSTRA parental cells (groups 3-8) as stimulators in the induction of cytotoxic lymphocytes against both the drug-altered and the original tumors. The data in tables 1 and 2 and text-figure 1 suggested the possibility that novel antigen(s) eliciting cell-mediated immunity would be on the LSTRA/DTIC. Therefore, studies on the cold-cell inhibition test were conducted (table 3). The 51Cr release by labeled LSTRA/DTIC in the presence of anti-LSTRA/DTIC immune spleen cells (secondary response) was not appreciably affected by the addition of unlabeled (inhibitor) LSTRA cells (groups 1, 3, and 5). When cold inhibitor LSTRA/DTIC cells were added to the attacker-target cell mixture , com petition was evidenced by a decrease in the 51Cr release (groups 2, 4,6, and 8). The differences were statistically significant (P 0 60 co
«
>-
I-
u
.,i-"
r.'
I-
- LSTRA - - LSTRA/DTIC .'-- RBL-5
I-
_.- NC57BL16
X
40
"
0 0
>- 20 u -;{( 0
0 5:1
20:1 80:1 A:I RATIO TEXT-FIGDRE 2.-Secondary response in vit.ro of C57BLj6Cr spleen cells 10 RBL-S lymphorna. Inhibitory effects of unlaheled cells added to attacker-target cell rnixture. Attacker:target cell ratio was 40: 1. A: I ratio=attacker:unlabeled cells ratio ; MSV -eS7 aRBL-5 = C57BLj6Cr rnice sensitized in vivo with Moloney murine sarco ma virus and cocultured in vitro with RBL-5 cells (RSC:stiInulator cell ratio was 20:1); LSTRA, LSTRA/DTIC, and RBL-5=unlabeled t.urnor cells; NC57BL/6=unlabeled normal spleen cells of C57BL/ 6er mice.
sponse was obtained in vitro against LSTRA/DTIC and M-MuLV-induced tumor of BALB/c origin. The presence of an immunodepressive virus (12) associated with LSTRA/D~rIC cells appears to have been ruled out in prelirninary studies where lirnited nu mbers of X-irradiated cells of the same tumor did not prevent primary sensitization in vitro against. normal, H-2-incOlTIpatible allogeneic spleen cells (Santoni A, TaramelliD: Unpublished data). Thus t.he lack of primary response in vitro could be ascribed to Iimited immu nogenicitv of the tumor for syngeneic l yrn phocytes. N evertheless, such an interpretation must be viewed with caution since a vigorous secondary response could be obtained readily in vitro. The data illustrated here seern consistent with previous observations (5), which showed cross-reactivity between LS1'RA and LSTRA/DTIC lines and appearanee of noble antigenicity on LSTRA cells after rreatment with DTIC in vivo. At.tacker splenocytes sensitized in vitro against LSTRA cells are equally cytotoxic against LS'TRA or LS'I'RA/D'I'IC targets (table 2, groups 3-8); this fact suggest.s that LSTRA and LSTRA/I)TIC possess cross-reacting antigen(s) possibly in co m parable amounts , When attacker splenocytes sensitized wit.h LSTRA/DrrIC were used, higher cytotoxic effects we re observed against both LS'-rRA and LS'l'RA/D]'IC lines. Nevertheless, killer effect.s were strikingly higher against LSTRA/DT~IC when corupared with those against the parentalline (table 1, groups 13-14; table 2, groups 9-14; anel text-fig. 1). This finding suggests that LSTRA/I)~rIC expresses additional antigen(s) not present in the paren tal lym phoma cells. Difference bet ween LSTRA and LsrrRA/D~rIC lines in their susceptibility to cell-mediated killing was ruled out by the followiug observations: a) No significant difference (P >0 .05, according to the Student's t-test) could be faund bei ween
J
NATL CANCER
I~ST
112
SANTONI, KINNEY, AND GOLDIN
51Cr release by parental and DTIC tumor lines exposed to anti-LSTRA attacker splenocytes (table 2, groups 38); b) target LSTRA or LSTRA/DTIC cells were equally susceptible to cell killing by allogeneic spleen cells sensitized in vitro against H-2 d alloantigens (Santoni A, Taramelli D: Unpublished observations). Further support for the presence of antigenicity on LSTRA/DTIC cells was provided by the cold-cell inhibition assay reported in table 3. Progressive inhibition of 51Cr release from labeled LSTRA/DTIC cells incubated with specifically sensitized splenocytes was provided by the increasing numbers of cold cells of the LSTRA/DTIC tumor subline. However, minimal or no inhibition was afforded by cold parental LSTRA cells in the same system. The specificity of cold-cell inhibition assay was supported by nonspecific inhibitory effects of the LSTRA/DTIC line being ruled out, as shown in text-figure 2. Both LSTRA and LSTRA/DTIC cells exerted minimal or no inhibitory effects in an in vitro systern of attacker-target cells, known to be unaffected specifically by the addition of cold LSTRA cells (11). Gur data seem to be consistent with the hypothesis that LSTRA/DTIC cells share tumor-associated antigens with LSTRA cells and express additional DMTA that are not detectable with the 51Cr-release test in the parentalline.
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9) (10)
(11)
(12)
REFERENCES (1) BONMASSAR E, BONMASSAR A, VADLAMUDI S, et al: Immunclog-
J
NATL CANCER INST
ical alteration of leukemic cells in vivo after treatment with an antitumor drug. Proc Natl Acad Sei USA 66:1089-1095, 1970 - - - : Antigenie changes of Ll210 leukemia in mice treated with 5-(3 ,3-dimethyl-l-triazeno)imidazole-4-carboxamide. Cancer Res 32: 1446-1450, 1972 NICOLIN A, BINI A, CORONETTI E, et al: Cellular immune response to a drug treated L5178Y lymphoma subline. Nature 251;654-655, 1974 NICOLIN A, BINI A, FRANCO P, et al: Cell-mediated response to a mouse leukemic subline antigenically alte red following drug treatment in vivo. Cancer Chemother Rep 58:325-330, 1974 HOUCHENS DP, BONMASSAR E, GASTON MR, et al: Drug-mediated immunogenic changes of virus-induced leukemia. Cancer Res 36:1347-1352,1976 WAGNER H, ROLLINGHOFF M: Cell-rnediated immunity in vitro against syngeneic mouse plasma tumour cells. Nature [New Biol] 241 :53-54, 1973 LUNDAK RL, RAlDT DJ: Cellular immune response against tumor cells. I. In vitro immunization of allogeneic and syngeneic mouse spleen cell suspensioris against DBA mastocytoma cells. Cell Immunol 9:60-66, 1973 PLATA F, CEROTTINI JC, BRUNNER RT: Primary and secondary in vitro generation of cytolytic T lymphocytes in the murine sarcoma virus system. Eur J Immunol 5:227-233, 1975 MISHELL TI, DUTTON RW; Immunization of dissociated spleen cell cultures from normal mice. J Exp Med 126:423-442, 1967 CANTY TG, WUNDERLICH JR: Quantitative in vitro assay of cytotoxic cellular immunity. J Natl Cancer Inst 45:761-772, 1970 HERBERMAN RB, AOKI T, NUNN M, et al: Specificity of 51Cr_ release cytotoxicity oflymphocytes immune to murine sarcoma virus. J Natl Cancer Inst 53: 1103-1111, 1974 BONNAItD GD, MANDER EK, CAMPBELL DA, et al: Immunosuppressive activity of a subline of the mouse EL-4 lymphoma. Evidence for minute virus of mice causing the inhibition. J Exp Med 143:187-205, 1976
VOL. 60, NO. 1, JANUARY 1978
