Proc. Natl. Acad. Sci. USA Vol. 74. No. 1, pp. 329-333, January 1977
Immunology
Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P815Y (tumor-associated antigens/tumor escape)
WILLIAM E. BIDDISON AND JON C. PALMER The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104
Communicated by Peter C. Nowell, September 24, 1976
ABSTRACT The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 103 tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.
Recent studies in humans and in animal model systems have demonstrated that many tumor-bearing hosts mount both cell-mediated and humoral immune responses to their tumors (1-3). Despite such ongoing antitumor immune responses, many syngeneic tumors are capable of continued growth leading to death of the host. The mechanisms by which tumor cells escape antitumor immune responses are thought to be complex, but may include: (a) antigenic modulation of tumor-associated antigens (4-6); (b) inhibition of cell-mediated immunity by serum blocking factors (7, 8); (c) specific and nonspecific immunosuppression of the hosts' immune responsiveness (9-11); (d) stimulation of tumor growth by the immune response (12); (e) location of the tumor in an immunologically "privileged site" (13); (f) lack of immunogenic determinants on the tumor cell membrane; and (g) tumor growth that proceeds more rapidly than does the induction and action of the antitumor immune response. Most previous studies of cell-mediated immunity to tumorassociated antigens have analyzed the immune capacities of cell populations obtained from various lymphoid organs or peripheral blood. However, the immune reactivities of the cells obtained from these areas, which may be distant from the tumor mass, may not accurately reflect events occurring within or surrounding the tumor (14). In addition, tumor cells maintained in tissue culture are usually used as target cells for assays of cell-mediated cytotoxicity (CMC) in vitro and for the presence of antibodies against tumors. It is possible that tumor cells in Abbreviations: CMC, cell-mediated cytotoxicity; B6, C57BL/6; B1O, C57BL/10; i.p., intraperitoneally; TAA, tumor-associated antigens. 329
tissue culture may lose and/or acquire some characteristics, and therefore, may no longer be representative of tumor cells in the tumor-bearing host (15). To assess more accurately the effect
of antitumor immune responses on tumor cells in situ, it is desirable to analyze the cellular and humoral immune responses to the tumor cells occurring within a progressively growing tumor mass.
MATERIALS AND METHODS Mice. Female DBA/2, C57BL/10 (B10), C57BL/6 (B6), and B10.D2 mice were obtained from the Jackson Laboratories, Bar Harbor, Me., and the Institute for Cancer Research, Philadelphia, Pa., and used at 8 weeks of age. Tumors. The P815Y (DBA/2, H-2d) mastocytoma, L1210 (DBA/2, H-2d) leukemia, and EL4 (B6, H-2b) lymphoma were maintained as ascites tumors in the syngeneic strain and cultured in vitro. Tumor-bearing mice used in this study were DBA/2 mice inoculated intraperitoneally (i.p.) with 103 viable P815Y cells grown in tissue culture and used at the fourth to tenth cell culture passage. Cell Separation Procedures. Peritoneal cells were harvested from normal and P815Y tumor-bearing DBA/2 mice by repeated flushing of the peritoneal cavity with phosphate-buffered saline, washed once with Fischer's modified LI medium (Schwartz BioResearch, Orangeburg, N.Y.), and counted by hemocytometer. Peritoneal cells were fractionated on the basis of cell size by unit gravity velocity sedimentation in a Sta-Put cell separation apparatus (Johns Scientific, Toronto, Ont.) as described by Miller and Phillips (16). Forty to 150 X 106 peritoneal cells in 20 ml of Li medium were loaded into the apparatus followed by a 1500-ml linear gradient of 5-25% fetal bovine serum in Li medium. After sedimentation for 3 hr at 40, 50-ml fractions were collected and cell concentrations determined in a Coulter Counter (Coulter Electronics, Hialeah, Fla.). In Vitro Induction of CMC. Cytotoxic lymphocytes reactive primarily against H-2d transplantation antigens present on P815Y were induced by culturing normal B6 spleen cells with mitomycin C-treated P815Y cells in Marbrook culture vessels as described (17). Assay for CMC. CMC against tumor target cells was measured by a 4-hr 51Cr-release assay as described in detail elsewhere (18). P815Y tumor target cells were obtained from cultures at the fourth to tenth cell culture passage. Cytotoxicity is expressed as percent specific lysis and is equal to experimental release minus spontaneous release divided by hypotonic release minus spontaneous release. All determinations were performed in triplicate, and the SEM rarely exceeded 5% of the mean. Assay for Cell Surface Immunoglobulin. Cell surface immunoglobulin was detected by the binding of fluoresceinconjugated rabbit antibody against mouse gamma globulin
330
Immunology: Biddison and Palmer
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Proc. Natl. Acad. Sci. USA 74 (1977)
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FIG. 1. Each data point represents the mean I SEM of the number of nucleated peritoneal cells present in DBA/2 mice at various times after i.p. inoculation of 103 viable P815Y tumor cells.
(Cappel Laboratories, Downingtown, Pa.) and by the binding of 125I-labeled rabbit antibody against mouse Fab as described (19).
RESULTS Tumor Growth. Normal DBA/2 mice were inoculated with 103 viable P815Y i.p., and at various times thereafter four to 20 mice were killed, their peritoneal cavities were flushed with phosphate-buffered saline, and tumor growth was evaluated by counting the number of nucleated peritoneal cells (Fig. 1). The mice developed a measurable ascitic tumor load by day 12 and all died within 22-28 days after tumor inoculation. Fractionation of Peritoneal Cells by Velocity Sedimentation. Normal DBA/2 peritoneal cells and peritoneal cells harvested from DBA/2 mice 8, 10, and 16 days after i.p. inoculation with 103 viable P815Y were fractionated by velocity sedimentation at unit gravity. Various pooled fractions and unfractionated cells were assayed for CMC against 51Cr-labeled P815Y at an effector cell:target cell ratio of 100:1. Normal DBA/2 peritoneal cells displayed a bimodal distribution in sedimentation rate, with one peak at 3.3 mm/hr and another peak between 5.3 and 6 mm/hr (Fig. 2a). No significant CMC against-P815Y was detected in any of the normal DBA/2 peritoneal cell fractions. Cytotoxic activity against P815Y was observed in fractions of day 8 tumor-bearer peritoneal cells that sedimented between 3.6 and 5.3 mm/hr (Fig. 2b). Day 10
FIG. 2. Kinetic studies of the sedimentation velocities of peritoneal cells harvested from normal DBA/2 mice (a) and DBA/2 mice 8 days (b), 10 days (c), and 16 days (d) after i.p. inoculation of 103 viable P815Y. A plot of the number of cells against sedimentation velocity in mm/hr is shown (0) for each of the peritoneal cell populations fractionated. GMC against P815Y by peritoneal cell fractions (shaded bars) and unfractionated peritoneal cells (open bars) is expressed as percent specific lysis.
tumor-bearer peritoneal cells (Fig. 2c) also displayed a bimodal distribution in sedimentation rate, but with peaks at 5.3 and 9.3 mm/hr. Strong CMC against P815Y was expressed by day 10 peritoneal cell populations that sedimented between 1.3-4.6 mm/hr and 4.6-6 mm/hr. Although detectable CMC to P815Y was not observed in unfractionated day 16 peritoneal cells, cytotoxic activity was present in peritoneal cell fractions that sedimented between 2 and 5.6 mm/hr (Fig. 2d). Specificity of CMC in Peritoneal Cell Populations of Tumor-bearing Mice. After velocity sedimentation at unit gravity, peritoneal cell populations from tumor-bearing mice were assayed for CMC against cultured P815Y and another DBA/2 tumor cell line, L1210. The results in Table 1 indicate Table 1. Specificity of CMC in peritoneal cell populations of tumor-bearing mice
Target cells
Effector cells Sedimentation rates (mm/hr) (peritoneal 9-11 6-8 5-6 0-4 cells)
P815Y L1210 P815Y Day 10 P.C.t L1210 P815Y
Day 12
0*
Day12
Dayl6P.C.t
Dayl6 Dayl6
0 0 6 0 0 0 0
L1210
Day 10 Day 10
DaylO Day 16
3 0 6 17 0 0 0 2
63 0 54 44 6 2 0 0
44 0 38 36 5 13 0 0
* Percent specific lysis values were measured at an effector cell:target cell ratio of 100:1. t Day 10 and day 16 peritoneal cells (P.C.) with sedimentation rates = 11-13 mm/hr were labeled with 5lCr and used as tumor target cells. Spontaneous and hypotonic release was, respectively: for day 10 P.C., 25% and 80%; for day 16 P.C., 22% and 80%; for P815Y, 13% and 88%; and for L1210, 15% and 84%.
Proc. Natl. Acad. Sci. USA 74 (1977)
Immunology: Biddison and Palmer Table 2. Susceptibility of tumor cells to CMC
cn 40-
331
b
a
Specific lysis, effectors: target
%
U
Effector cells
Target cells
w 0.
Day 10 P.C. (4-6 mm/hr)* Day 10 P.C. (2.6-4 mm/hr)*
Day 16 P.C. (2-4.6 mm/hr)* B6 spleen anti-P815Y
P815Y Day 10 P.C.t
54 41
36 33
16 14
Day 16 P.C.t
8
5
2
23 28
18 13 0 2
13 6 0 0
P815Y
Day 10 P.C. Day 16P.C. P815Y Day 10 P.C. Dayl6P.C. P815Y Day 10 P.C. Day 16 P.C. EL4
40
100:1 30:1 10:1
0 7 7 0 72 67 68 8
2 0 58 33 51 6
0 0 29 20
21 3
Peritoneal cells (P.C.) from day 10 and day 16 tumor-bearing mice simultaneously fractionated by velocity sedimentation at unit gravity. The indicated fractions were pooled and assayed for CMC against 51Cr-labeled P815Y and tumor cells obtained from the P.C. populations in a single assay. t Day 10 and day 16 P.C. used as 51Cr-labeled target cells in the CMC assay were cells with sedimentation rates = 11-13 mm/hr. *
were
that peritoneal cell populations that exhibited CMC against P815Y expressed little or no CMC against L1210. It was also of interest to determine if effector cells in peritoneal cell populations of tumor-bearing mice that could lyse cultured P815Y could also kill tumor cells present in these same animals. Day 10 and day 16 peritoneal cells that sedimented between 11 and 13 mm/hr were labeled with 51Cr and used as target cells since these fractions contained maximum percentages of P815Y tumor cells. As judged by morphology after Wright-Giemsa staining and by neutral red staining (20), these fractions from day 10 and day 16 peritoneal cells contained 80% and 95% P815Y and 17% and 3% macrophages, respectively. Furthermore, when 103 of these day 16 peritoneal cells were inoculated i.p. into normal DBA/2 mice, all of the recipients died bearing large ascitic tumors within 19-28 days. As shown in Table 1, day 10 peritoneal cell populations that displayed CMC against cultured P815Y also displayed a similar amount of CMC against day 10 peritoneal cell tumor cell targets. In contrast, day 16 peritoneal cell populations active in the CMC assay against cultured P815Y target cells were unable to cause cytolysis of day 16 tumor cell targets. Susceptibility of Day 16 Tumor Cells to CMC. The absence of CMC against day 16 tumor cell targets by day 16 effector cells suggested that day 16 tumor cells were unable to be rec-
ognized and killed by effector cells in the tumor-bearing host. To test this hypothesis, peritoneal cells from both day 10 and day 16 tumor-bearing animals were assayed for CMC against P815Y, day 10, and day 16 tumor cell targets (Table 2). The results indicated that cultured P815Y and day 10 tumor cell targets were similar in their susceptibility to lysis by these effector cells, but the day 16 tumor cell targets were much less susceptible to lysis by these effectors. The entire experiment was repeated and yielded essentially the same results. To test the possibility that the reduced CMC against day 16 tumor cells was due to resistance to lysis or to nonspecific suppression of effector cell function, we assayed these tumor target cells with effector cells directed primarily against H-2d antigenic determinants. B6 spleen cell (H-2b) effectors, obtained
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FIG. 3. Inhibition of CMC against 56Cr-labeled target cells by various numbers of unlabeled P815Y cells (0), day 16 tumor cells (A), and L1210 tumor cells (O). Effector cells were day 10 peritoneal cells with sedimentation rate = 3.3-6 mm/hr (a) and day 16 peritoneal cells with sedimentation rate = 2.3-4.6 mm/hr (b) added to yield an effector cell:labeled target cell ratio of 100:1 and 200:1, respectively. (0) Percent specific lysis in the absence of unlabeled inhibitor cells. Spontaneous and hypotonic release for P815Y was 11% and 81%, respectively.
by in vitro culture with mitomycin C-treated P815Y, were assayed for CMC against P815Y, EL4 (H-2b), day 10, and day 16 tumor cell targets (Table 2). The results demonstrated that day 16 tumor cells were as susceptible as day 10 tumor cells and cultured P815Y cells to CMC by allogeneic effector cells. Ability of Day 16 Tumor Cells to Inhibit CMC against P815Y. The absence of CMC against day 16 tumor cells could have resulted from the number of determinants on the tumor cell surface required for effector cell recognition being below the level required for detectable CMC (21). Therefore, the capacity of day 16 tumor cells to competitively inhibit CMC against cultured P815Y by effector cells obtained from day 10 and day 16 peritoneal cell populations was determined. The data in Fig. 3 indicate that a 4- to 64-fold excess of unlabeled day 16 tumor cells and unlabeled L1210 cells caused only a marginal inhibition of CMC against P815Y mediated by both day 10 (Fig. 3a) and day 16 (Fig. 3b) peritoneal cell effector cell populations. However, a marked inhibition of CMC against cultured P815Y occurred when a 4- to 64-fold excess of unlabeled P815Y cells was added to day 10 effector cells and labeled P815Y target cells (Fig. Sa). A similar effect was observed when inhibition of day 16 effector cell activity was analyzed (Fig. 3b). These data indicate that day 16 tumor cells do not display a significant number of cell surface determinants that are recognized by effector cells directed against tumor-associated antigens (TAA) on cultured P815Y. Assay for Immunoglobulin on the Surface of Tumor Cells. The absence of TAA on day 16 tumor cells as detected by CMC could have resulted from the presence of antibody molecules specifically bound to TAA determinants. Such cell-bound antibody could have effectively "blocked" the recognition and cytotoxic functions of effector cells that were specific for these determinants (22). Therefore, cell surface immunoglobulin on day 16 tumor cells was assayed by both immunofluorescence and a highly sensitive radioimmunoassay (Table 3). This radioimmunoassay is capable of detecting 0.1 ng of specific antibody (19). The radioimmunoassay and the immunofluorescence assay revealed that no detectable immunoglobulin was present on P815Y and day 16 tumor cells. In addition, day 16 ascitic fluid contained no more antibody capable of binding to P815Y than was present in normal DBA/2 serum. Also, incubation of cultured P815Y with day 16 ascitic fluid did not "block" syngeneic CMC to these target cells (data not shown).
332
Immunology: Biddison and Palmer
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 3. Assay for immunoglobulin on the surface of P815Y and day 16 tumor cells
Cells P815Y Day 1.6 peritoneal cells (11-13 mm/hr) P815Y + anti-H-2d serum (1:80)t P815Y + normal DBA/2serum(1:5) P815Y + day 16 ascitic fluid (1: 5)
Bound % Cells stained with 1 25I-labeled antibody fluoresceinantibody* (cpm ± SEM)t
Immunology
Development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of ascitic mastocytoma P815Y (tumor-associated antigens/tumor escape)
WILLIAM E. BIDDISON AND JON C. PALMER The Wistar Institute of Anatomy and Biology, 36th Street at Spruce, Philadelphia, Pennsylvania 19104
Communicated by Peter C. Nowell, September 24, 1976
ABSTRACT The immune reactivity to tumor cells within a progressively growing tumor mass in the syngeneic host has been analyzed by studying the cell-mediated cytolytic response of DBA/2 mice to the ascitic mastocytoma P815Y. Peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity. Cell-mediated cytotoxicity of fractionated and unfractionated cells was measured by 51Cr-release from tumor target cells. The cell separation procedure revealed significant levels of specific cell-mediated cytotoxicity to P815Y within peritoneal cell populations at 8-16 days after tumor cell inoculation. Tumor cells purified from the peritoneal cell populations of mice injected with 103 tumor cells 10 days previously were as susceptible to syngeneic and allogeneic cell-mediated cytotoxicity as P815Y grown in vitro. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to cytolysis by syngeneic, but not allogeneic, effector cells. In addition, day 16 tumor cells did not inhibit syngeneic cell-mediated cytotoxicity against P815Y grown in vitro. Immunoglobulin was not detected on day 16 tumor cells and no circulating antibody to P815Y was found in the ascitic fluid of day 16 tumor-bearing mice. These results indicate that tumor cells may escape immune attack by loss of expression of cell surface tumor-associated antigens in the absence of circulating antibody against tumor.
Recent studies in humans and in animal model systems have demonstrated that many tumor-bearing hosts mount both cell-mediated and humoral immune responses to their tumors (1-3). Despite such ongoing antitumor immune responses, many syngeneic tumors are capable of continued growth leading to death of the host. The mechanisms by which tumor cells escape antitumor immune responses are thought to be complex, but may include: (a) antigenic modulation of tumor-associated antigens (4-6); (b) inhibition of cell-mediated immunity by serum blocking factors (7, 8); (c) specific and nonspecific immunosuppression of the hosts' immune responsiveness (9-11); (d) stimulation of tumor growth by the immune response (12); (e) location of the tumor in an immunologically "privileged site" (13); (f) lack of immunogenic determinants on the tumor cell membrane; and (g) tumor growth that proceeds more rapidly than does the induction and action of the antitumor immune response. Most previous studies of cell-mediated immunity to tumorassociated antigens have analyzed the immune capacities of cell populations obtained from various lymphoid organs or peripheral blood. However, the immune reactivities of the cells obtained from these areas, which may be distant from the tumor mass, may not accurately reflect events occurring within or surrounding the tumor (14). In addition, tumor cells maintained in tissue culture are usually used as target cells for assays of cell-mediated cytotoxicity (CMC) in vitro and for the presence of antibodies against tumors. It is possible that tumor cells in Abbreviations: CMC, cell-mediated cytotoxicity; B6, C57BL/6; B1O, C57BL/10; i.p., intraperitoneally; TAA, tumor-associated antigens. 329
tissue culture may lose and/or acquire some characteristics, and therefore, may no longer be representative of tumor cells in the tumor-bearing host (15). To assess more accurately the effect
of antitumor immune responses on tumor cells in situ, it is desirable to analyze the cellular and humoral immune responses to the tumor cells occurring within a progressively growing tumor mass.
MATERIALS AND METHODS Mice. Female DBA/2, C57BL/10 (B10), C57BL/6 (B6), and B10.D2 mice were obtained from the Jackson Laboratories, Bar Harbor, Me., and the Institute for Cancer Research, Philadelphia, Pa., and used at 8 weeks of age. Tumors. The P815Y (DBA/2, H-2d) mastocytoma, L1210 (DBA/2, H-2d) leukemia, and EL4 (B6, H-2b) lymphoma were maintained as ascites tumors in the syngeneic strain and cultured in vitro. Tumor-bearing mice used in this study were DBA/2 mice inoculated intraperitoneally (i.p.) with 103 viable P815Y cells grown in tissue culture and used at the fourth to tenth cell culture passage. Cell Separation Procedures. Peritoneal cells were harvested from normal and P815Y tumor-bearing DBA/2 mice by repeated flushing of the peritoneal cavity with phosphate-buffered saline, washed once with Fischer's modified LI medium (Schwartz BioResearch, Orangeburg, N.Y.), and counted by hemocytometer. Peritoneal cells were fractionated on the basis of cell size by unit gravity velocity sedimentation in a Sta-Put cell separation apparatus (Johns Scientific, Toronto, Ont.) as described by Miller and Phillips (16). Forty to 150 X 106 peritoneal cells in 20 ml of Li medium were loaded into the apparatus followed by a 1500-ml linear gradient of 5-25% fetal bovine serum in Li medium. After sedimentation for 3 hr at 40, 50-ml fractions were collected and cell concentrations determined in a Coulter Counter (Coulter Electronics, Hialeah, Fla.). In Vitro Induction of CMC. Cytotoxic lymphocytes reactive primarily against H-2d transplantation antigens present on P815Y were induced by culturing normal B6 spleen cells with mitomycin C-treated P815Y cells in Marbrook culture vessels as described (17). Assay for CMC. CMC against tumor target cells was measured by a 4-hr 51Cr-release assay as described in detail elsewhere (18). P815Y tumor target cells were obtained from cultures at the fourth to tenth cell culture passage. Cytotoxicity is expressed as percent specific lysis and is equal to experimental release minus spontaneous release divided by hypotonic release minus spontaneous release. All determinations were performed in triplicate, and the SEM rarely exceeded 5% of the mean. Assay for Cell Surface Immunoglobulin. Cell surface immunoglobulin was detected by the binding of fluoresceinconjugated rabbit antibody against mouse gamma globulin
330
Immunology: Biddison and Palmer
1. . \ . *
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Proc. Natl. Acad. Sci. USA 74 (1977)
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IL. 0
z
107-
I-
0
8 4 20 16 12 DAYS AFTER TUMOR INOCULATION
FIG. 1. Each data point represents the mean I SEM of the number of nucleated peritoneal cells present in DBA/2 mice at various times after i.p. inoculation of 103 viable P815Y tumor cells.
(Cappel Laboratories, Downingtown, Pa.) and by the binding of 125I-labeled rabbit antibody against mouse Fab as described (19).
RESULTS Tumor Growth. Normal DBA/2 mice were inoculated with 103 viable P815Y i.p., and at various times thereafter four to 20 mice were killed, their peritoneal cavities were flushed with phosphate-buffered saline, and tumor growth was evaluated by counting the number of nucleated peritoneal cells (Fig. 1). The mice developed a measurable ascitic tumor load by day 12 and all died within 22-28 days after tumor inoculation. Fractionation of Peritoneal Cells by Velocity Sedimentation. Normal DBA/2 peritoneal cells and peritoneal cells harvested from DBA/2 mice 8, 10, and 16 days after i.p. inoculation with 103 viable P815Y were fractionated by velocity sedimentation at unit gravity. Various pooled fractions and unfractionated cells were assayed for CMC against 51Cr-labeled P815Y at an effector cell:target cell ratio of 100:1. Normal DBA/2 peritoneal cells displayed a bimodal distribution in sedimentation rate, with one peak at 3.3 mm/hr and another peak between 5.3 and 6 mm/hr (Fig. 2a). No significant CMC against-P815Y was detected in any of the normal DBA/2 peritoneal cell fractions. Cytotoxic activity against P815Y was observed in fractions of day 8 tumor-bearer peritoneal cells that sedimented between 3.6 and 5.3 mm/hr (Fig. 2b). Day 10
FIG. 2. Kinetic studies of the sedimentation velocities of peritoneal cells harvested from normal DBA/2 mice (a) and DBA/2 mice 8 days (b), 10 days (c), and 16 days (d) after i.p. inoculation of 103 viable P815Y. A plot of the number of cells against sedimentation velocity in mm/hr is shown (0) for each of the peritoneal cell populations fractionated. GMC against P815Y by peritoneal cell fractions (shaded bars) and unfractionated peritoneal cells (open bars) is expressed as percent specific lysis.
tumor-bearer peritoneal cells (Fig. 2c) also displayed a bimodal distribution in sedimentation rate, but with peaks at 5.3 and 9.3 mm/hr. Strong CMC against P815Y was expressed by day 10 peritoneal cell populations that sedimented between 1.3-4.6 mm/hr and 4.6-6 mm/hr. Although detectable CMC to P815Y was not observed in unfractionated day 16 peritoneal cells, cytotoxic activity was present in peritoneal cell fractions that sedimented between 2 and 5.6 mm/hr (Fig. 2d). Specificity of CMC in Peritoneal Cell Populations of Tumor-bearing Mice. After velocity sedimentation at unit gravity, peritoneal cell populations from tumor-bearing mice were assayed for CMC against cultured P815Y and another DBA/2 tumor cell line, L1210. The results in Table 1 indicate Table 1. Specificity of CMC in peritoneal cell populations of tumor-bearing mice
Target cells
Effector cells Sedimentation rates (mm/hr) (peritoneal 9-11 6-8 5-6 0-4 cells)
P815Y L1210 P815Y Day 10 P.C.t L1210 P815Y
Day 12
0*
Day12
Dayl6P.C.t
Dayl6 Dayl6
0 0 6 0 0 0 0
L1210
Day 10 Day 10
DaylO Day 16
3 0 6 17 0 0 0 2
63 0 54 44 6 2 0 0
44 0 38 36 5 13 0 0
* Percent specific lysis values were measured at an effector cell:target cell ratio of 100:1. t Day 10 and day 16 peritoneal cells (P.C.) with sedimentation rates = 11-13 mm/hr were labeled with 5lCr and used as tumor target cells. Spontaneous and hypotonic release was, respectively: for day 10 P.C., 25% and 80%; for day 16 P.C., 22% and 80%; for P815Y, 13% and 88%; and for L1210, 15% and 84%.
Proc. Natl. Acad. Sci. USA 74 (1977)
Immunology: Biddison and Palmer Table 2. Susceptibility of tumor cells to CMC
cn 40-
331
b
a
Specific lysis, effectors: target
%
U
Effector cells
Target cells
w 0.
Day 10 P.C. (4-6 mm/hr)* Day 10 P.C. (2.6-4 mm/hr)*
Day 16 P.C. (2-4.6 mm/hr)* B6 spleen anti-P815Y
P815Y Day 10 P.C.t
54 41
36 33
16 14
Day 16 P.C.t
8
5
2
23 28
18 13 0 2
13 6 0 0
P815Y
Day 10 P.C. Day 16P.C. P815Y Day 10 P.C. Dayl6P.C. P815Y Day 10 P.C. Day 16 P.C. EL4
40
100:1 30:1 10:1
0 7 7 0 72 67 68 8
2 0 58 33 51 6
0 0 29 20
21 3
Peritoneal cells (P.C.) from day 10 and day 16 tumor-bearing mice simultaneously fractionated by velocity sedimentation at unit gravity. The indicated fractions were pooled and assayed for CMC against 51Cr-labeled P815Y and tumor cells obtained from the P.C. populations in a single assay. t Day 10 and day 16 P.C. used as 51Cr-labeled target cells in the CMC assay were cells with sedimentation rates = 11-13 mm/hr. *
were
that peritoneal cell populations that exhibited CMC against P815Y expressed little or no CMC against L1210. It was also of interest to determine if effector cells in peritoneal cell populations of tumor-bearing mice that could lyse cultured P815Y could also kill tumor cells present in these same animals. Day 10 and day 16 peritoneal cells that sedimented between 11 and 13 mm/hr were labeled with 51Cr and used as target cells since these fractions contained maximum percentages of P815Y tumor cells. As judged by morphology after Wright-Giemsa staining and by neutral red staining (20), these fractions from day 10 and day 16 peritoneal cells contained 80% and 95% P815Y and 17% and 3% macrophages, respectively. Furthermore, when 103 of these day 16 peritoneal cells were inoculated i.p. into normal DBA/2 mice, all of the recipients died bearing large ascitic tumors within 19-28 days. As shown in Table 1, day 10 peritoneal cell populations that displayed CMC against cultured P815Y also displayed a similar amount of CMC against day 10 peritoneal cell tumor cell targets. In contrast, day 16 peritoneal cell populations active in the CMC assay against cultured P815Y target cells were unable to cause cytolysis of day 16 tumor cell targets. Susceptibility of Day 16 Tumor Cells to CMC. The absence of CMC against day 16 tumor cell targets by day 16 effector cells suggested that day 16 tumor cells were unable to be rec-
ognized and killed by effector cells in the tumor-bearing host. To test this hypothesis, peritoneal cells from both day 10 and day 16 tumor-bearing animals were assayed for CMC against P815Y, day 10, and day 16 tumor cell targets (Table 2). The results indicated that cultured P815Y and day 10 tumor cell targets were similar in their susceptibility to lysis by these effector cells, but the day 16 tumor cell targets were much less susceptible to lysis by these effectors. The entire experiment was repeated and yielded essentially the same results. To test the possibility that the reduced CMC against day 16 tumor cells was due to resistance to lysis or to nonspecific suppression of effector cell function, we assayed these tumor target cells with effector cells directed primarily against H-2d antigenic determinants. B6 spleen cell (H-2b) effectors, obtained
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TARGET CELLS
FIG. 3. Inhibition of CMC against 56Cr-labeled target cells by various numbers of unlabeled P815Y cells (0), day 16 tumor cells (A), and L1210 tumor cells (O). Effector cells were day 10 peritoneal cells with sedimentation rate = 3.3-6 mm/hr (a) and day 16 peritoneal cells with sedimentation rate = 2.3-4.6 mm/hr (b) added to yield an effector cell:labeled target cell ratio of 100:1 and 200:1, respectively. (0) Percent specific lysis in the absence of unlabeled inhibitor cells. Spontaneous and hypotonic release for P815Y was 11% and 81%, respectively.
by in vitro culture with mitomycin C-treated P815Y, were assayed for CMC against P815Y, EL4 (H-2b), day 10, and day 16 tumor cell targets (Table 2). The results demonstrated that day 16 tumor cells were as susceptible as day 10 tumor cells and cultured P815Y cells to CMC by allogeneic effector cells. Ability of Day 16 Tumor Cells to Inhibit CMC against P815Y. The absence of CMC against day 16 tumor cells could have resulted from the number of determinants on the tumor cell surface required for effector cell recognition being below the level required for detectable CMC (21). Therefore, the capacity of day 16 tumor cells to competitively inhibit CMC against cultured P815Y by effector cells obtained from day 10 and day 16 peritoneal cell populations was determined. The data in Fig. 3 indicate that a 4- to 64-fold excess of unlabeled day 16 tumor cells and unlabeled L1210 cells caused only a marginal inhibition of CMC against P815Y mediated by both day 10 (Fig. 3a) and day 16 (Fig. 3b) peritoneal cell effector cell populations. However, a marked inhibition of CMC against cultured P815Y occurred when a 4- to 64-fold excess of unlabeled P815Y cells was added to day 10 effector cells and labeled P815Y target cells (Fig. Sa). A similar effect was observed when inhibition of day 16 effector cell activity was analyzed (Fig. 3b). These data indicate that day 16 tumor cells do not display a significant number of cell surface determinants that are recognized by effector cells directed against tumor-associated antigens (TAA) on cultured P815Y. Assay for Immunoglobulin on the Surface of Tumor Cells. The absence of TAA on day 16 tumor cells as detected by CMC could have resulted from the presence of antibody molecules specifically bound to TAA determinants. Such cell-bound antibody could have effectively "blocked" the recognition and cytotoxic functions of effector cells that were specific for these determinants (22). Therefore, cell surface immunoglobulin on day 16 tumor cells was assayed by both immunofluorescence and a highly sensitive radioimmunoassay (Table 3). This radioimmunoassay is capable of detecting 0.1 ng of specific antibody (19). The radioimmunoassay and the immunofluorescence assay revealed that no detectable immunoglobulin was present on P815Y and day 16 tumor cells. In addition, day 16 ascitic fluid contained no more antibody capable of binding to P815Y than was present in normal DBA/2 serum. Also, incubation of cultured P815Y with day 16 ascitic fluid did not "block" syngeneic CMC to these target cells (data not shown).
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Immunology: Biddison and Palmer
Proc. Natl. Acad. Sci. USA 74 (1977)
Table 3. Assay for immunoglobulin on the surface of P815Y and day 16 tumor cells
Cells P815Y Day 1.6 peritoneal cells (11-13 mm/hr) P815Y + anti-H-2d serum (1:80)t P815Y + normal DBA/2serum(1:5) P815Y + day 16 ascitic fluid (1: 5)
Bound % Cells stained with 1 25I-labeled antibody fluoresceinantibody* (cpm ± SEM)t
