development of tumor cell resistance to syngeneic cell-mediated cytotoxicity during growth of...

Development of Tumor Cell Resistance to Syngeneic Cell-Mediated Cytotoxicity duringGrowth of Ascitic Mastocytoma P815YAuthor(s): William E. Biddison and Jon C. PalmerSource: Proceedings of the National Academy of Sciences of the United States of America,Vol. 74, No. 1 (Jan., 1977), pp. 329-333Published by: National Academy of SciencesStable URL: http://www.jstor.org/stable/66570 .

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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 respon- siveness (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 pro- ceeds more rapidly than does the induction and action of the antitumor immune response.

Most previous studies of cell-mediated immunity to tumor- associated antigens have analyzed the immune capacities of cell populations obtained from various lymphoid organs or pe- ripheral blood. However, the immnune reactivities of the cells obtained from these areas, which miay 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.

tissue culture may lose and/or acquire some characteristics, and therefore, may no longer be representative of tumor cells in the turnor-bearing host (15). To assess more accurately the effect of antitumor immune responses on tumor cells in situ, it is de- sirable to analyze the cellular and humoral immune responses to the tumor cells occurring within a progressively growing turnor 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, Philadel- phia, Pa., and used at 8 weeks of age.

Tumors. The P815Y (DBA/2, H-2d) mastocytoma, L1210 (D:BA/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 10 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 desicribed by Miller and Phillips (16). Forty to 150 X 106 peri- tonweal 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 4?, 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 P8 15Y were induced by culturing normal B6 spleen cells with mitomycin C-treated P815Y cells in Marbrook culture vessels as (lescribed (17).

Assay for CMC. CMC against tumor target cells was measured by a 4-hr 5lCr-release assay as described in detail else- where (18). P815Y tumor target cells were obtained from cul- tures 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 im- mutnoglobulin was detected by the binding of fluorescein- conjugated rabbit antibody against mouse gamma globulin

329



330 Immunology: Biddison and Palmer Proc. Natl. Acad. Sci. USA 74 (1977)

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(Cappel Laboratories, Downingtown, Pa.) and by the binding of 1251-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 Sedimen- tation. 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

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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 (-) for each of the peritoneal cell populations fractionated. CMC against P815Y by peritoneal cell fractions (shaded bars) and unfractionated peritoneal cells (open bars) is expressed as percent specific lysis.

tumor-hearer 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

Effector cells Sedimentation rates (mm/hr)

(peritoneal Target cells cells) 9-11 6-8 5-6 0-4

P815Y Day 12 0* 3 63 44 L1210 Dayl12 0 0 0 0 P815Y Dayo 1 0 6 54 38 Day 10 P.C.t Day 10 6 17 44 36 L1210 Dayl10 0 u 6 5 P815Y Day 16 0 0 2 13 Dayl6P.C.t Dayl6 0 0 0 0 L1210 Dayi16 0 2 0 0

* Percent specific lysis values were measured at an effector cell:target cell ratio of 100:1.

tgDay 10 and day 16 peritoneal cells (P.C.) with sedimentation rates = 11-13 mm/hr were labeled with 51Cr 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%.



Immunology: Biddison and Palmer Proc. Natl. Acad. Sci. USA 74 (1977) 331

Table 2. Susceptibility of tumor cells to CMC

% Specific lysis, effectors: target

Effector cells Target cells 100:1 30:1 10:1

Day 10 P.C. P815Y 54 36 16 (4-6 mm/hr)* Day 10 P.C.t 41 33 14

Dayl6P.C.t 8 5 2

Day 10 P.C. P815Y 23 18 13 (2.6-4 mm/hr)* Day 10 P.C. 28 13 6

Day 16 P.C. 0 0 0

Dayl6P.C. P815Y 7 2 0 (2-4.6 mm/hr)* Day 10 P.C. 7 2 0

Day 16P.C. 0 0 0

B6 spleen P815Y 72 58 29 anti-P815Y Day 10 P.C. 67 33 20

Day 16 P.C. 68 51 21 EL4 8 6 3

* Peritoneal cells (P.C.) from day 10 and day 16 tumor-bearing mice were 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.

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 5ICr 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. Further- more, 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 recognized 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 1'815Y 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 5'Cr-labeled target cells by various numbers of unlabeled P815Y cells (0), day 16 tumor cells (A&), anid L1210 tumor cells (o3). 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. 3a). 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 immunofluo- re scence 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 Bound stained with "25H-labeled fluorescein- antibody

Cells antibody* (cpm ? SEM)t

P815Y <1 29 ? 1 Day 16 peritoneal cells

(11-13mm/hr) <1 44? 8 P815Y + anti-H-2d

serum (1:80)t 97 2992 ? 215 P815Y + normal

DBA/2 serum (1:5) <1 93 ? 9 P815Y + day 16

ascitic fluid (1:5) <1 114 ? 5

* Fluorescein-conjugated rabbit antibody against mouse gamma globulin was used.

t The radioimmunoassay was performed with 4 x 104 cpm of 1251- labeled rabbit antibody against mouse Fab per 2 x 105 cells.

t Antiserum against H-2d was prepared by immunization of B10 mice with spleen cells obtained from congenic B1O.D2 mice and produced 50% cytotoxicity of L1210 (H-2d) cells at a dilution of 1:15 in the presence of guinea pig complement.

These results indicate that the resistance of day 16 tumor cells to CMC could not have been mediated by "blocking" immunoglobulin molecules bound to TAA on day 16 tumor cells.

DISCUSSION This study has shown that the host's immune reactivity to tumor cells within a progressively growing tumor mass can be evaluated in an ascitic tumor system. A cell separation procedure in which peritoneal cells from P815Y tumor-bearing hosts were fractionated by velocity sedimentation at unit gravity revealed that CMC to P815Y tumor target cells was present in populations of peritoneal cells at 8-16 days after tumor cell inoculation. Preliminary characterization of the effector cells that express CMC to P815Y in these peritoneal cell populations has indicated that they are thymus-derived lymphocytes (T cells) (W. E. Biddison, in preparation).

The specificity of CMC to P815Y expressed by the tumor- bearer peritoneal cells was shown by the absence of significant CMC to another DBA/2 tumor, L1210, and the absence of CMC to P815Y by normal DBA/2 peritoneal cells. In addition, effector cells obtained from peritoneal cell populations from mice inoculated 10 days previously with tumor cells expressed similar levels of CMC against cultured P815Y and against tumor cells separated from day 10 peritoneal cells. However, tumor cells obtained from mice 16 days after tumor inoculation were resistant to CMC by both day 10 and day 16 effector cells.

The resistance of day 16 tumor cells to CMC by both day 10 and day 16 effector cells strongly suggested that the day 16 tumor cells did not display any cell surface determinants that were recognized by these effector cells. Further support for this hypothesis was obtained from results that demonstrated that day 16 tumor cells were unable to specifically inhibit the CMC against cultured P815Y expressed by day 10 and day 16 effector cells. The resistance of day 16 tumor cells to CMC by tumor- bearer effector cells could not have been a result of nonspecific suppression of effector cell function or inability to release measurable quantities of 51Cr when killed, since cultured P815Y, day 10, and day 16 tumor cell targets were equally susceptible to lysis by effector cells reactive to H-2 transplantation antigens.

To our knowledge, this is the first report that demonstrates

that tumor cells within a progressively growing tumor mass "lose" surface expression of TAA capable of being recognized by host cytotoxic effector cells. It is possible that the antigens are present on the tumor cell surface but are "masked" by substances other than immunoglobulin (23). Alternatively, resistance to CMC directed against TAA could have been caused by rapid shedding of these determinants from the plasma membrane into the surrounding medium. If tumor cells were shedding large quantities of TAA into their immediate surroundings, these cells might be expected to competitively inhibit CMC against tumor cells grown in vitro (8). However, day 16 tumor cells were unable to inhibit CMC to cultured P815Y (Fig. 3). Therefore, rapid shedding of cell surface determinants probably could not explain the absence of surface TAA expression on these tumor cells. In addition, the inability of day 16 tumor cells to inhibit the CMC of day 10 or day 16 effector cells against cultured P815Y would argue against a specific suppression of effector cell function by the day 16 tumor cell population.

Immunoselection of tumor cells with very low densities of surface TAA by the immune response of the host against its own tumor cells (24) would also result in the observed absence of surface TAA expression on tumor cells obtained from animals in later phases of tumor growth. However, P815Y tumor cells obtained from animals in later phases of tumor growth and passaged 7 days in vitro displayed surface TAA as measured by CMC (data not shown). Therefore, immunoselection that resulted in the complete elimination of tumor cells capable of expressing surface TAA recognized by cytotoxic effector cells could not have occurred.

A decrease in the expression of cell surface antigens on tumor cells can also be induced by antigenic modulation (25), a process that may be important for tumor cell escape from immune recognition. Antigenic modulation in the thymus-leukemia (TL) antigen system involves the binding of antibody to cell surface antigens, resulting in the disappearance of free TL antigens from the cell surface, as detected by the inability of the modulated cells to be lysed by antibody and guinea pig complement (26). Antigenic modulation is a reversible process, since re-expression of surface antigens can occur if modulated cells are permitted to metabolize and divide in the absence of antibody (6, 27).

In the present study, antigenic modulation may be respon- sible for the absence on day 16 tumor cells of TAA recognized by cytotoxic effector cells. To date, antigenic modulation, as studied both in vivo and in vitro, has been observed to occur only in the presence of excess antibody (5, 6, 25-27). However, no significant quantities of circulating antibody to P815Y were found in day 16 ascitic fluid. Attempts to detect circulating antibody to P815Y in the sera of tumor-bearing mice and mice immunized to reject repeated challenges of P815Y cells have so far been unsuccessful. In addition, incubation of cultured P815Y with day 16 ascitic fluid did not "block" syngeneic CMC against these targets. The concentrations of circulating antibody capable of inducing antigenic modulation when cells are con- tinuously exposed to the antibody are not known. It is possible that very low concentrations of circulating antibody could be effective under these conditions. Finally, the decreased expression of cell surface antigens such as TAA may be the result of local growth conditions, including cell density in the tumor mass. Investigations of these possibilities should increase our understanding of the mechanisms of tumor escape from immune attack and the biology of the tumor-host relationship.

We are grateful to Dr. Lionel A. Manson for incisive discussions during the course of this investigation. This work was supported by



Immunology: Biddison and Palmer Proc. Natl. Acad. Sci. USA 74 (1977) 333

USPHS Research Grants CA-07973 and CA-10815 from the National Cancer Institute and Grant RR-05540 from the Division of Research Resources.

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