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Expression of the differentiation antigen of activated T and B lymphocytes (ACA-1) on cells of lymphoid and nonlymphoid tumors
CELLULAR
IMMUNOLOGY
&5,412-418
(1984)
Expression of the Differentiation Antigen of Activated B Lymphocytes (ACA-1) on Cells of Lymphoid and Nonlymphoid Tumors
T and
V. G. NESTERENKO,G. P. YERMAKOV, L. N. FONTALIN, AND E. I. RUBAKOVA Gamaleya Institute of Epidemiology and Microbiology, USSR Academy of Medical Science, Gamaleya St. 18, Moscow 123098, USSR Received October 20, 1983; accepted January 29, 1984 In cytotoxicity and indirect immunofluorescence tests an antiserum to ACA-1 (activated cell antigen) reacted with 58-100% of actively proliferating cells from tumors of lymphoid (EL4 T lymphoma, MOPC 104E plasmacytoma) and nonlymphoid origin (AH-22 hepatoma, Sa- 1 and MCh- 11 sarcomas, F2 mammary cancer). Absorption of anti-ACA- 1 serum with tumor cells sharply reduced its activity both against the cells of all these neoplasms and against normal activated T and B lymphocytes. Absorption with proliferating murine cells from the brain of embryos and the retina of neonates or with similar (nonproliferating) cells from adult mice did not affect the activity of the antiserum. It is concluded that ACA-1 is expressed on actively proliferating cells of the tumors studied.
INTRODUCTION
In the last few years the impression has been prevalent in immunology that differentiation of cells is associated with a change in their surface antigenic characteristics (l-3). Antigenic markers have been identified on various subpopulations of normal T and B lymphocytes (1, 2, 4, 5). Several authors have found some markers on lymphocytes stimulated with nonspecific mitogens or antigens (6-l 5). Previously we have detected a special ACA-1’ antigen on the surface of activated, but not resting, T and B cells ( 16- 19). The aim of this study was to explore actively proliferating cells from tumors of various histogenesis for the presence of ACA-1. Indeed, we succeeded in revealing this antigen on the cell surface in neoplasms of lymphoid and nonlymphoid origin. MATERIALS
AND
METHODS
Animals. Male and female C57BL/6 (H-2b), C57BL/10sI, (H-2b), C3H (H-2’), A/Sri (H-2”), BALB/c (H-2d), CBA (H-2k), and (CBA X C57BL/6)FI (H-2k/b) mice weighing 18-20 g were obtained from the Stolbovaya Animal Breeding Center, USSR Academy of Medical Science. ’ Abbreviations used: ACA- 1, activated cell antigen; EDTA, ethylenediaminetetraacetic acid, PFC, plaqueforming cells; SBBC, sheep red blood cells; T-, T lymphocytes activated with allogeneic transplantation antigens; C, complement; ATS, rabbit antiserum against murine T lymphocytes; CTI, cytotoxicity index. 412 0008-8749184 $3.00 Copyrieht 0 1984 by Academic Press, Inc. All rishts of swroducdon in any fm mcrved.
EXPRESSION
OF ACA-I
ON TUMOR
CELLS
473
Cell suspensions. Thymus, lymph node, spleen, and brain cell suspensions were prepared in cold medium 199 using a glass homogenizer. To obtain retinal cells, isolated retinas were incubated in 0.004% EDTA in Tyrode’s solution without Ca2+ or Mg2+ at 37°C for 10 min; cell suspensions were then aspirated repeatedly through injection needles of decreasing diameter. Before use all cells were washed three times. Tumor cells. Ascites forms of Sa-1 (H-23, MCh-11 (H-2b), and EL-4 (H-2b) tumors were kindly made available to us by B. D. Brondz (National Oncological Center, USSR Acad. Med. Sci.); an ascites AH-22 (H-2k) tumor, by Y. A. Rovensky (Nat. One. Center); cells of F2 clonal line derived from transplantable mammary carcinoma of GR mice (20), by I. N. Kryukova (Nat. One. Center); and MOPC 104E (H-2d) plasmacytoma, by E. V. Sidorova (Gamaleya Institute). Serial passages were carried out by injecting intraperitoneally normal recipients with tumor cells (5 X lo6 to 10 X 1O6 per mouse) at intervals of 8 to 13 days. AH-22 hepatoma was passaged in C3H mice; Sa- 1 sarcoma, in the A/Sri strain; EL-4 T lymphoma and MCh- 11 sarcoma, in C57BL/6 and C57BL/lOs, mice respectively; MOPC 104E plasmacytoma, in BALB/c animals; and F2 cells were maintained in vitro (20). Plaque assays. Plaque-forming cells (PFC) producing 19 S antibodies to sheep red blood cells (SRBC) were estimated according to Jerne and Nordin (21). T lymphocytes activated with allogeneic transplantation antigen (Tad) were obtained by the following method: 1OSallogeneic thymocytes were injected intravenously into lethally irradiated recipients and after 3 days cell suspensions were prepared from their spleens. Viable splenocytes were T cells of the donor phenotype and included 30 to 60% of mediumsized lymphocytes and blasts (22,23). CBA-anti-C57BL/6 and CBA-anti-BALB/c Tati were used for the experiments. Anti-ACA-1 seyuw1.A rabbit antiserum to ACA-1 was obtained and evaluated as described previously ( 16- 19). Briefly, rabbits were immunized with Tti and the immune sera were absorbed with erythrocytes, serum, liver, thymus, spleen, and lymph node cells from normal mice. The effect of the antiserum on Tti was measured in a complement (C)-dependent lymphocytotoxicity test (6,23). Its capacity of inhibiting PFC was determined in the presence of rabbit C using a suspension of spleen cells from mice immunized with SRBC (19,23). Percentage inhibition of PFC was calculated according to the formula ((a - @/a) X 100, where a was the PFC number upon treatment of spleen cells with C only, and b was the PFC number in the suspension treated with the antiserum plus C. Rabbit antiserum to murine T lymphocytes (ATS). ATS was obtained as described previously (23). It lysed 100% of thymocytes, 38% of splenocytes, 6 1% of lymph node cells, 3% of marrow cells, and did not affect (CBA X C57BL/6)FI PFC-producing antibodies to SRBC or the immune response to thymus-independent Vi antigen of Salmonella typhi (23). Cytotoxicity assays. Cytotoxicity of the antiserum for tumor cells was assayed as follows: 0.05 ml of thrice-washed cells (1 X 106/ml) and 0.05 ml of nontoxic rabbit serum (as a source of C) were added to 0.15 ml of each dilution of the antiserum. After a 45-min incubation at 37”C, cell viability was tested with 0.25 ml of 0.1% trypan blue plus 0.1% eosin. The cytotoxicity index (CD) was expressed for each antiserum dilution as ((c - d)/( 100 - d)) X 100, where c was the percentage of nonviable cells upon treatment with antiserum plus C, and d was the percentage of nonviable cells in the control suspension treated only with C.
NESTERENKO
474
ET AL.
We also performed the indirect immunofluorescence test to estimate the binding of the antiserum to target cells (24). Fluorescein-labeled goat antibodies to rabbit IgG were kindly supplied for this purpose by K. L. Shakhanina (Gamaleya Institute). Statistical analysis. For CT1 and inhibition of PFC arithmetic means were calculated. Student’s t test (P B 0.05) was used for calculating confidence limits. RESULTS The effect of anti-ACA- 1 serum on the cells of various neoplasms is shown in Figs. 1 and 2. The antiserum diluted 1:4 to 1: 16 lysed 64% of Sa- 1, 89% of AH-22, 80% of EL4, 89% of MCh-11, 100% of MOPC 104E, and 76% of F2 cells. Its additional absorption with lymphocytes (equal proportions of thymus, spleen, and lymph node cells) or liver cells from normal (CBA X C57BL/6)F1 mice (4.5 X lo8 cells/ml of antiserum) did not reduce CT1 (data not shown), while absorption with Sa-1, AH22, MCh-11, F2, or EL-4 cells led to a considerable decrease in cytotoxic activity (Figs. 1, 2).
100 a
“‘1
b
30 I
FIG. 1. Cytotoxicity of anti-ACA- 1 serum for Sa- 1, AH-22, EG4, and MCh- 11 tumor cells. Abscissa: dilution of antiserum; ordinate: cytotoxicity index (%). Target cells: (a) Sa-1 (six experiments); (b) AH-22 (eight experiments); (c) EL-4 (eight experiments); (d) MCh-11 (five experiments). (0 0) Effect of antiserum before additional absorption; absorption with tumor cells is designated as follows: (X X) Sa-I; (0 0) AH-22; (0 0) EG4, (0 - - - 0) MB-1 1. Absorption was carried out at room temperature for 1 hr using 3.3 X 10s tumor cells per 1 ml of antiserum.
EXPRESSION
OF ACA-1 ON TUMOR
475
CELLS
FIG. 2. Cytotoxicity of anti-ACA-1 serum for MOPC 104E and F2 tumor cells. Target cells: (a) F2 (two experiments); (b) MOPC 104E (three experiments). (0 0) Effect of antiserum before additional absorption; (A A) absorption with F2. For other designations see the legend to Fig. 1.
In the indirect immunofluorescence test anti-ACA-1 serum stained 58% of Sa-1, 84% of AH-22, 80% of MCh-11, and 83% of EL4 cells. It should be noted that it exhibited practically no capacity for reacting with normal lymphocytes either in this test or in cytotoxicity assays (16-19). The above evidence seemed to suggest that ACA-1 was expressed on actively proliferating cells of the tumors studied in our experiments (just as it is present on activated T and B lymphocytes). The data shown in Figs. 3 and 4 supported this suggestion. Anti-ACA- 1 serum inhibited PFC numbers by 95%. Additional absorption of the antiserum with Sa-1, AH-22, MCh-11, F2, or EL-4 cells reduced inhibition by 86-lOO%, while absorption with lymphocytes or liver cells from nonimmunized mice was ineffective (Fig. 3). Similar results were obtained in the lymphocytotoxicity test using Tact. In 1:5 to 1: 10 dilutions anti-ACA- 1 serum lolled 44% of CBA-antiC57BL/6 and 35% of CBA-anti-BALB/c T,&. Upon additional absorption with Sa1, AH-22, MCh- 11, or EL-4 cells, its cytotoxicity for CBA-anti-C57BL/6 and CBA-
5
l
*
-
1
2
3
4
s
0
7
B
FIG. 3. Effect of anti-ACA-1 serum on PFC. Results of 10 experiments. Ordinate: percentage inhibition. (1) Before additional absorption; absorption with different cells is designated as follows: (2) Sa-1; (3) AH-22; (4) EG4; (5) MCh-11; (6) F2; (7) normal lymphocytes; (8) liver cells. PFC were assayed in (CBA X C57BL/6)FI spleens 4 days after iv immunization with 5 X IO* SRBC. The antiserum was diluted 1:50 and absorbed as described in the legend to Fig. 1. Normal cells were used at a ratio of 4.5 X 10’ per 1 ml of antiserum.
416
NESTERENKO 60
la
ET AL. So b
FIG. 4. Cytotoxicity of anti-ACA-1 serum for T-. Results of eight experiments. Target cells: (a) CBAanti-C57BL/6 T,; (b) CBA-anti-BALB/c T,. For other designations see the legend to Fig. 1.
anti-BALB/c T, fell by 82-100% and 66-98%, respectively (Fig. 4). Additional absorption with lymphocytes or liver cells from normal mice had practically no influence on cytotoxic activity. In order to confirm the specificity ,of the absorption of antiserum by tumor cells, a control series of experiments was performed using ATS (Fig. 5). It was absorbed with (CBA X C57BL/6)FI thymocytes and EL-4 T lymphoma cells known to carry MTLA (antigens reacting with ATS). ATS diluted 1: 100 lysed 100% of such cells, but upon absorption its activity fell by 99 and 88%, respectively. On the other hand, when normal murine liver cells, Sa-1 cells, or AH-22 cells were used for absorption, ATS, which was not cytotoxic for these cells, retained its activity. Therefore, at the cell/antiserum ratio chosen for our studies the cytotoxic effect of ATS was not influenced by nonspecific absorption. In further experiments anti-ACA-1 serum was absorbed with murine brain cells from embryos or adult animals, or with retinal cells from neonates or adult mice. As shown in Fig. 6, the inhibitory effect of the antiserum on PFC remained virtually unaltered after such absorption. 100'
60
60
40
20 _
--;"
'4s
‘132
'6.
f/128
FIG. 5. Cytotoxic activity OF ATS against (CBA X C57BL/6)F1 thymocytes as r&&ted by additional absorption with tumor eelis. Results of three experiments. Abscksaz dilution of antkrum; ordinate: cytotoxicity index (‘9%).(0 0) Before absorption; absorption with different c&s is designated as follm (X X) Sa-1; (0-m) AH-22; (Oo);‘EL-4; (0 0) thymocytes; (W n ) liver c&k from normal mice. For details of the absorption procedure see the legends to Figs. 1 and 3.
EXPRESSION
“io
OF ACA-1 ON TUMOR
“00
'40
'40
CELLS
"So
477
‘420
6. Effect of anti-ACA-1 serum on PFC before and after additional absorption with murine retinal (a) and brain (b) cells. Results of five experiments. Abscissa: dilution of antiserum; ordinate: percentage inhibition. (V V) Before absorption; absorption with different cells is designated as follows: (0 Cl) brain cells from CBA embryos aged 14 days; (m n ) brain cells from adult CBA mice weighing 18-20 0) retinal cells taken 1 or 2 days atIer birth from CBA neonates; (0 0) retinal cells from I%;(0 adult CBA mice weighing 18-20 g. Absorption was carried out at room temperature for I hr using 2 X 10’ cells per 1 ml of antiserum. FIG.
DISCUSSION Using the cytotoxicity assay and the immunofluorescence test, we have established that anti-ACA- 1 serum reacts with 58-100% of cells from tumors of different histogenesis (malignancies of T- and B-lymphocyte derivation, sarcomas, hepatoma, and mammary carcinoma). Absorption with cells of these neoplasms sharply reduces the effect of anti-ACA-1 serum both on tumor cells (Figs. 1 and 2) and on activated B and T lymphocytes (Figs. 3 and 4). Control experiments (Fig. 5) have shown that tumor cells do not alfect the cytotoxic activity of ATS via nonspecific absorption. Therefore, loss of activity of anti-ACA-1 serum upon its absorption with tumor cells cannot be explained by a high nonspecific absorptive capacity of the latter cells. These data allow us to conclude that ACA- 1 is not only present on activated murine T and B cells (16- 19), but seems to find expression also on actively proliferating tumor cells of nonlymphoid origin (Sa-1, AH-22, MCh-11, F2) and those of T and B classes (EL-4, MOPC 104E). Previously ( 16- 19) we have described the differences between ACA- 1 and the antigens known to be carried by nonstimulated (H-2, MTLA, MBLA, Thy- 1, Ig, etc.) and activated (Ala- 1, PC- 1, etc.) lymphocytes. The expression of ACA-1 is probably not related to the functioning of oncomaviruses, since it has been detected on F2 cells lacking structural proteins of the oncomavirus C type (20). We have also attempted to detect ACA-1 on normal proliferating cells of nonlymphoid origin. Most brain cells in murine embryos aged 1 l- 15 days and the majority of retinal cells in neonates l-3 days after birth are known to be proliferating, while similar cells of adult mice are practically incapable of division (25-27). Therefore we absorbed anti-ACA-1 serum with retinal cells from newborn and adult mice, and with brain cells from embryos and adult animals. When the antiserum was tested on activated B lymphocytes (PFC), we found that absorption with any one of the four types of cells had no influence on its inhibitory activity. Evidently ACA-1 is absent (or its concentration is insufficient) on proliferating cells of the embryonic brain and the neonatal retina, as well as on nonproliferating cerebral and retinal cells of adult
478
NESTERENKO
ET AL.
mice. It may be assumed that expression of ACA-1 is limited to activated T and B lymphocytes and the tumor cells studied. The reasons for such limitation remain unclear, but a possible explanation is that ACA-l+ and ACA-I- cells evolve along different lines. REFERENCES 1. Cantor, H., and Boyse, E. A., Contemp. Top. Immunobiol. 7, 41, 1977. Greaves, M. F., Cancer Rex 41, 4752, 1981. 3. Nesterenko, V. G., Usp. Sovi. Biol. (Moscow) 5, 211, 1980. 4. Raff, M. C., Transplant Rev. 6, 52, 1971. 5. Reinherz, E. L., and Schlossman, S. F., Cancer Res. 41, 4767, 1981. 6. Nesterenko, V. G., and Kovalchuk, L. V., Byull. Eks. Biol. Med. (Moscow) 7, 836, 1976. 7. Bluming, A., Lynch, M. J., Kavanah, M., and Ehiroya, R., J. Immunol. 114, 717, 1975. 8. Kimura, A. K., and Wigzell, H., Contemp. Top. Mol. Immunol. 6, 209, 1977. 9. Sullivan, R. A., Berke, G., and Amos, D. B., Transplantation 16, 388, 1973. 10. Takahashi, T., Old, L. J., and Boyse, E. A., J. Exp. Med. 131, 1325, 1970. 11. Fenney, A. J., and Hiimmerling, U., Immunogenetics (N.Y.) 3,369, 1976. 12. Bach, F. H., Alter, B. J., Widmer, M. B., Segall, M., and Dunlap, B., Zmmunol. Rev. 54, 6, 1981. 13. Thierry, H., Nadler, L. M., Pesando, J. M., Reinherz, E. L., Schlossman, S. F., and Ritz, J., Cell. Immunol. 64, 192, 1981. 14. Sutherland, R., Deha, D., Schneider, C., Newman, R., Remshead, J., and Greaves, M. F., Proc. Nat. Acad. Sci. U.S.A. 78,4515, 1981. 15. Goding, J. W., and Harris, A. W., Proc. Nat. Acad. Sci. U.S.A. 78, 4530, 1981. 16. Nesterenko, V. G., Fontalin, L. N., and Novikova, T. K., In “Advances in Comparative Leukemia Research 1979” (B. A. Lapin and D. S. Yohn, Eds.), pp. 414-418. USSR Acad. Med. Sci./IEP & T, Moscow, 1980. 17. Nesterenko, V. G., and Gruner, Sh., ByuN. Eks. Biol. Med. (Moscow) 6, 708, 1980. 18. Nesterenko, V. G., Novikova, T. K., Fontalin, L. N., Wechnik, E., Rubakova, E. I., Gruner, Sh., and Sidorova, E. V., Byull. Ekes.Biol. Med. (Moscow) 10, 449, 1980. 19. Nesterenko, V. G., Novikova, T. K., Fontalin, L. N., Rubakova, E. I., Gruner, Sh., Wechnik, E., and Sidorova, E. V., Cell. Immunol. 79, 253, 1983. 20. Beriashvili, M. M., Kryukova, I. N., and Shaginian, 0. V., Vopr. Virusol. (Moscow) 2, 221, 1981. 21. Jeme, N. K., and Nordin, A. A., Science 140,405, 1963. 22, Sprent, J., and Miller, J. A. F. T., Cell. Immunol. 3, 213, 1972. 23. Nesterenko, V. G., Kraskina, N. A., Rubakova, E. I., and Gruner, Sh., CeZl. Zmmunol. 69,215, 1982. 24. Nesterenko, V. G., and Yermakov, G. P., Byull. Eks. Biol. Med. (Moscow) 10, 449, 1980. 25. Sidman, R. L., “The Structure of the Eye,” pp. 487-506. Academic Press, New York/London, 1961. 26. Korr, H., Adv. Anat. EmbryoZ. Cell Biol. 61, No. 1, 1980. 27. Reznikov, K. Y., “Proliferation of Cerebral Cells in Vertebrates during Normal Development and after Brain Injury.” Science Publ., Moscow, 1981. 2.
IMMUNOLOGY
&5,412-418
(1984)
Expression of the Differentiation Antigen of Activated B Lymphocytes (ACA-1) on Cells of Lymphoid and Nonlymphoid Tumors
T and
V. G. NESTERENKO,G. P. YERMAKOV, L. N. FONTALIN, AND E. I. RUBAKOVA Gamaleya Institute of Epidemiology and Microbiology, USSR Academy of Medical Science, Gamaleya St. 18, Moscow 123098, USSR Received October 20, 1983; accepted January 29, 1984 In cytotoxicity and indirect immunofluorescence tests an antiserum to ACA-1 (activated cell antigen) reacted with 58-100% of actively proliferating cells from tumors of lymphoid (EL4 T lymphoma, MOPC 104E plasmacytoma) and nonlymphoid origin (AH-22 hepatoma, Sa- 1 and MCh- 11 sarcomas, F2 mammary cancer). Absorption of anti-ACA- 1 serum with tumor cells sharply reduced its activity both against the cells of all these neoplasms and against normal activated T and B lymphocytes. Absorption with proliferating murine cells from the brain of embryos and the retina of neonates or with similar (nonproliferating) cells from adult mice did not affect the activity of the antiserum. It is concluded that ACA-1 is expressed on actively proliferating cells of the tumors studied.
INTRODUCTION
In the last few years the impression has been prevalent in immunology that differentiation of cells is associated with a change in their surface antigenic characteristics (l-3). Antigenic markers have been identified on various subpopulations of normal T and B lymphocytes (1, 2, 4, 5). Several authors have found some markers on lymphocytes stimulated with nonspecific mitogens or antigens (6-l 5). Previously we have detected a special ACA-1’ antigen on the surface of activated, but not resting, T and B cells ( 16- 19). The aim of this study was to explore actively proliferating cells from tumors of various histogenesis for the presence of ACA-1. Indeed, we succeeded in revealing this antigen on the cell surface in neoplasms of lymphoid and nonlymphoid origin. MATERIALS
AND
METHODS
Animals. Male and female C57BL/6 (H-2b), C57BL/10sI, (H-2b), C3H (H-2’), A/Sri (H-2”), BALB/c (H-2d), CBA (H-2k), and (CBA X C57BL/6)FI (H-2k/b) mice weighing 18-20 g were obtained from the Stolbovaya Animal Breeding Center, USSR Academy of Medical Science. ’ Abbreviations used: ACA- 1, activated cell antigen; EDTA, ethylenediaminetetraacetic acid, PFC, plaqueforming cells; SBBC, sheep red blood cells; T-, T lymphocytes activated with allogeneic transplantation antigens; C, complement; ATS, rabbit antiserum against murine T lymphocytes; CTI, cytotoxicity index. 412 0008-8749184 $3.00 Copyrieht 0 1984 by Academic Press, Inc. All rishts of swroducdon in any fm mcrved.
EXPRESSION
OF ACA-I
ON TUMOR
CELLS
473
Cell suspensions. Thymus, lymph node, spleen, and brain cell suspensions were prepared in cold medium 199 using a glass homogenizer. To obtain retinal cells, isolated retinas were incubated in 0.004% EDTA in Tyrode’s solution without Ca2+ or Mg2+ at 37°C for 10 min; cell suspensions were then aspirated repeatedly through injection needles of decreasing diameter. Before use all cells were washed three times. Tumor cells. Ascites forms of Sa-1 (H-23, MCh-11 (H-2b), and EL-4 (H-2b) tumors were kindly made available to us by B. D. Brondz (National Oncological Center, USSR Acad. Med. Sci.); an ascites AH-22 (H-2k) tumor, by Y. A. Rovensky (Nat. One. Center); cells of F2 clonal line derived from transplantable mammary carcinoma of GR mice (20), by I. N. Kryukova (Nat. One. Center); and MOPC 104E (H-2d) plasmacytoma, by E. V. Sidorova (Gamaleya Institute). Serial passages were carried out by injecting intraperitoneally normal recipients with tumor cells (5 X lo6 to 10 X 1O6 per mouse) at intervals of 8 to 13 days. AH-22 hepatoma was passaged in C3H mice; Sa- 1 sarcoma, in the A/Sri strain; EL-4 T lymphoma and MCh- 11 sarcoma, in C57BL/6 and C57BL/lOs, mice respectively; MOPC 104E plasmacytoma, in BALB/c animals; and F2 cells were maintained in vitro (20). Plaque assays. Plaque-forming cells (PFC) producing 19 S antibodies to sheep red blood cells (SRBC) were estimated according to Jerne and Nordin (21). T lymphocytes activated with allogeneic transplantation antigen (Tad) were obtained by the following method: 1OSallogeneic thymocytes were injected intravenously into lethally irradiated recipients and after 3 days cell suspensions were prepared from their spleens. Viable splenocytes were T cells of the donor phenotype and included 30 to 60% of mediumsized lymphocytes and blasts (22,23). CBA-anti-C57BL/6 and CBA-anti-BALB/c Tati were used for the experiments. Anti-ACA-1 seyuw1.A rabbit antiserum to ACA-1 was obtained and evaluated as described previously ( 16- 19). Briefly, rabbits were immunized with Tti and the immune sera were absorbed with erythrocytes, serum, liver, thymus, spleen, and lymph node cells from normal mice. The effect of the antiserum on Tti was measured in a complement (C)-dependent lymphocytotoxicity test (6,23). Its capacity of inhibiting PFC was determined in the presence of rabbit C using a suspension of spleen cells from mice immunized with SRBC (19,23). Percentage inhibition of PFC was calculated according to the formula ((a - @/a) X 100, where a was the PFC number upon treatment of spleen cells with C only, and b was the PFC number in the suspension treated with the antiserum plus C. Rabbit antiserum to murine T lymphocytes (ATS). ATS was obtained as described previously (23). It lysed 100% of thymocytes, 38% of splenocytes, 6 1% of lymph node cells, 3% of marrow cells, and did not affect (CBA X C57BL/6)FI PFC-producing antibodies to SRBC or the immune response to thymus-independent Vi antigen of Salmonella typhi (23). Cytotoxicity assays. Cytotoxicity of the antiserum for tumor cells was assayed as follows: 0.05 ml of thrice-washed cells (1 X 106/ml) and 0.05 ml of nontoxic rabbit serum (as a source of C) were added to 0.15 ml of each dilution of the antiserum. After a 45-min incubation at 37”C, cell viability was tested with 0.25 ml of 0.1% trypan blue plus 0.1% eosin. The cytotoxicity index (CD) was expressed for each antiserum dilution as ((c - d)/( 100 - d)) X 100, where c was the percentage of nonviable cells upon treatment with antiserum plus C, and d was the percentage of nonviable cells in the control suspension treated only with C.
NESTERENKO
474
ET AL.
We also performed the indirect immunofluorescence test to estimate the binding of the antiserum to target cells (24). Fluorescein-labeled goat antibodies to rabbit IgG were kindly supplied for this purpose by K. L. Shakhanina (Gamaleya Institute). Statistical analysis. For CT1 and inhibition of PFC arithmetic means were calculated. Student’s t test (P B 0.05) was used for calculating confidence limits. RESULTS The effect of anti-ACA- 1 serum on the cells of various neoplasms is shown in Figs. 1 and 2. The antiserum diluted 1:4 to 1: 16 lysed 64% of Sa- 1, 89% of AH-22, 80% of EL4, 89% of MCh-11, 100% of MOPC 104E, and 76% of F2 cells. Its additional absorption with lymphocytes (equal proportions of thymus, spleen, and lymph node cells) or liver cells from normal (CBA X C57BL/6)F1 mice (4.5 X lo8 cells/ml of antiserum) did not reduce CT1 (data not shown), while absorption with Sa-1, AH22, MCh-11, F2, or EL-4 cells led to a considerable decrease in cytotoxic activity (Figs. 1, 2).
100 a
“‘1
b
30 I
FIG. 1. Cytotoxicity of anti-ACA- 1 serum for Sa- 1, AH-22, EG4, and MCh- 11 tumor cells. Abscissa: dilution of antiserum; ordinate: cytotoxicity index (%). Target cells: (a) Sa-1 (six experiments); (b) AH-22 (eight experiments); (c) EL-4 (eight experiments); (d) MCh-11 (five experiments). (0 0) Effect of antiserum before additional absorption; absorption with tumor cells is designated as follows: (X X) Sa-I; (0 0) AH-22; (0 0) EG4, (0 - - - 0) MB-1 1. Absorption was carried out at room temperature for 1 hr using 3.3 X 10s tumor cells per 1 ml of antiserum.
EXPRESSION
OF ACA-1 ON TUMOR
475
CELLS
FIG. 2. Cytotoxicity of anti-ACA-1 serum for MOPC 104E and F2 tumor cells. Target cells: (a) F2 (two experiments); (b) MOPC 104E (three experiments). (0 0) Effect of antiserum before additional absorption; (A A) absorption with F2. For other designations see the legend to Fig. 1.
In the indirect immunofluorescence test anti-ACA-1 serum stained 58% of Sa-1, 84% of AH-22, 80% of MCh-11, and 83% of EL4 cells. It should be noted that it exhibited practically no capacity for reacting with normal lymphocytes either in this test or in cytotoxicity assays (16-19). The above evidence seemed to suggest that ACA-1 was expressed on actively proliferating cells of the tumors studied in our experiments (just as it is present on activated T and B lymphocytes). The data shown in Figs. 3 and 4 supported this suggestion. Anti-ACA- 1 serum inhibited PFC numbers by 95%. Additional absorption of the antiserum with Sa-1, AH-22, MCh-11, F2, or EL-4 cells reduced inhibition by 86-lOO%, while absorption with lymphocytes or liver cells from nonimmunized mice was ineffective (Fig. 3). Similar results were obtained in the lymphocytotoxicity test using Tact. In 1:5 to 1: 10 dilutions anti-ACA- 1 serum lolled 44% of CBA-antiC57BL/6 and 35% of CBA-anti-BALB/c T,&. Upon additional absorption with Sa1, AH-22, MCh- 11, or EL-4 cells, its cytotoxicity for CBA-anti-C57BL/6 and CBA-
5
l
*
-
1
2
3
4
s
0
7
B
FIG. 3. Effect of anti-ACA-1 serum on PFC. Results of 10 experiments. Ordinate: percentage inhibition. (1) Before additional absorption; absorption with different cells is designated as follows: (2) Sa-1; (3) AH-22; (4) EG4; (5) MCh-11; (6) F2; (7) normal lymphocytes; (8) liver cells. PFC were assayed in (CBA X C57BL/6)FI spleens 4 days after iv immunization with 5 X IO* SRBC. The antiserum was diluted 1:50 and absorbed as described in the legend to Fig. 1. Normal cells were used at a ratio of 4.5 X 10’ per 1 ml of antiserum.
416
NESTERENKO 60
la
ET AL. So b
FIG. 4. Cytotoxicity of anti-ACA-1 serum for T-. Results of eight experiments. Target cells: (a) CBAanti-C57BL/6 T,; (b) CBA-anti-BALB/c T,. For other designations see the legend to Fig. 1.
anti-BALB/c T, fell by 82-100% and 66-98%, respectively (Fig. 4). Additional absorption with lymphocytes or liver cells from normal mice had practically no influence on cytotoxic activity. In order to confirm the specificity ,of the absorption of antiserum by tumor cells, a control series of experiments was performed using ATS (Fig. 5). It was absorbed with (CBA X C57BL/6)FI thymocytes and EL-4 T lymphoma cells known to carry MTLA (antigens reacting with ATS). ATS diluted 1: 100 lysed 100% of such cells, but upon absorption its activity fell by 99 and 88%, respectively. On the other hand, when normal murine liver cells, Sa-1 cells, or AH-22 cells were used for absorption, ATS, which was not cytotoxic for these cells, retained its activity. Therefore, at the cell/antiserum ratio chosen for our studies the cytotoxic effect of ATS was not influenced by nonspecific absorption. In further experiments anti-ACA-1 serum was absorbed with murine brain cells from embryos or adult animals, or with retinal cells from neonates or adult mice. As shown in Fig. 6, the inhibitory effect of the antiserum on PFC remained virtually unaltered after such absorption. 100'
60
60
40
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FIG. 5. Cytotoxic activity OF ATS against (CBA X C57BL/6)F1 thymocytes as r&&ted by additional absorption with tumor eelis. Results of three experiments. Abscksaz dilution of antkrum; ordinate: cytotoxicity index (‘9%).(0 0) Before absorption; absorption with different c&s is designated as follm (X X) Sa-1; (0-m) AH-22; (Oo);‘EL-4; (0 0) thymocytes; (W n ) liver c&k from normal mice. For details of the absorption procedure see the legends to Figs. 1 and 3.
EXPRESSION
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OF ACA-1 ON TUMOR
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6. Effect of anti-ACA-1 serum on PFC before and after additional absorption with murine retinal (a) and brain (b) cells. Results of five experiments. Abscissa: dilution of antiserum; ordinate: percentage inhibition. (V V) Before absorption; absorption with different cells is designated as follows: (0 Cl) brain cells from CBA embryos aged 14 days; (m n ) brain cells from adult CBA mice weighing 18-20 0) retinal cells taken 1 or 2 days atIer birth from CBA neonates; (0 0) retinal cells from I%;(0 adult CBA mice weighing 18-20 g. Absorption was carried out at room temperature for I hr using 2 X 10’ cells per 1 ml of antiserum. FIG.
DISCUSSION Using the cytotoxicity assay and the immunofluorescence test, we have established that anti-ACA- 1 serum reacts with 58-100% of cells from tumors of different histogenesis (malignancies of T- and B-lymphocyte derivation, sarcomas, hepatoma, and mammary carcinoma). Absorption with cells of these neoplasms sharply reduces the effect of anti-ACA-1 serum both on tumor cells (Figs. 1 and 2) and on activated B and T lymphocytes (Figs. 3 and 4). Control experiments (Fig. 5) have shown that tumor cells do not alfect the cytotoxic activity of ATS via nonspecific absorption. Therefore, loss of activity of anti-ACA-1 serum upon its absorption with tumor cells cannot be explained by a high nonspecific absorptive capacity of the latter cells. These data allow us to conclude that ACA- 1 is not only present on activated murine T and B cells (16- 19), but seems to find expression also on actively proliferating tumor cells of nonlymphoid origin (Sa-1, AH-22, MCh-11, F2) and those of T and B classes (EL-4, MOPC 104E). Previously ( 16- 19) we have described the differences between ACA- 1 and the antigens known to be carried by nonstimulated (H-2, MTLA, MBLA, Thy- 1, Ig, etc.) and activated (Ala- 1, PC- 1, etc.) lymphocytes. The expression of ACA-1 is probably not related to the functioning of oncomaviruses, since it has been detected on F2 cells lacking structural proteins of the oncomavirus C type (20). We have also attempted to detect ACA-1 on normal proliferating cells of nonlymphoid origin. Most brain cells in murine embryos aged 1 l- 15 days and the majority of retinal cells in neonates l-3 days after birth are known to be proliferating, while similar cells of adult mice are practically incapable of division (25-27). Therefore we absorbed anti-ACA-1 serum with retinal cells from newborn and adult mice, and with brain cells from embryos and adult animals. When the antiserum was tested on activated B lymphocytes (PFC), we found that absorption with any one of the four types of cells had no influence on its inhibitory activity. Evidently ACA-1 is absent (or its concentration is insufficient) on proliferating cells of the embryonic brain and the neonatal retina, as well as on nonproliferating cerebral and retinal cells of adult
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mice. It may be assumed that expression of ACA-1 is limited to activated T and B lymphocytes and the tumor cells studied. The reasons for such limitation remain unclear, but a possible explanation is that ACA-l+ and ACA-I- cells evolve along different lines. REFERENCES 1. Cantor, H., and Boyse, E. A., Contemp. Top. Immunobiol. 7, 41, 1977. Greaves, M. F., Cancer Rex 41, 4752, 1981. 3. Nesterenko, V. G., Usp. Sovi. Biol. (Moscow) 5, 211, 1980. 4. Raff, M. C., Transplant Rev. 6, 52, 1971. 5. Reinherz, E. L., and Schlossman, S. F., Cancer Res. 41, 4767, 1981. 6. Nesterenko, V. G., and Kovalchuk, L. V., Byull. Eks. Biol. Med. (Moscow) 7, 836, 1976. 7. Bluming, A., Lynch, M. J., Kavanah, M., and Ehiroya, R., J. Immunol. 114, 717, 1975. 8. Kimura, A. K., and Wigzell, H., Contemp. Top. Mol. Immunol. 6, 209, 1977. 9. Sullivan, R. A., Berke, G., and Amos, D. B., Transplantation 16, 388, 1973. 10. Takahashi, T., Old, L. J., and Boyse, E. A., J. Exp. Med. 131, 1325, 1970. 11. Fenney, A. J., and Hiimmerling, U., Immunogenetics (N.Y.) 3,369, 1976. 12. Bach, F. H., Alter, B. J., Widmer, M. B., Segall, M., and Dunlap, B., Zmmunol. Rev. 54, 6, 1981. 13. Thierry, H., Nadler, L. M., Pesando, J. M., Reinherz, E. L., Schlossman, S. F., and Ritz, J., Cell. Immunol. 64, 192, 1981. 14. Sutherland, R., Deha, D., Schneider, C., Newman, R., Remshead, J., and Greaves, M. F., Proc. Nat. Acad. Sci. U.S.A. 78,4515, 1981. 15. Goding, J. W., and Harris, A. W., Proc. Nat. Acad. Sci. U.S.A. 78, 4530, 1981. 16. Nesterenko, V. G., Fontalin, L. N., and Novikova, T. K., In “Advances in Comparative Leukemia Research 1979” (B. A. Lapin and D. S. Yohn, Eds.), pp. 414-418. USSR Acad. Med. Sci./IEP & T, Moscow, 1980. 17. Nesterenko, V. G., and Gruner, Sh., ByuN. Eks. Biol. Med. (Moscow) 6, 708, 1980. 18. Nesterenko, V. G., Novikova, T. K., Fontalin, L. N., Wechnik, E., Rubakova, E. I., Gruner, Sh., and Sidorova, E. V., Byull. Ekes.Biol. Med. (Moscow) 10, 449, 1980. 19. Nesterenko, V. G., Novikova, T. K., Fontalin, L. N., Rubakova, E. I., Gruner, Sh., Wechnik, E., and Sidorova, E. V., Cell. Immunol. 79, 253, 1983. 20. Beriashvili, M. M., Kryukova, I. N., and Shaginian, 0. V., Vopr. Virusol. (Moscow) 2, 221, 1981. 21. Jeme, N. K., and Nordin, A. A., Science 140,405, 1963. 22, Sprent, J., and Miller, J. A. F. T., Cell. Immunol. 3, 213, 1972. 23. Nesterenko, V. G., Kraskina, N. A., Rubakova, E. I., and Gruner, Sh., CeZl. Zmmunol. 69,215, 1982. 24. Nesterenko, V. G., and Yermakov, G. P., Byull. Eks. Biol. Med. (Moscow) 10, 449, 1980. 25. Sidman, R. L., “The Structure of the Eye,” pp. 487-506. Academic Press, New York/London, 1961. 26. Korr, H., Adv. Anat. EmbryoZ. Cell Biol. 61, No. 1, 1980. 27. Reznikov, K. Y., “Proliferation of Cerebral Cells in Vertebrates during Normal Development and after Brain Injury.” Science Publ., Moscow, 1981. 2.