394
Brain Research, 88 (1975) 394-402 ((:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Nervous system antigen-2 (NS-2), an antigenic cell surface component expressed on a murine glioblastoma
MELITTA SCHACHNER ANDTHOMAS B. CARNOW Department of Neuropathology, Harvard Medical School and Department of Neuroscience, Children's Hospital Medical Center, Boston, Mass. 02115 (U.S.A.)
(Accepted January 27th, 1975)
Interest in the surface of nerve cells stems from its possible significance for morphogenesis and differentiationS,6,s,9,15,87-39. The elucidation o f functional mechanisms requires the knowledge of neural cell surfaces in molecular terms. A search for nervous system-specific cell surface markers has been greatly aided by the availability of neural tumors and the application of serological techniques 1,2,xn,21,z3,2s. Nervous system antigen-1 (NS-1), the first new cell surface component (or components) to be reported from this laboratory, is not only unique to nervous tissue but is present predominantly on a particular subclass within the nervous system, the glial cell population 28. The present communication deals with the characterization of a second nervous system-specific antigen, NS-2, which by several criteria is distinct from NS-1. Glioblastoma (kindly given to us by Dr. S. Sato) was induced with methylcholanthrene in the C57BL/6 mouse strain 20. The biochemical, serological and morphological characteristics of this tumor are consistent with the view that it is of nervous system origin 20,za. It was maintained in C57BL/6J males by injecting between 1 × 106 and 1 × 107 tumor cells into the peritoneal cavity at passage intervals of 2-3 weeks. The nodules of solid tumor that formed on the peritoneal lining contained between 20-40 ~ live cells (as judged by the criterion of trypan blue exclusion) and were used for immunizations and preparation of single cell suspensions for cytotoxicity tests as described below. For immunization, two adult New Zealand white female rabbits were injected subcutaneously and intradermally with 1 × 108 tumor cells washed 3 times in Earle's Balanced Salt Solution (EBSS) in complete Freund's adjuvant on day 0; 2 × l0 s tumor cells in incomplete Freund's adjuvant subcutaneously and intradermally on day 31; 5 × 10s tumor cells intraperitoneally on day 62 and 1 × 10s tumor cells intraperitoneally on day 97. Tumor cells for immunization were not subjected to any enzymatic treatment. On day 104 the rabbits were bled and subsequent immunizations were carried out at intervals of 2-3 weeks. The rabbits were bled 7 days after each immunization. The antiserum from the first bleeding which had most activity for the glioblastoma and least for C57BL/6J thymocytes and lymph node cells was
395 chosen for further studies. No neurological symptoms were observed in the animals during a total immunization period of 9 months. Bulk absorptions of crude rabbit anti-glioblastoma antiserum were performed to remove non-neural rabbit anti-mouse antibodies. Antiserum (1:3 diluted with test medium) was absorbed 2-3 times for 30 min at room temperature with equal volumes of washed particulate fraction of liver, spleen, kidney and thymus from adult C57BL/6J mice and (C3H/HeJ x C57BL/6J)F1 hybrids. In vivo absorptions were also carried out by injecting intraperitoneally 1 ml of unabsorbed rabbit antiserum into a C57BL/6J female and exsanguinating the animal 4.5 h later. The cytotoxicity test, a serological test designed for the detection exclusively of cell surface antigens, was carried out as described in detail previously21,2~. Briefly, for the preparation of single cell suspensions with a high percentage of viability, mechanically dispersed cells from the solid tumor mass were subjected at a concentration of 5 × 107 cells per ml to trypsin and DNAse I treatment (0.25 70 and 0.025 ~o in EBSS, respectively, from Worthington Biochemical Co.) for 8 min at 37 °C. Cells were washed three times with EBSS containing 10~ immune precipitated fetal-bovine serum (Gibco). After these procedures the glioblastoma consisted of single cells of which more than 90 70 were viable, as judged by trypan blue exclusion. For the cytotoxicity test, 1 x 105 tumor cells were incubated with varying dilutions of antiserum in a volume of 0.05 ml for 30 min at room temperature, washed once with 1 ml of test medium (Medium 199 containing 2 700of immune precipitated fetalbovine serum) and incubated for 30 rain at 37 °C with 0.05 ml of complement. Guinea pig serum was used as a source of complement at an initial dilution of 1 : 4 with test medium. Antiserum activity was determined by calculating the percentage of dead cells (as judged by trypan blue uptake) in the total cell suspension. Cross-reactivity of NS-2 antigen between glioblastoma as the target indicator cell and other tissues was measured by test-absorption of anti-NS-2 antiserum with washed particulate fraction of tissue homogenates under the conditions described previously2z,26. Antiserum at a concentration of 2-4 serial dilutions below the titer endpoint was incubated for 30 min at 0 °C with particulate fraction of tissue homogenate that had been washed three times and which contained mostly membrane fragments and a few whole cells without processes. The tissue homogenates were not subjected to enzymatic treatment. The ratio of serum to packed tissue homogenate was I :1. The residual activity of the absorbed serum was tested in the cytotoxicity test as outlined above. Blocking capacity of anti-NS-1 antibody for the attachment of anti-NS-2 antibody to brain tissue homogenate was measured according to the method of Boyse et a1.1°. 0.04 ml of washed particulate fraction of washed C57BL/6J brain homogenate was incubated for 30 min at 0 °C in antibody excess of anti-NS-1 antiserum. 0.5 ml of (C57BL/6J x DBA/ 2)F1 anti-glioma G26 (1:10 diluted) and with normal, non-hyperimmune C57BL/6J serum (0.5 ml, 1:10diluted). The two samples were washed 3 times in the cold with 1 ml of test medium, incubated for 30 min at 0 °C with 0.05 ml of anti-NS-2 antiserum (1 : 9 diluted) and tested for residual cytotoxic activity on glioblastoma as described. Indirect immunofluorescence was carried out as described in ref. 23. Fluores-
396 ceinated goat antiserum to rabbit immunoglobulin (Antibodies, Inc.) was used at a dilution of 1:10. Before bulk absorptions with non-neural tissues, rabbit anti-glioblastoma antiserum had a cytotoxic titer of 1:500 on glioblastoma, 1:400 on C57BL/6J thymocytes, and 1:100 on C57BL/6J lymph node cells. After absorption, the antiserum had an approximate titer of 1:100 on glioblastoma and no detectable titer on C57BL/ 6J thymocytes and lymph node cells. The in vivo absorbed antiserum had a titer of approximately 1:24 on glioblastoma and no detectable titer on C57BL/6J thymocytes and lymph node cells. When tested by the indirect immunoflorescence method more than 95 % of the viable glioblastoma cells showed ring fluorescence, whereas more than 95 % of C57BL/6J lymph node cells or thymocytes showed no fluorescence. The titer endpoint of the antiserum on glioblastoma was at a serum dilution of about 1:60. For this particular antiserum the indirect immunofluorescence technique was less sensitive than the more quantitative cytotoxicity assay method. The preabsorbed antiserum was then further test-absorbed with various tissues of the C57BL/6J strain and assayed for residual cytotoxic activity on glioblastoma (Fig. l). The index of residual cytotoxic activity is inversely proportional to the expression of antigen on a particular tissue : high residual activity is indicative of low antigen expression, whereas low residual activity reveals presence of antigen on the absorbing tissue. Except for the glioblastoma and brain, none of the tested mouse
>95-
o >50-
IJJ 0¢:
lid
Brain Research, 88 (1975) 394-402 ((:) Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
Nervous system antigen-2 (NS-2), an antigenic cell surface component expressed on a murine glioblastoma
MELITTA SCHACHNER ANDTHOMAS B. CARNOW Department of Neuropathology, Harvard Medical School and Department of Neuroscience, Children's Hospital Medical Center, Boston, Mass. 02115 (U.S.A.)
(Accepted January 27th, 1975)
Interest in the surface of nerve cells stems from its possible significance for morphogenesis and differentiationS,6,s,9,15,87-39. The elucidation o f functional mechanisms requires the knowledge of neural cell surfaces in molecular terms. A search for nervous system-specific cell surface markers has been greatly aided by the availability of neural tumors and the application of serological techniques 1,2,xn,21,z3,2s. Nervous system antigen-1 (NS-1), the first new cell surface component (or components) to be reported from this laboratory, is not only unique to nervous tissue but is present predominantly on a particular subclass within the nervous system, the glial cell population 28. The present communication deals with the characterization of a second nervous system-specific antigen, NS-2, which by several criteria is distinct from NS-1. Glioblastoma (kindly given to us by Dr. S. Sato) was induced with methylcholanthrene in the C57BL/6 mouse strain 20. The biochemical, serological and morphological characteristics of this tumor are consistent with the view that it is of nervous system origin 20,za. It was maintained in C57BL/6J males by injecting between 1 × 106 and 1 × 107 tumor cells into the peritoneal cavity at passage intervals of 2-3 weeks. The nodules of solid tumor that formed on the peritoneal lining contained between 20-40 ~ live cells (as judged by the criterion of trypan blue exclusion) and were used for immunizations and preparation of single cell suspensions for cytotoxicity tests as described below. For immunization, two adult New Zealand white female rabbits were injected subcutaneously and intradermally with 1 × 108 tumor cells washed 3 times in Earle's Balanced Salt Solution (EBSS) in complete Freund's adjuvant on day 0; 2 × l0 s tumor cells in incomplete Freund's adjuvant subcutaneously and intradermally on day 31; 5 × 10s tumor cells intraperitoneally on day 62 and 1 × 10s tumor cells intraperitoneally on day 97. Tumor cells for immunization were not subjected to any enzymatic treatment. On day 104 the rabbits were bled and subsequent immunizations were carried out at intervals of 2-3 weeks. The rabbits were bled 7 days after each immunization. The antiserum from the first bleeding which had most activity for the glioblastoma and least for C57BL/6J thymocytes and lymph node cells was
395 chosen for further studies. No neurological symptoms were observed in the animals during a total immunization period of 9 months. Bulk absorptions of crude rabbit anti-glioblastoma antiserum were performed to remove non-neural rabbit anti-mouse antibodies. Antiserum (1:3 diluted with test medium) was absorbed 2-3 times for 30 min at room temperature with equal volumes of washed particulate fraction of liver, spleen, kidney and thymus from adult C57BL/6J mice and (C3H/HeJ x C57BL/6J)F1 hybrids. In vivo absorptions were also carried out by injecting intraperitoneally 1 ml of unabsorbed rabbit antiserum into a C57BL/6J female and exsanguinating the animal 4.5 h later. The cytotoxicity test, a serological test designed for the detection exclusively of cell surface antigens, was carried out as described in detail previously21,2~. Briefly, for the preparation of single cell suspensions with a high percentage of viability, mechanically dispersed cells from the solid tumor mass were subjected at a concentration of 5 × 107 cells per ml to trypsin and DNAse I treatment (0.25 70 and 0.025 ~o in EBSS, respectively, from Worthington Biochemical Co.) for 8 min at 37 °C. Cells were washed three times with EBSS containing 10~ immune precipitated fetal-bovine serum (Gibco). After these procedures the glioblastoma consisted of single cells of which more than 90 70 were viable, as judged by trypan blue exclusion. For the cytotoxicity test, 1 x 105 tumor cells were incubated with varying dilutions of antiserum in a volume of 0.05 ml for 30 min at room temperature, washed once with 1 ml of test medium (Medium 199 containing 2 700of immune precipitated fetalbovine serum) and incubated for 30 rain at 37 °C with 0.05 ml of complement. Guinea pig serum was used as a source of complement at an initial dilution of 1 : 4 with test medium. Antiserum activity was determined by calculating the percentage of dead cells (as judged by trypan blue uptake) in the total cell suspension. Cross-reactivity of NS-2 antigen between glioblastoma as the target indicator cell and other tissues was measured by test-absorption of anti-NS-2 antiserum with washed particulate fraction of tissue homogenates under the conditions described previously2z,26. Antiserum at a concentration of 2-4 serial dilutions below the titer endpoint was incubated for 30 min at 0 °C with particulate fraction of tissue homogenate that had been washed three times and which contained mostly membrane fragments and a few whole cells without processes. The tissue homogenates were not subjected to enzymatic treatment. The ratio of serum to packed tissue homogenate was I :1. The residual activity of the absorbed serum was tested in the cytotoxicity test as outlined above. Blocking capacity of anti-NS-1 antibody for the attachment of anti-NS-2 antibody to brain tissue homogenate was measured according to the method of Boyse et a1.1°. 0.04 ml of washed particulate fraction of washed C57BL/6J brain homogenate was incubated for 30 min at 0 °C in antibody excess of anti-NS-1 antiserum. 0.5 ml of (C57BL/6J x DBA/ 2)F1 anti-glioma G26 (1:10 diluted) and with normal, non-hyperimmune C57BL/6J serum (0.5 ml, 1:10diluted). The two samples were washed 3 times in the cold with 1 ml of test medium, incubated for 30 min at 0 °C with 0.05 ml of anti-NS-2 antiserum (1 : 9 diluted) and tested for residual cytotoxic activity on glioblastoma as described. Indirect immunofluorescence was carried out as described in ref. 23. Fluores-
396 ceinated goat antiserum to rabbit immunoglobulin (Antibodies, Inc.) was used at a dilution of 1:10. Before bulk absorptions with non-neural tissues, rabbit anti-glioblastoma antiserum had a cytotoxic titer of 1:500 on glioblastoma, 1:400 on C57BL/6J thymocytes, and 1:100 on C57BL/6J lymph node cells. After absorption, the antiserum had an approximate titer of 1:100 on glioblastoma and no detectable titer on C57BL/ 6J thymocytes and lymph node cells. The in vivo absorbed antiserum had a titer of approximately 1:24 on glioblastoma and no detectable titer on C57BL/6J thymocytes and lymph node cells. When tested by the indirect immunoflorescence method more than 95 % of the viable glioblastoma cells showed ring fluorescence, whereas more than 95 % of C57BL/6J lymph node cells or thymocytes showed no fluorescence. The titer endpoint of the antiserum on glioblastoma was at a serum dilution of about 1:60. For this particular antiserum the indirect immunofluorescence technique was less sensitive than the more quantitative cytotoxicity assay method. The preabsorbed antiserum was then further test-absorbed with various tissues of the C57BL/6J strain and assayed for residual cytotoxic activity on glioblastoma (Fig. l). The index of residual cytotoxic activity is inversely proportional to the expression of antigen on a particular tissue : high residual activity is indicative of low antigen expression, whereas low residual activity reveals presence of antigen on the absorbing tissue. Except for the glioblastoma and brain, none of the tested mouse
>95-
o >50-
IJJ 0¢:
lid
