Clin. exp. Immunol. (1975) 21, 267-277.
ANTIGENIC DETERMINANTS COMMON TO ESTABLISHED HUMAN B-CELL LINES, BUT NOT SHARED BY HUMAN T-CELL LINES (MOLT AND SOMMER) YUMIKO TAKADA,* A. TAKADA* AND J. MINOWADAt * Springville Laboratories, and t Department of Immunology and Immunochemistry, Roswell Park Memorial Institute, Springville, New York, U.S.A. (Revised 10 December 1974) SUMMARY
Rabbits were immunized with two kinds of human B-cell lines (SOMMER-B, and RPMI number 1788 cells) to get anti-B-cell sera. Sera were absorbed with human red blood cells, human liver, human brain and human T cells (MOLT-4 and SOMMER-T cells). Cytotoxicity of absorbed sera was not only tested against B cells which were used for immunization but also tested against unrelated B cells, including B35M cells derived from Burkitt lymphoma specimens. Results indicated that established human B cells had antigenic determinants which were shared with allogeneic human B cells, but not shared with non-lymphoid cells or tissues, nor with established human T cells. INTRODUCTION Lymphoid cells have been classified into bone marrow-derived cells, B cells and thymusderived cells, T cells. These two populations of lymphocytes carry different antigenic markers. The murine thymus has H-2 antigens, TL antigens (Boyse, Old & Luell, 1963) 0-antigens (Reif & Allen, 1964a, b), Ly antigens (Stuck et al., 1964; Old, Boyse & Stockert, 1965) and other alloantigens such as H-14 (Popp, 1969). On the other hand, B cells bear readily detectable immunoglobulins (Ig) on their surface (Tanigaki et al., 1966; Raff, 1970; Rabellino et al., 1971; Jondal, Holm & Wigzell, 1972). Besides this marker, mouse Blymphocyte antigen, MBLA, which is detected by a rabbit anti-mouse lymphocyte absorbed with thymocytes (Raff, Nase & Mitchison, 1971), PC-l (Takahashi, Old & Boyse, 1970) expressed on plasma cells, and Ly-4 (Snell et al., 1973) have been described to be present on murine B cells. Furthermore, the population of B lymphocytes seems to be heterogeneous (Playfair & Purves, 1971). Laskov, Rabinowitz & Schlesinger (1973) suggested that there might be three different antigens, B 1, B2 and My, on cells of B-cell lineage and that antigenbinding precursor cells bore Bi antigen and Ig, while antibody-producing cells might acquire B2 and My in addition to BI, but lose 1g. Compared with the mouse system, specific Correspondence: Dr Yumiko Takada, Second Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu City, Japan.
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antigens on human T and B lymphocytes were not so clearly demonstrated. Recently Ablin, Baird & Morris (1972), Ablin & Morris (1973) and Smith et al. (1973) have shown that human T cells have specific antigens. Although human B cells have surface Ig, receptors for complement (Nussenzweig et al., 1971; Shevach et al., 1973) and for antigenantibody complexes (Shevach et al., 1973; Dickler & Kunkel, 1972), it has not been shown if human B lymphocytes have specific antigens not shared with T lymphocytes. One of the difficulties of showing B cell-specific antigens is the absence of the pure population of T cells. The foetal thymus tissue is still contaminated with circulating blood cells and other epithelial cells which might have B-cell characteristics. The other is that of eliminating the possible involvement of antigens specific to individuals in making heteroantibodies. Recently, Minowada, Ohnuma & Moore (1972), and Minowada & Moore (1974) have succeeded in establishing permanent cell lines (MOLT and SOMMER) from the peripheral blood of patients with acute lymphoblastic leukaemia. MOLT cells did not produce Ig, and did not have receptors for Ig or the Fc portion of Ig, but bound sheep red blood cells (SRBC) upon exposure. The formation of spontaneous rosettes with SRBC is considered to be a marker for T cells (Jondal et al., 1972; Wybran, Fudenberg & Sleisinger, 1971; Wybran, Carr & Fudenberg, 1972). The original culture of SOMMER cells gave rise to two populations. One, called SOMMER-B cells showed B-cell characteristics, and had forty-six chromosomes without markers (Huang et al., 1974). The other, called SOMMER-T cells, had T-cell characteristics, formed SRBC rosettes and karyologically had two typical markers (Huang et al., 1974). Since SOMMER-T and SOMMER-B cells were obtained from the same person, differences in antigenic markers between these two kinds of cells must be due to the difference of the origin of these cells, bone marrow-derived or thymusderived, thus being organ-specific. In the present studies, we prepared antisera in rabbits against two kinds of B cells, RPMI number 1788 and SOMMER-B cells. Cytotoxicity against various B cells was tested by using these antisera. Results indicate that there might be antigenic determinants on B cells which are shared with allogeneic B cells but not shared with autologous or allogeneic T cells. MATERIALS AND METHODS Cell lines MOLT-4 cells and SOMMER-T cells, both T-cell lines, were obtained as reported previously (Minowada et al., 1972, 1974). SOMMER-B cells were obtained from the person with acute lymphoblastic leukaemia, from whom SOMMER-T cells were also obtained. RPMI number 1788 cells were from healthy male adults, and B35M cells, from Burkitt lymphoma specimen. They were grown at 370C in a suspension culture using the nutrient medium RPMI number 1640 supplemented with 10% (v/v) heat-inactivated foetal calf serum. Immunization SOMMER-B cells (I 5 x 107) or RPMI number 1788 cells (I 5 x 107) was mixed with 1 ml of Freund's complete adjuvant (FCA) and 0 5 ml was injected into bilateral footpads, the rest (1 ml) being injected into a neck muscle. One week later, 2 x 107 cells were injected intravenously, and another week later 1 2 x 107 cells with FCA were injected into footpads and neck muscle. Blood was drawn 2 weeks after the last injection. After separation, serum was inactivated by heating at 56°C for 30 min.
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Cytotoxicity Cytotoxicity tests were performed using the same method as described previously (Takada et al., 1974). Briefly, 0.1 ml of cell suspension containing 2 x 106 viable cells per millilitre of IX Eagle's solution was mixed with 0 1 ml of either antiserum or normal rabbit serum (NRS) and the mixture was kept at 20'C for 30 min. Then 0- 1 ml of rabbit complement was added to each tube and incubated at 370C for 30 min. After incubation viability was tested by trypan blue exclusion tests. Viability (percentage live cells) was calculated by dividing the percentage of viable cells in a tube with antiserum by the percentage of viable cells in a tube with NRS, multiplied by 100. The percentage of dead cells was obtained by subtracting the percentage of live cells from 100.
Absorption One gram of human brain (cortex, mainly grey matter) or human liver, provided by Dr U. Kim of our Institute, was homogenized in 3 ml of saline in a Potter homogenizer. The homogenate was centrifuged for 20 min at 3000 rev/min. The supernatant was drawn and 3 ml of saline was added to the residue and mixed vigorously before the mixture was centrifuged again. After the second centrifugation, the pellet was mixed with 3 ml of antiserum.
Absorption of anti-RPMI number 1788 serum Three millilitres of the serum was absorbed with human liver four times, then 3 ml of the liver-absorbed serum was mixed with 1 x 107 live MOLT-4 cells. After leaving the mixture for 30 min at 20'C, it was centrifuged for 20 min at 3000 rev/min. The supernatant was either used for cytotoxicity tests or further mixed with 1 x 107 live MOLT-4 cells. To know the possible involvement of anti-surface Ig antibody in cytotoxicity studies, twice liver-absorbed serum (1 ml) was mixed with 1 ml of human serum, and kept at 20'C for 30 min with constant gentle shaking. Then the mixture was centrifuged at 3000 rev/min for 20 min to obtain the supernatant.
Absorption of anti-SOMMER-B serum Three millilitres of anti-SOMMER-B serum was added to 3 ml of packed human red blood cells and mixed well. The mixture was kept at 20°C for 30 min, then centrifuged for 20 min at 3000 rev/min. The supernatant was again mixed with 3 ml of packed human red blood cells, and incubation and centrifugation were repeated. The supernatant thus obtained further absorbed twice with human liver (1 g of liver per 3 ml of serum) and four times with human brain (1 g brain per 3 ml of serum). The absorbed serum (3 ml) was mixed with 1 x 107 SOMMER-T cells, and kept for 30 min at 20°C. Then the mixture was centrifuged for 20 min at 3000 rev/min to get the supernatant, which was used either for cytotoxicity tests or further absorption with SOMMER-T cells.
Immunofluorescence Rabbit anti-RPMI number 1788 or SOMMER-B serum (3 ml) was absorbed four times with liver and with 1 x 107 SOMMER-T cells. Sera were then centrifuged at 15,000 rev/min for 1 hour by using T1-50 rotor of Beckman's ultracentrifuge (L-4). The supernatant was mixed with either RPMI number 1788, SOMMER-B cells, or human peripheral lymphocytes fractionated by Ficoll-Isopaque. 0 3 ml of antiserum was mixed with 5 x 106 cells and kept at 37°C for 30 min, washed three times with RPMI 1640 media. Cell pellets were then added
Yumiko Takada, A. Takada and J. Minowada
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with each 0-1 ml of 10-times diluted goat-anti-rabbit Ig conjugated with fluorescein (FITC) (Behring Diagnostics, New Jersey, U.S.A.). The mixture was kept at 20'C for 30 min, and washed thoroughly with media. A drop of cell suspension thus obtained was transferred to a slide glass and fluorescing cells were photographed by using Leitz fluorescence microscope and Kodak Ektachrome high speed film (ASA164). RESULTS Cytotoxicity against B35M cells of antiserum to RPMI number 1788 Anti-RPMI number 1788 serum was absorbed with human liver quite extensively (four times). Then it was absorbed twice with MOLT-4 cells. Since RPMI number 1788 cells have surface immunoglobulins, the injection of these cells into rabbit might have resulted in the production of anti-Ig antibody which would kill cells in the presence of complement due to antibody-mediated cytotoxicity. To exclude the possibility of the involvement of antibodymediated cytotoxicity, twice liver-absorbed serum was mixed with an equal volume of human serum, kept for 30 min at room temperature, and centrifuged for 20 min at 3000 rev/min. Since antiserum was diluted twice by the addition of normal serum, the adjustment was made in drawing the curve in Fig. 1. Cytotoxicity of anti-RPMI number 1788 serum against B35M cells was absorbed to some extent with human liver and to a small extent with MOLT-4 cells. Further absorption with MOLT-4 cells did not change cytotoxicity of the serum any more. Antisurface Ig antibody did not have any effect on the cytotoxicity in the present
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Cytotoxicity against SOMMER-B cells of antiserum to RPMI number 1788 cells The absorption schedule was the same as used in the previous experiment. Target cells were SOMMER-B cells. The results of cytotoxicity tests are shown in Fig. 2. Cytotoxicity of anti-RPMI number 1788 serum against SOMMER-B cells was absorbed with human liver to some extent. The further absorption with MOLT-4 cells resulted in a small decrease in cytotoxicity, but additional absorption of the serum with MOLT-4 cells did not change the cytotoxic activity of the serum. The data shown in Figs 1 and 2 indicate that cytotoxicity of anti-B-cell serum against two kinds of allogeneic B-cells was not completely absorbed with extensive absorptions with human liver and human T-cell line (MOLT-4). 90S A
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Cytotoxicity against B35M cells of antiserum to SOMMER-B cells Anti-SOMMER-B serum was sequentially absorbed with human red blood cells (twice), human liver (twice), human brain (four times), and then SOMMER-T cells (twice). Results shown in Fig. 3 indicate that cytotoxicity of anti-SOMMER-B serum against B35M cells was slightly absorbed with human red blood cells and human liver, then to some extent with human brain. Lines E and F in Fig. 3 are almost identical, indicating that no antibody against B35M cells was removed with human brain any more. Further absorption of the serum with SOMMER-T cells once or twice did not change the cytotoxicity of antiSOMMER-B-cell serum against B35M cells. Cytotoxicity against SOMMER-B cells of antiserum to SOMMER-B cells Antiserum against SOMMER-B cells was sequentially absorbed with human red blood
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cells (twice), human liver (twice), human brain (four times) and SOMMER-T cells (four times). Target cells of cytotoxicity tests were SOMMER-B cells. Fig. 4 shows the results. Anti-SOMMER-B-cell activity was somewhat absorbed with human red blood cells (Lines B and C). Further absorption with human liver decreased cytotoxic activity of the serum (Lines D and E). Absorption of the serum with human brain further removed antiSOMMER-B activity from the serum (Lines F), but four times absorption did not result in any significant decrease in anti-SOMMER-B-cell activity (Line G). Anti-SOMMER-Bcell serum which was absorbed with human red blood cells, human liver, and human brain, still had cytotoxicity against SOMMER-B cells (Line H), and further absorption with SOMMER-T cells did not change the cytotoxic activity any more. Cytotoxicity against RPMI number 1788 cells of antiserum to RPMI number 1788 absorbed with RPMI number 1788 cells Although we have shown that anti-B-cell activity of serum could not be abolished by the absorption with human T-cell lines, it seems important to show that anti-B-cell activity would be abolished by the absorption with human B cells, especially by using homologous materials. Fig. 5 shows the results of experiments in which rabbit anti-RPMI number 1788 serum was absorbed four times with human liver and then with 1 x 107 live RPMI number 1788 cells. One absorption with RPMI number 1788 cells of the liver-absorbed serum resulted in the presence of some activity remaining only up to four times dilution. Absorption three times with RPMI number 1788 cells resulted in complete abolishment of cytotoxicity against RPMI number 1788 cells. These results indicate that the cytotoxicity
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remaining after the absorptions with RBC, liver, brain and T cells shown in the preceding results was due to the antibody against B cells, not due to the presence of cytotoxic substances unrelated to antibody.
Immunofluorescence of B cells with anti-B-cell antibody coupled with FITC-conjugated goat anti-rabbit immunoglobulin Although it is shown that the cytotoxicity against B cells was due to the presence of anti-B-cell antibodies, it may be important to show directly the attachment of anti-B-cell antibodies on B cells. Anti-SOMMER-B or RPMI 1788 serum was mixed with either SOMMER-B, RPMI number 1788, or human peripheral lymphocytes. Fig. 6a and b show the fluorescence of RPMI number 1788 and SOMMER-B cells stained with anti-RPMI number 1788 serum and FITC-conjugated goat anti-rabbit immunoglobulin, respectively. As shown here, cells show fluorescence, but capping or ring formation typical of live lymphocytes is not shown in many cells. Since established cultured B cells are not roundshaped even under regular microscopic observation in contrast to fresh peripheral lymphocytes, and have many protrusions, antigen-antibody complexes may not smoothly migrate to the pole. Fluorescence on cultured cells looks patchy, thus indicating some migration of
FIG. 6(a). Immunofluorescence of RPMI number 1788 cells. Cells were incubated with antiRPMI number 1788 serum absorbed four times with human liver and SOMMER-T cells, then mixed with goat anti-rabbit immunoglobulin conjugated with FITC. (b) Immunofluorescence of SOMMER-B cells. Cells were incubated with anti-RPML number 1788 serum. (c) Immunofluorescence of human peripheral lymphocytes. Peripheral lymphocytes were incubated with anti-SOMMER-B-cell serum absorbed four times with human liver and SOMMER-T cells, then mixed with FITC-conjugated goat anti-rabbit 1g.
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antigen-antibody complexes on the membrane. On the other hand, Fig. 6c shows the fluorescence on the membrane of fresh human peripheral lymphocytes. Typical ring formation is seen on lymphocytes. DISCUSSION Much attention has been given to the classification of human lymphocytes into two categories, B and T cells. Since Wilson & Nossal (1971) suggested that acute lymphoblastic leukaemia might be a leukaemia of T cells and chronic lymphatic leukaemia might be that of B cells, many investigators have studied the possible role of T and B cells, in leukaemogenesis. Recently Minowada et al. (1972, 1974) succeeded in the establishment of two lymphoid cell lines (called MOLT-4 and SOMMER) from the peripheral blood of patients of acute lymphoblastic leukaemia. MOLT-4 cells did not have immunoglobulins on the surface, nor receptors for antigen-antibody complex, but bound SRBC to form rosettes. Electronmicroscopically, MOLT-4 cells had smooth surface characteristics of T-cells (Polliack et al., 1973). The original culture of SOMMER cells gave rise to two populations. One called SOMMER-B cells, showed B-cell characteristics and had forty-six chromosomes without markers (Huang et al., 1974). The other, called SOMMER-T cells, had T-cell characteristics, formed SRBC rosettes and karyologically had two typical markers. The isolation of SOMMER-B and -T cells provided a unique opportunity to study antigens specific to B cells but not shared with T cells. Furthermore, since both SOMMER-B and -T cells were established from the same person, alloantigens such as HL-A must be identical and the difference in antigenic determinants between B and T cells can be considered to be due to the difference in the origins of these cells. In the present experiment, we injected B cells established from a normal person (RPMI number 1788) or SOMMER-B cells into rabbits and obtained respective antisera. Cytotoxic tests of anti-RPMI number 1788 serum were performed against SOMMER-B cells and B35M cells (obtained from a patient of Burkitt lymphoma) in order to study antigenic determinants common to established B cells. Figs 1 and 2 indicate that after extensive absorptions of antiRPMI number 1788 serum with human liver there still remained antibody against both SOMMER-B and B35M cells. Further absorptions with MOLT-4 cells resulted in slight, if any, decrease in cytotoxic activity. The cytotoxicity was not mediated by anti-surface Ig antibody because human serum containing Ig did not remove anti-B-cell activity. Therefore, antiserum against RPMI number 1788 after absorptions contained antibody against two kinds of B cells (SOMMER-B and B35M) but not against human T cells. Then we used anti-SOMMER-B-cell serum against B35M and SOMMER-B itself. Figs 3 and 4 indicate the results of experiments in which the antiserum was extensively absorbed with human red blood cells, human liver, human brain and SOMMER-T cells, and then cytotoxicity was tested against B35M and SOMMER-B cells. It is shown that after exhaustive absorptions with non-B cells or non-lymphoid tissues there still remained cytotoxicity not only against allogeneic B cells (B35M) but also against autologous B cells (SOMMER-B). Fig. 4 indicates that these surface antigens on SOMMER-B cells were not expressed on SOMMER-T cells because of the failure of absorption with SOMMER-T cells. Since SOMMER-B and -T cells were obtained from the same person, it may be concluded that SOMMER-B cells have antigenic determinants specific to and common among established B cells, but not shared with established human T cells. Some of the antigenic
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determinants on SOMMER-B cells might be leukaemia-specific, but those shared with other established B cells must be B cell-specific because antibody against SOMMER-B cells after absorptions was not only cytotoxic to cells established from patients with leukaemia or lymphoma (B35M) but also to cells established from a normal person (RPMI number 1788, data not shown). Also antibody against RPMI number 1788 cells was cytotoxic to SOMMER-B cells after exhaustive absorptions. As to the absorption experiments utilizing human brain, it has been shown (Takada et al., 1974) that human brain shares antigenic determinants with established human T cells (MOLT-4). The present experiments showed that only small extent of cytotoxicity against B cells was removed after exhaustive absorptions of anti-B sera with human brain. This observation may indicate that established human B cells have small amounts of antigenic determinants shared with human brain, and that these determinants should be human-specific, since human liver could absorb as well (Figs 1 and 2). Recently, several Ir-associated (Ia) mouse lymphocyte alloantigens have been demonstrated (Sachs & Cone, 1973; Lozner et al., 1974). Ia antigens were shown to be present only on B cells (called fi antigens). The present B cell-specific antigens may be ft equivalents in humans. Although we showed that B cell-specific antigens were common among allogeneic B cells, it does not imply that they are identical. Since antibodies were formed in rabbits, rabbits may not have recognized subtle differences in the structures of the present antigens between individuals. Rabbits, for example, hardly recognize the differences between different HL-A antigens. Purified anti-B-cell antibody may serve as a tool to study the biological functions of the B cell-specific antigens. Recent reports concerning the relationship between Ia and Fc receptor may be extremely interesting in this respect (Dickler & Sachs, 1974). We are currently working on the possible link between B cell-specific antigen and Fc receptor on cultured B cells. ACKNOWLEDGMENTS
Excellent technical help by Harry Babbitt is greatly appreciated. We acknowledge encouragement by Dr Julian L. Ambrus, Director of the Springville Laboratories, throughout the present work. SOMMER cells were provided by Dr G. E. Moore, Denver, Colorado, U.S.A. This work was supported in part by PHS grant number CA 14413 from the National Cancer Institute, number AI-08899 from the National Institute of Allergy and Infectious Disease, and G.R.S.G. number RR-05648-08. REFERENCES ABLIN, R.J., BAIRD, W.M. & MORRIS, A.J. (1972) Tissue and species-specific antibodies in antithymocyte globulin. Transplantation, 13, 306. ABLIN, R.J. & MORRIS, A.J. (1973) Thymus-specific antigens on human thymocytes and on thymicderived lymphocytes. Transplantation, 15, 415. BoYSE, E.A., OLD, L.J. & LUELL, S. (1963) Antigenic properties of experiment leukemias. 1I. Immunological studies in vivo with C57B1/6 radiation-induced leukemias. J. nat. Cancer Inst. 31, 987. DICKLER, H.B. & KUNKEL, H.G. (1972) Interaction of aggregated y-globulin with B lymphocytes. J. exp. Med. 136, 191. DICKLER, H.B. & SACHS, D.H. (1974) Evidence for identity or close association of the Fc receptor of B lymphocytes and alloantigens determined by the Ir region of the H-2 complex. J. exp. Med. 140, 779. HUANG, C.C., Hou, Y., WOODS, L.K., MOORE, G.E. & MINOWADA, J. (1974) Cytogenetic study of human
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lymphoid cell lines with characterization of thymus-derived lymphocytes J. nat. Cancer Inst. 53, 665. JONDAL, M., HOLM, G. & WIGZELL, H. (1972) Surface markers on human T and B lymphocytes. I. A large population of lymphocytes forming nonimmune rosettes with sheep red blood cells. J. exp. Med. 136, 207. LASKOV, R., RABINOWITZ, R. & SCHLESINGER, M. (1973) Antigenic characterization of murine rosette and plaque forming cells. Immunology, 24, 939. LOZNER, E.C., SACHS, D.H., SHEARER, G.M. & TERRY, W.D. (1974) B-cell alloantigens determined by the H-2 linked Ir region are associated with mixed lymphocyte culture stimulation. Science, 183, 757. MINOWADA, J. & MOORE, G.E. (1974) T-lymphocyte cell lines derived from patients with acute lymphoblastic leukaemia. Proceedings of the VI International Symposium on Comparative Leukemia Research. (In press.) MINOWADA, J., OHNUMA, T. & MOORE, G.E. (1972) Rosette forming human lymphoid cell line. I. Establishment and evidence for origin of thymus-derived lymphocytes. J. nat. Cancer Inst. 49, 891. NuSSENZWEIG, V., BIANCO, C., DUKOR, P. & EDEN, A. (1971) Receptors for C3 on B lymphocytes. Possible role in the immune response. Progr. Immunol. 1, 73. OLD, L.J., BOYSE, E.A. & STOCKERT, E. (1965) The G (Gross) leukaemia antigen. Cancer Res. 25, 813. PLAYFAIR, J.H.L. & PURVES, E.C. (1971) Separate thymus dependent and thymus independent antibody forming cell precursors. Nature: New Biology, 231, 149. POLLIACK, A., LAMPEN, N., CLARKSON, B.D., DEHARVEN, E., BENTWICH, Z., SIEGAL, F.P. & KUNKEL, H.G. (1973) Identification of human B and T lymphocytes by scanning electronmicroscopy. J. exp. Med. 138, 607. Popp, D.M. (1969) Histocompatibility-14. Correlation of the isoantigens Rho and R-Z locus. Transplantation, 7, 233. RABELLINO, E., COLON, S., GREY, H.M. & UNANUE, E.R. (1971) Immunoglobulins on the surface of lymphocytes. I. Distribution and quantitation. J. exp. Med. 133, 156. RAFF, M.C. (1970) Two distinct populations of peripheral lymphocytes in mice distinguishable by immunofluorescence. Immunology, 19, 637. RAFF, M.C., NASE, S. & MITCHISON, N.A. (1971) Mouse specific bone marrow-derived lymphocyte antigen as a marker for thymus independent lymphocyte. Nature: New Biology, 230, 50. REIF, A.E. & ALLEN, J.M.V. (1964a) Immunological distinction of AKR thymocytes. Nature (Lond.), 203, 886. REIF, A.E. & ALLEN, J.M.V. (1964b) The AKR thymic antigen and its distribution in leukemias and nervous tissues. J. exp. Med. 120, 413. SACHS, D.H. & CONE, J.L. (1973) A mouse B-cell alloantigen determined by gene(s) linked to the major histocompatibility complex. J. exp. Med. 138, 1289. SHEVACH, G.M., HERBERMAN, R., FRANK, M.M. & GREEN, I. (1972) Receptors for complement and immunoglobulins on human leukemic cells and human lymphoblastoid cell lines. J. clin. Invest. 51, 1933. SMITH, R.W., TERRY, W.D., BUELL, D.N. & SELL, K.W. (1973) An antigenic marker for human thymic lymphocytes. J. Immunol. 110, 884. SNELL, G.D., CHERRY, M., MCKENZIE, I.F.C. & BAILEY, D.W. (1973) Ly-4, a new locus determining a lymphocyte cell-surface alloantigen in mice. Proc. nat. Acad. Sci. (Wash.), 70, 1108. STUCK, B., BOYSE, E.A., OLD, L.J. & CARSWELL, E.A. (1964) ML. A new antigen found in leukemias and mammary tumours of the mouse. Nature (Lond.), 203, 1033. TAKADA, A., TAKADA, Y., ITO, U. & MINOWADA, J. (1974) Shared antigenic determinants between human brain and human T-cell line. Clin. exp. Immunol. 18, 491. TAKAHASHI, T., OLD, L.J. & BOYSE, E.A. (1970) Surface alloantigens of plasma cells. J. exp. Med. 131, 1325. TANIGAKI, N., YAGI, Y., MOORE, G.E. & PRESSMAN, D. (1966) Immunoglobulin production in human leukemia cell lines. J. Immunol. 97, 634. WILSON, J.D. & NosSAL, G.J.V. (1971) Identification of human T and B lymphocytes in normal peripheral blood and in chronic lymphatic leukemia. Lancet, ii, 788. WYBRAN, J., CARR, M.C. & FUDENBERG, H.H. (1972) The human rosette-forming cell as a marker of population of thymus-derived cells. J. clin. Invest. 51, 2537. WYBRAN, J.H., FUDENBERG, H.H. & SLEISINGER, M.H. (1971) Rosette formation. A test for cellular immunity. Clin. Res. 19, 568.
ANTIGENIC DETERMINANTS COMMON TO ESTABLISHED HUMAN B-CELL LINES, BUT NOT SHARED BY HUMAN T-CELL LINES (MOLT AND SOMMER) YUMIKO TAKADA,* A. TAKADA* AND J. MINOWADAt * Springville Laboratories, and t Department of Immunology and Immunochemistry, Roswell Park Memorial Institute, Springville, New York, U.S.A. (Revised 10 December 1974) SUMMARY
Rabbits were immunized with two kinds of human B-cell lines (SOMMER-B, and RPMI number 1788 cells) to get anti-B-cell sera. Sera were absorbed with human red blood cells, human liver, human brain and human T cells (MOLT-4 and SOMMER-T cells). Cytotoxicity of absorbed sera was not only tested against B cells which were used for immunization but also tested against unrelated B cells, including B35M cells derived from Burkitt lymphoma specimens. Results indicated that established human B cells had antigenic determinants which were shared with allogeneic human B cells, but not shared with non-lymphoid cells or tissues, nor with established human T cells. INTRODUCTION Lymphoid cells have been classified into bone marrow-derived cells, B cells and thymusderived cells, T cells. These two populations of lymphocytes carry different antigenic markers. The murine thymus has H-2 antigens, TL antigens (Boyse, Old & Luell, 1963) 0-antigens (Reif & Allen, 1964a, b), Ly antigens (Stuck et al., 1964; Old, Boyse & Stockert, 1965) and other alloantigens such as H-14 (Popp, 1969). On the other hand, B cells bear readily detectable immunoglobulins (Ig) on their surface (Tanigaki et al., 1966; Raff, 1970; Rabellino et al., 1971; Jondal, Holm & Wigzell, 1972). Besides this marker, mouse Blymphocyte antigen, MBLA, which is detected by a rabbit anti-mouse lymphocyte absorbed with thymocytes (Raff, Nase & Mitchison, 1971), PC-l (Takahashi, Old & Boyse, 1970) expressed on plasma cells, and Ly-4 (Snell et al., 1973) have been described to be present on murine B cells. Furthermore, the population of B lymphocytes seems to be heterogeneous (Playfair & Purves, 1971). Laskov, Rabinowitz & Schlesinger (1973) suggested that there might be three different antigens, B 1, B2 and My, on cells of B-cell lineage and that antigenbinding precursor cells bore Bi antigen and Ig, while antibody-producing cells might acquire B2 and My in addition to BI, but lose 1g. Compared with the mouse system, specific Correspondence: Dr Yumiko Takada, Second Department of Physiology, Hamamatsu University, School of Medicine, Hamamatsu City, Japan.
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antigens on human T and B lymphocytes were not so clearly demonstrated. Recently Ablin, Baird & Morris (1972), Ablin & Morris (1973) and Smith et al. (1973) have shown that human T cells have specific antigens. Although human B cells have surface Ig, receptors for complement (Nussenzweig et al., 1971; Shevach et al., 1973) and for antigenantibody complexes (Shevach et al., 1973; Dickler & Kunkel, 1972), it has not been shown if human B lymphocytes have specific antigens not shared with T lymphocytes. One of the difficulties of showing B cell-specific antigens is the absence of the pure population of T cells. The foetal thymus tissue is still contaminated with circulating blood cells and other epithelial cells which might have B-cell characteristics. The other is that of eliminating the possible involvement of antigens specific to individuals in making heteroantibodies. Recently, Minowada, Ohnuma & Moore (1972), and Minowada & Moore (1974) have succeeded in establishing permanent cell lines (MOLT and SOMMER) from the peripheral blood of patients with acute lymphoblastic leukaemia. MOLT cells did not produce Ig, and did not have receptors for Ig or the Fc portion of Ig, but bound sheep red blood cells (SRBC) upon exposure. The formation of spontaneous rosettes with SRBC is considered to be a marker for T cells (Jondal et al., 1972; Wybran, Fudenberg & Sleisinger, 1971; Wybran, Carr & Fudenberg, 1972). The original culture of SOMMER cells gave rise to two populations. One, called SOMMER-B cells showed B-cell characteristics, and had forty-six chromosomes without markers (Huang et al., 1974). The other, called SOMMER-T cells, had T-cell characteristics, formed SRBC rosettes and karyologically had two typical markers (Huang et al., 1974). Since SOMMER-T and SOMMER-B cells were obtained from the same person, differences in antigenic markers between these two kinds of cells must be due to the difference of the origin of these cells, bone marrow-derived or thymusderived, thus being organ-specific. In the present studies, we prepared antisera in rabbits against two kinds of B cells, RPMI number 1788 and SOMMER-B cells. Cytotoxicity against various B cells was tested by using these antisera. Results indicate that there might be antigenic determinants on B cells which are shared with allogeneic B cells but not shared with autologous or allogeneic T cells. MATERIALS AND METHODS Cell lines MOLT-4 cells and SOMMER-T cells, both T-cell lines, were obtained as reported previously (Minowada et al., 1972, 1974). SOMMER-B cells were obtained from the person with acute lymphoblastic leukaemia, from whom SOMMER-T cells were also obtained. RPMI number 1788 cells were from healthy male adults, and B35M cells, from Burkitt lymphoma specimen. They were grown at 370C in a suspension culture using the nutrient medium RPMI number 1640 supplemented with 10% (v/v) heat-inactivated foetal calf serum. Immunization SOMMER-B cells (I 5 x 107) or RPMI number 1788 cells (I 5 x 107) was mixed with 1 ml of Freund's complete adjuvant (FCA) and 0 5 ml was injected into bilateral footpads, the rest (1 ml) being injected into a neck muscle. One week later, 2 x 107 cells were injected intravenously, and another week later 1 2 x 107 cells with FCA were injected into footpads and neck muscle. Blood was drawn 2 weeks after the last injection. After separation, serum was inactivated by heating at 56°C for 30 min.
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Cytotoxicity Cytotoxicity tests were performed using the same method as described previously (Takada et al., 1974). Briefly, 0.1 ml of cell suspension containing 2 x 106 viable cells per millilitre of IX Eagle's solution was mixed with 0 1 ml of either antiserum or normal rabbit serum (NRS) and the mixture was kept at 20'C for 30 min. Then 0- 1 ml of rabbit complement was added to each tube and incubated at 370C for 30 min. After incubation viability was tested by trypan blue exclusion tests. Viability (percentage live cells) was calculated by dividing the percentage of viable cells in a tube with antiserum by the percentage of viable cells in a tube with NRS, multiplied by 100. The percentage of dead cells was obtained by subtracting the percentage of live cells from 100.
Absorption One gram of human brain (cortex, mainly grey matter) or human liver, provided by Dr U. Kim of our Institute, was homogenized in 3 ml of saline in a Potter homogenizer. The homogenate was centrifuged for 20 min at 3000 rev/min. The supernatant was drawn and 3 ml of saline was added to the residue and mixed vigorously before the mixture was centrifuged again. After the second centrifugation, the pellet was mixed with 3 ml of antiserum.
Absorption of anti-RPMI number 1788 serum Three millilitres of the serum was absorbed with human liver four times, then 3 ml of the liver-absorbed serum was mixed with 1 x 107 live MOLT-4 cells. After leaving the mixture for 30 min at 20'C, it was centrifuged for 20 min at 3000 rev/min. The supernatant was either used for cytotoxicity tests or further mixed with 1 x 107 live MOLT-4 cells. To know the possible involvement of anti-surface Ig antibody in cytotoxicity studies, twice liver-absorbed serum (1 ml) was mixed with 1 ml of human serum, and kept at 20'C for 30 min with constant gentle shaking. Then the mixture was centrifuged at 3000 rev/min for 20 min to obtain the supernatant.
Absorption of anti-SOMMER-B serum Three millilitres of anti-SOMMER-B serum was added to 3 ml of packed human red blood cells and mixed well. The mixture was kept at 20°C for 30 min, then centrifuged for 20 min at 3000 rev/min. The supernatant was again mixed with 3 ml of packed human red blood cells, and incubation and centrifugation were repeated. The supernatant thus obtained further absorbed twice with human liver (1 g of liver per 3 ml of serum) and four times with human brain (1 g brain per 3 ml of serum). The absorbed serum (3 ml) was mixed with 1 x 107 SOMMER-T cells, and kept for 30 min at 20°C. Then the mixture was centrifuged for 20 min at 3000 rev/min to get the supernatant, which was used either for cytotoxicity tests or further absorption with SOMMER-T cells.
Immunofluorescence Rabbit anti-RPMI number 1788 or SOMMER-B serum (3 ml) was absorbed four times with liver and with 1 x 107 SOMMER-T cells. Sera were then centrifuged at 15,000 rev/min for 1 hour by using T1-50 rotor of Beckman's ultracentrifuge (L-4). The supernatant was mixed with either RPMI number 1788, SOMMER-B cells, or human peripheral lymphocytes fractionated by Ficoll-Isopaque. 0 3 ml of antiserum was mixed with 5 x 106 cells and kept at 37°C for 30 min, washed three times with RPMI 1640 media. Cell pellets were then added
Yumiko Takada, A. Takada and J. Minowada
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with each 0-1 ml of 10-times diluted goat-anti-rabbit Ig conjugated with fluorescein (FITC) (Behring Diagnostics, New Jersey, U.S.A.). The mixture was kept at 20'C for 30 min, and washed thoroughly with media. A drop of cell suspension thus obtained was transferred to a slide glass and fluorescing cells were photographed by using Leitz fluorescence microscope and Kodak Ektachrome high speed film (ASA164). RESULTS Cytotoxicity against B35M cells of antiserum to RPMI number 1788 Anti-RPMI number 1788 serum was absorbed with human liver quite extensively (four times). Then it was absorbed twice with MOLT-4 cells. Since RPMI number 1788 cells have surface immunoglobulins, the injection of these cells into rabbit might have resulted in the production of anti-Ig antibody which would kill cells in the presence of complement due to antibody-mediated cytotoxicity. To exclude the possibility of the involvement of antibodymediated cytotoxicity, twice liver-absorbed serum was mixed with an equal volume of human serum, kept for 30 min at room temperature, and centrifuged for 20 min at 3000 rev/min. Since antiserum was diluted twice by the addition of normal serum, the adjustment was made in drawing the curve in Fig. 1. Cytotoxicity of anti-RPMI number 1788 serum against B35M cells was absorbed to some extent with human liver and to a small extent with MOLT-4 cells. Further absorption with MOLT-4 cells did not change cytotoxicity of the serum any more. Antisurface Ig antibody did not have any effect on the cytotoxicity in the present
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Antigenic determinants on T- and B-cell lines
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Cytotoxicity against SOMMER-B cells of antiserum to RPMI number 1788 cells The absorption schedule was the same as used in the previous experiment. Target cells were SOMMER-B cells. The results of cytotoxicity tests are shown in Fig. 2. Cytotoxicity of anti-RPMI number 1788 serum against SOMMER-B cells was absorbed with human liver to some extent. The further absorption with MOLT-4 cells resulted in a small decrease in cytotoxicity, but additional absorption of the serum with MOLT-4 cells did not change the cytotoxic activity of the serum. The data shown in Figs 1 and 2 indicate that cytotoxicity of anti-B-cell serum against two kinds of allogeneic B-cells was not completely absorbed with extensive absorptions with human liver and human T-cell line (MOLT-4). 90S A
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Cytotoxicity against B35M cells of antiserum to SOMMER-B cells Anti-SOMMER-B serum was sequentially absorbed with human red blood cells (twice), human liver (twice), human brain (four times), and then SOMMER-T cells (twice). Results shown in Fig. 3 indicate that cytotoxicity of anti-SOMMER-B serum against B35M cells was slightly absorbed with human red blood cells and human liver, then to some extent with human brain. Lines E and F in Fig. 3 are almost identical, indicating that no antibody against B35M cells was removed with human brain any more. Further absorption of the serum with SOMMER-T cells once or twice did not change the cytotoxicity of antiSOMMER-B-cell serum against B35M cells. Cytotoxicity against SOMMER-B cells of antiserum to SOMMER-B cells Antiserum against SOMMER-B cells was sequentially absorbed with human red blood
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cells (twice), human liver (twice), human brain (four times) and SOMMER-T cells (four times). Target cells of cytotoxicity tests were SOMMER-B cells. Fig. 4 shows the results. Anti-SOMMER-B-cell activity was somewhat absorbed with human red blood cells (Lines B and C). Further absorption with human liver decreased cytotoxic activity of the serum (Lines D and E). Absorption of the serum with human brain further removed antiSOMMER-B activity from the serum (Lines F), but four times absorption did not result in any significant decrease in anti-SOMMER-B-cell activity (Line G). Anti-SOMMER-Bcell serum which was absorbed with human red blood cells, human liver, and human brain, still had cytotoxicity against SOMMER-B cells (Line H), and further absorption with SOMMER-T cells did not change the cytotoxic activity any more. Cytotoxicity against RPMI number 1788 cells of antiserum to RPMI number 1788 absorbed with RPMI number 1788 cells Although we have shown that anti-B-cell activity of serum could not be abolished by the absorption with human T-cell lines, it seems important to show that anti-B-cell activity would be abolished by the absorption with human B cells, especially by using homologous materials. Fig. 5 shows the results of experiments in which rabbit anti-RPMI number 1788 serum was absorbed four times with human liver and then with 1 x 107 live RPMI number 1788 cells. One absorption with RPMI number 1788 cells of the liver-absorbed serum resulted in the presence of some activity remaining only up to four times dilution. Absorption three times with RPMI number 1788 cells resulted in complete abolishment of cytotoxicity against RPMI number 1788 cells. These results indicate that the cytotoxicity
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remaining after the absorptions with RBC, liver, brain and T cells shown in the preceding results was due to the antibody against B cells, not due to the presence of cytotoxic substances unrelated to antibody.
Immunofluorescence of B cells with anti-B-cell antibody coupled with FITC-conjugated goat anti-rabbit immunoglobulin Although it is shown that the cytotoxicity against B cells was due to the presence of anti-B-cell antibodies, it may be important to show directly the attachment of anti-B-cell antibodies on B cells. Anti-SOMMER-B or RPMI 1788 serum was mixed with either SOMMER-B, RPMI number 1788, or human peripheral lymphocytes. Fig. 6a and b show the fluorescence of RPMI number 1788 and SOMMER-B cells stained with anti-RPMI number 1788 serum and FITC-conjugated goat anti-rabbit immunoglobulin, respectively. As shown here, cells show fluorescence, but capping or ring formation typical of live lymphocytes is not shown in many cells. Since established cultured B cells are not roundshaped even under regular microscopic observation in contrast to fresh peripheral lymphocytes, and have many protrusions, antigen-antibody complexes may not smoothly migrate to the pole. Fluorescence on cultured cells looks patchy, thus indicating some migration of
FIG. 6(a). Immunofluorescence of RPMI number 1788 cells. Cells were incubated with antiRPMI number 1788 serum absorbed four times with human liver and SOMMER-T cells, then mixed with goat anti-rabbit immunoglobulin conjugated with FITC. (b) Immunofluorescence of SOMMER-B cells. Cells were incubated with anti-RPML number 1788 serum. (c) Immunofluorescence of human peripheral lymphocytes. Peripheral lymphocytes were incubated with anti-SOMMER-B-cell serum absorbed four times with human liver and SOMMER-T cells, then mixed with FITC-conjugated goat anti-rabbit 1g.
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antigen-antibody complexes on the membrane. On the other hand, Fig. 6c shows the fluorescence on the membrane of fresh human peripheral lymphocytes. Typical ring formation is seen on lymphocytes. DISCUSSION Much attention has been given to the classification of human lymphocytes into two categories, B and T cells. Since Wilson & Nossal (1971) suggested that acute lymphoblastic leukaemia might be a leukaemia of T cells and chronic lymphatic leukaemia might be that of B cells, many investigators have studied the possible role of T and B cells, in leukaemogenesis. Recently Minowada et al. (1972, 1974) succeeded in the establishment of two lymphoid cell lines (called MOLT-4 and SOMMER) from the peripheral blood of patients of acute lymphoblastic leukaemia. MOLT-4 cells did not have immunoglobulins on the surface, nor receptors for antigen-antibody complex, but bound SRBC to form rosettes. Electronmicroscopically, MOLT-4 cells had smooth surface characteristics of T-cells (Polliack et al., 1973). The original culture of SOMMER cells gave rise to two populations. One called SOMMER-B cells, showed B-cell characteristics and had forty-six chromosomes without markers (Huang et al., 1974). The other, called SOMMER-T cells, had T-cell characteristics, formed SRBC rosettes and karyologically had two typical markers. The isolation of SOMMER-B and -T cells provided a unique opportunity to study antigens specific to B cells but not shared with T cells. Furthermore, since both SOMMER-B and -T cells were established from the same person, alloantigens such as HL-A must be identical and the difference in antigenic determinants between B and T cells can be considered to be due to the difference in the origins of these cells. In the present experiment, we injected B cells established from a normal person (RPMI number 1788) or SOMMER-B cells into rabbits and obtained respective antisera. Cytotoxic tests of anti-RPMI number 1788 serum were performed against SOMMER-B cells and B35M cells (obtained from a patient of Burkitt lymphoma) in order to study antigenic determinants common to established B cells. Figs 1 and 2 indicate that after extensive absorptions of antiRPMI number 1788 serum with human liver there still remained antibody against both SOMMER-B and B35M cells. Further absorptions with MOLT-4 cells resulted in slight, if any, decrease in cytotoxic activity. The cytotoxicity was not mediated by anti-surface Ig antibody because human serum containing Ig did not remove anti-B-cell activity. Therefore, antiserum against RPMI number 1788 after absorptions contained antibody against two kinds of B cells (SOMMER-B and B35M) but not against human T cells. Then we used anti-SOMMER-B-cell serum against B35M and SOMMER-B itself. Figs 3 and 4 indicate the results of experiments in which the antiserum was extensively absorbed with human red blood cells, human liver, human brain and SOMMER-T cells, and then cytotoxicity was tested against B35M and SOMMER-B cells. It is shown that after exhaustive absorptions with non-B cells or non-lymphoid tissues there still remained cytotoxicity not only against allogeneic B cells (B35M) but also against autologous B cells (SOMMER-B). Fig. 4 indicates that these surface antigens on SOMMER-B cells were not expressed on SOMMER-T cells because of the failure of absorption with SOMMER-T cells. Since SOMMER-B and -T cells were obtained from the same person, it may be concluded that SOMMER-B cells have antigenic determinants specific to and common among established B cells, but not shared with established human T cells. Some of the antigenic
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Yumiko Takada, A. Takada and J. Minowada
determinants on SOMMER-B cells might be leukaemia-specific, but those shared with other established B cells must be B cell-specific because antibody against SOMMER-B cells after absorptions was not only cytotoxic to cells established from patients with leukaemia or lymphoma (B35M) but also to cells established from a normal person (RPMI number 1788, data not shown). Also antibody against RPMI number 1788 cells was cytotoxic to SOMMER-B cells after exhaustive absorptions. As to the absorption experiments utilizing human brain, it has been shown (Takada et al., 1974) that human brain shares antigenic determinants with established human T cells (MOLT-4). The present experiments showed that only small extent of cytotoxicity against B cells was removed after exhaustive absorptions of anti-B sera with human brain. This observation may indicate that established human B cells have small amounts of antigenic determinants shared with human brain, and that these determinants should be human-specific, since human liver could absorb as well (Figs 1 and 2). Recently, several Ir-associated (Ia) mouse lymphocyte alloantigens have been demonstrated (Sachs & Cone, 1973; Lozner et al., 1974). Ia antigens were shown to be present only on B cells (called fi antigens). The present B cell-specific antigens may be ft equivalents in humans. Although we showed that B cell-specific antigens were common among allogeneic B cells, it does not imply that they are identical. Since antibodies were formed in rabbits, rabbits may not have recognized subtle differences in the structures of the present antigens between individuals. Rabbits, for example, hardly recognize the differences between different HL-A antigens. Purified anti-B-cell antibody may serve as a tool to study the biological functions of the B cell-specific antigens. Recent reports concerning the relationship between Ia and Fc receptor may be extremely interesting in this respect (Dickler & Sachs, 1974). We are currently working on the possible link between B cell-specific antigen and Fc receptor on cultured B cells. ACKNOWLEDGMENTS
Excellent technical help by Harry Babbitt is greatly appreciated. We acknowledge encouragement by Dr Julian L. Ambrus, Director of the Springville Laboratories, throughout the present work. SOMMER cells were provided by Dr G. E. Moore, Denver, Colorado, U.S.A. This work was supported in part by PHS grant number CA 14413 from the National Cancer Institute, number AI-08899 from the National Institute of Allergy and Infectious Disease, and G.R.S.G. number RR-05648-08. REFERENCES ABLIN, R.J., BAIRD, W.M. & MORRIS, A.J. (1972) Tissue and species-specific antibodies in antithymocyte globulin. Transplantation, 13, 306. ABLIN, R.J. & MORRIS, A.J. (1973) Thymus-specific antigens on human thymocytes and on thymicderived lymphocytes. Transplantation, 15, 415. BoYSE, E.A., OLD, L.J. & LUELL, S. (1963) Antigenic properties of experiment leukemias. 1I. Immunological studies in vivo with C57B1/6 radiation-induced leukemias. J. nat. Cancer Inst. 31, 987. DICKLER, H.B. & KUNKEL, H.G. (1972) Interaction of aggregated y-globulin with B lymphocytes. J. exp. Med. 136, 191. DICKLER, H.B. & SACHS, D.H. (1974) Evidence for identity or close association of the Fc receptor of B lymphocytes and alloantigens determined by the Ir region of the H-2 complex. J. exp. Med. 140, 779. HUANG, C.C., Hou, Y., WOODS, L.K., MOORE, G.E. & MINOWADA, J. (1974) Cytogenetic study of human
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