Summary Hybridomas secreting anti‐Thy‐1 antibodies were produced by fusing cells of the mouse myeloma line P3‐NSI/1‐Ag4‐1 (NS‐1) with spleen cells from AKR/J mice immunized with C3H/Di thymus cells and by subsequent growth in tissue culture and selection of the hybrid cells. Two permanent hybridomas, 1B5 and 1aG4/C5, secreting antibodies of IgG3 subclass were isolated by repeated cloning of cells by dilution and in soft agar. Growth of the hybrid cell colonies depended on the presence of feeder cells; spleen cells at 1‐2 X 106/ml were most effective, then thymus cells at 1‐4 X 106/ml and peritoneal cells at a concentration 1‐2 orders of magnitude lower. The two hybridomas were grown in vitro or in vivo and their products were further analysed. In tissue culture in serum‐free medium under the optimum conditions the supernatant from hybridoma 1B5 contained 0.07 mg/ml of antibodies and that from hybridoma 1aG4/C5 had 0.26 mg/ml of antibodies, whereas ascites 1B5 contained 3.6 mg/ml and ascites 1aG4/C5 4.4 mg/ml of antibodies. A very low electrophoretic mobility of both antibodies facilitated their isolation. The specificity of the antibodies was tested in the cytotoxicity assay in the presence of complement and by the binding of isotopically labelled antibodies to thymus cells from A/Ph mice and other Thy‐1.2+ strains and A. Thy‐1.1 and AKR/J mice. Antibodies of clone 1aG4/C5 were specific for Thy‐1.2+ cells, whereas antobodies of clone 1B5 at higher concentrations also reacted with Thy‐1.1+ cells from the thymus and lymph nodes. Both antibodies killed more than 95% thymus cells and 60‐70% lymph node cells in the cytotoxicity assay. The specificity of antibodies for T lymphocytes was confirmed in the functional test in which the antibodies eliminated the response of spleen cells to Concanavalin A but did not affect the response to lipopolysaccharide in the presence of complement.

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Significant inhibition of spermatogenesis and appearance of antibodies against spermatogenic cells identified by cytotoxicity and immunofluorescence reactions were observed in mice of inbred strains 129/Sv and BALB/c and in albino guinea pigs after syngeneic, allogeneic, and xenogeneic immunization with mouse F9 embryonic carcinoma cells and Freund's complete adjuvant. A similar syngeneic immunization with PYS-2 cells was ineffective. Appropriate absorption experiments confirmed the similarity between the antigens ofF9 and spermatogenic cells and the absence of such a similarity with antigens ofPYS-2 cells. These results support the hypothesis that the oncofetal F9 antigens represent spermatogenic differentiation antigens and thus play an essential role in spermatogenic cell differentiation.The F9 cell line is a stable line of mouse embryonic carcinoma cells (ECC). F9 cells carry surface cell antigens (called F9) that are expressed on all ECC tested, as well as on male germ cells and cleavage stage embryos (1). After day 10 of development, the F9 antigens are detectable only on spermatogenic cells (2-4). On the basis of these findings, the F9 antigens may be included among the so-called differentiation antigens (5). In contrast to other differentiation antigens, however, the antigens of germ cells do not cause autotolerance because they are sequestered from the circulatory elements of the immune system by the blood-testis barrier. One of several experimental approaches to a test of the role of F9 antigens would be the induction of aspermatogenesis by the immunization of adult animals with F9 antigens. We report here the results ofsyngeneic, allogeneic, and xenogeneic immunization of adult mice and guinea pigs with F9 cells and Freund's complete adjuvant (FCA); the results show inhibition of spermatogenesis and appearance of specific immunofluorescent and cytotoxic antibodies to F9 and to spermatogenic cells. (GIBCO) in an atmosphere of 5% CO2 (1). MATERIALS AND METHODS AnimalsImmunization Procedures. Six adult 129/Sv males were injected with three doses ofF9 cells (having received 100 Gy from an x-ray source) in 0.1 ml of phosphate-buffered saline plus 0.1 ml of FCA (two doses of 1 x 107 F9 cells given 2 weeks apart, the third dose of 6.5 x 106 cells 6 weeks later). The immunizing material was injected both into the hind foot pads and subcutaneously in the region of the axillary lymph nodes. The mice were bled and their testes were removed and weighed 4 weeks after the last injection. Similarly, five adult 129/Sv males were immunized with PYS-2 cells plus FCA. A group of 10 untreated males served as controls. Solid tumors were induced with F9 cells in the BALB/c males. Cell suspensions from the irradiated (100 Gy from an x-ray source) tumor tissue were used for allogeneic immunization of 22 adult BALB/c males with three or six intracutaneous injections of 6 X 107 ECC plus FCA each in 2-week intervals. Unilateral castration was performed in five males 8 days after the third injection, and in an additio...