The Distribution of Rabbit Anti-Mouse Thymus Globulin After Injection into Mice ∗

Abstract

The immunosuppressive action of heterologous antilymphoid sera (ALS) has been well established in the laboratory in various species of animal, and prolongation of survival of allografts of skin (1), kidney (2), and liver (3) and xenografts of tumor (4) has been achieved with their use. Such observations have led to the clinical trial of ALS as an adjunct to more conventional immunosuppressive therapy in the grafting of organs in man (5, 6). Although theories have been formulated as to the mechanism of the immunosuppressive action (7), no definitive explanation has been found. We considered that a study of the distribution of heterologous antilymphoid globulin in vivo after intravenous injection might be contributory in this regard and the distributions of radio labeled gamma globulin and IgG prepared from heterologous antithymus serum were investigated after their parenteral injection. Presented here is a report of the results of these experiments.

Materials and Methods. Rabbit anti-mouse thymus sera (RAMTS) were prepared by injecting intravenously each of three New Zealand rabbits (average weight 2 kg) with suspensions of 1 × 10⁹ viable thymus cells obtained from neonatal mice. Each rabbit was injected twice at a 2-weekly interval and bled 1 week after the second injection. The sera were pooled and heated at 56° for 30 min. The initial titer of lymphocytotoxicity in vitro for murine lymphocytes was 1:4096 when tested by the method described by Terasaki and associates (8) and titrated to less than 10% kill. After repeated absorptions with murine red cells (1 vol erythrocytes: 4 vol serum) to remove in vitro he-magglutinating and hemolyzing activity, and a single absorption with a homogenate of murine kidney (1 vol kidney homogenate: 4 vol serum), the gamma globulin (ATG) was precipitated with 40% ammonium sulfate.

In preparation for labeling with radioactive iodine, one portion of the precipitate was reconstituted in sodium phosphate buffer, 0.05 M, pH 7.0, to the original volume of the serum and then dialyzed against the same buffer. At this stage the lymphocytotoxic titer of the globulin (ATG) was 1:4096 when tested in vitro against murine lymphocytes from the peripheral blood and titrated to less than 10% kill. After reconstitution in 0.1 M potassium phosphate buffer (pH 8.0) another portion of the precipitate was dialyzed against this same buffer prior to the separation of the IgG (ATIgG) by column chromatography using diethylaminoethanol (DEAE) cellulose (9). The immunoelectrophoretic pattern of this fraction showed the presence of a single band of precipitation located in the position normally ascribed to IgG.