The preparation and testing of horse antidog and antihuman antilymphoid plasma or serum and its protein fractions. (1967)

Horses were immunized against dog or human lymphoid tissue with multiple subcutaneous injections of fresh lymph nodes, thymus, and spleen. The equine antibody response, as gauged by the titer of leukoagglutinating antibodies, was small when the immunizing dose of donor cells ranged from 0.5 to 19 billion. When this dose was increased to as much as 200 billion spleen cells, the titers rose rapidly to as high as 1:16,384. With in vitro testing, the antibody tended to agglutinate equally the white cells present in white cell pack and pure lymphocyte suspensions obtained from the thoracic duct. A weak cross species agglutination was observed, indicating a lack of absolute species specificity. Furthermore, the ability to agglutinate white cells of different members of the lymphoid donor species was variable, suggesting a degree of individual specificity. An undesired collateral response was a rise in the horse hemagglutinin titer.

Absorption of the immune horse serum with the red cells or serum from the species that had donated the lymphoid tissue for immunization did not alter the antiwhite cell titer. In contrast, repeated absorption with either kidney or liver resulted in a titer loss of approximately 90 per cent. This finding indicates that most of the antigens in the lymphocyte are represented in other tissues, and that the antiserums are capable of reacting with other than lymphoid tissues.

Fractionation of the antilymphoid antibody was carried out with ammonium sulfate precipitation. The most effective concentration was at 0.4 saturation, which when done twice allowed recovery of approximately half of the antibody with a minimum of contamination with extraneous horse protein. Further studies with column chromatography, electrophoresis, and immunoelectrophoresis demonstrated the presence of agglutinating antibodies in the gamma G, T equine, and beta globulin fractions.

Various antilymphoid products were tested in vivo. In dogs, unabsorbed antilymphoid plasma, antilymphoid serum which was partially absorbed with red cells, antilymphoid serum which was completely absorbed with red cells, serum, liver, or kidney, and the raw globulin precipitated from absorbed antiserum all had a lymphopenic effect. With the progressive stages of refinement, the morbidity from therapy was reduced. Inasmuch as immune horse globulin was well tolerated in dogs, a comparable product was developed and used for 7 patients who were prospective recipients of renal homografts. In both dogs and man, a low titer response of precipitin antibodies to normal horse serum was documented.

In the lymphoid tissues of the treated animals small lymphocytes were replaced by large and medium-sized proliferating cells with pyroninophilic cytoplasm, and germinal center formation was common. These findings are compatible with Levey and Medawar's hypothesis that at least one action of antilymphoid agents is to stimulate transformation of lymphocytes and that its immunosuppressive effect is not dependent upon lymphocyte killing.

Although changes in blood urea nitrogen could be produced only by giving antilymphoid serum intravenously, there was a high incidence of histologic renal damage with various antilymphoid agents after all routes of administration. The renal injury consisted of dense accumulations of horse and dog gamma globulin, together with complement, along the subepithelial aspects of the renal glomerular capillary basement membranes. Once developed, these deposits persisted for at least 28 days after injections were stopped. The incidence of these lesions was highest when the antilymphoid serum was given intravenously. Prior absorption of the serum with dog kidney and liver did not prevent the renal deposits from occurring.