ALLOGYRAFT SURVIVAL IN MICE

The isolation and partial purification fro:m calf thymus of a soluble lymphocytopoietic factor termed thymosin has been reported.' When administered to normal mice, this factor stimulated lymphocytopoiesis as reflected by an increase ini peripheral lymphoid tissue weight and augmented incorporation of labeled precursors into the DNA and protein of lymphoid cells.'-3 Thymosin also accelerated lymphoid tissue regeneration in mice exposed to whole-body X irradiation,4 restored immunological competence to spleen cells from neonatally thymectomized mice as measured by a graft-versus-host response assay,5 and reduced the incidence of wasting and death in neonatally thymectomized mice.6 In studies with lethally irradiated, thymectomized mice injected with syngeneic bone marrow cells, it has been found that thymosin increased the number of plaque-forming cells per spleen.7 In view of the previously demonstrated immunosuppressive properties of antithymocyte sera,8 9 experiments were designed to examine the effect on skin allograft survival in mice of a thymosin fraction and an antiserum prepared to this soluble fraction of calf thymus. Materials and Methods.-Preparation of thymosin and of antithymosin serum: Thymosin was prepared from calf thymus according to the method of Goldstein, Slater, and White;' the acetone-insoluble, relatively heat-stable fraction (fraction 3) was used in these experiments. The antithymosin serum (ATS) was prepared in male white New Zealand rabbits (2.5-3.5 kg). The thymosin fraction was dissolved in saline in a concentration of 30 mg protein/ml; protein analyses were done by the Lowry method.'0 Two milliliters of the thymosin solution were emulsifiedi with an equal volume of complete Freund's adjuvant (Difco) and injected intradermally in four sites into each-rabbit. After 2 weeks, additional injections of 2 ml of thymosin (60 mg protein) without adjuvant were given subcutaneously to each rabbit every other day for 6 days. The animals were bled 1 week after the last injection. All the sera were pooled and stored at -20?C. Normal rabbit sera (NRS) were obtained from nonsensitized rabbits. Allograft procedures; effect of thymosin: The effect of thymosin on skill allograft survival was studied in 8-9-week-old male B,0D2/Sn mice receiving skin from C57BL/6 mice. The recipients were divided into six groups of 10-15 mice each. One week prior to grafting and throughout the period of the experiment, three experimental groups received daily subcutaneous injections of 0.5, 1.0, and 4.0 mg of thymosin, respectively. Three control groups received either 2.0 mg of calf liver extract (prepared in the same manner as thymosin), 4.0 mg bovine serum albumin (BSA, Sigma Chemical Co., St. Louis, Mo.), or saline. Circular skin grafts (1.0 cm) were performed according to the method of Billingham and Medawar." The grafts were evaluated daily by visual and tactile inspection after the plaster casts were removed on the sixth day after grafting. One week after all first-set skin allografts were rejected, during which time injections of thymosin, calf liver extract, BSA, or saliiie were continued, seconld-set skin allografts were applied to the same mice. Injections were continued until second-set allografts were rejected (plasters were removed on the fifth day after grafting). Total white cell and lymphocyte counts were done weekly on four mice from each group. White cell counts and lymphocyte counts were done daily