To determine the region of human tumor necrosis factor-alpha (TNF-alpha), essential for cytotoxic activity against mouse L-M cells, single amino-acid-substituted TNF-alpha mutant proteins (muteins) were produced in Escherichia coli by protein engineering techniques. An expression plasmid for TNF-alpha was mutagenized by passage through an E. coli mutD5 mutator strain and by oligonucleotide-directed mutagenesis. Approximately 100 single amino-acid-substituted TNF-alpha muteins were produced and assayed for cytotoxic activity. The cytotoxic activities of purified TNF-alpha muteins, e.g. TNF-31T, -32Y, -82D, -85H, -115L, -141Y, -144K and -146E, were less than 1% of that of parent TNF-alpha. These results indicate that the integrity of at least four distinct regions of the TNF-alpha molecule is required for full biological activity. These regions are designated as follows: region I, from position 30 to 32; region II, from position 82 to 89; region III, from position 115 to 117; region IV, from position 141 to 146. In addition, TNF-141Y could not completely compete with parent TNF-alpha for binding to the receptor. This demonstrates that region IV, and at least aspartic acid at position 141, must be involved in the TNF receptor binding site.

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Tolerance induction with rabbit gamma-globulin was employed as a probe for age-dependent changes in suppressor capacity of SJL lymphoid cells. The tolerant state was assessed by loss of cooperative capacity and by infectious tolerance. The supply of precursor cells was assessed by thymectomy and by treatment with colchicine and cyclophosphamide, which have been reported to eliminate suppressor cells. Thymectomy, 16-18 days before tolerance induction, did not affect antibody response or tolerance inducibility; thymectomy, 33 days before tolerance induction, reduced both antibody response and tolerance inducibility. Colchicine, injected together with aggregate-freed rabbit gamma-globulin, inhibited tolerance induction partially in 35-day-old mice and completely in 106-day-old mice. Colchicine, given to younger mice, thymectomized 17 days before tolerance induction, prevented tolerance induction completely. A low dose of cyclophosphamide interfered with tolerance induction in older, but not in younger mice. A high dose of cyclophosphamide interfered with tolerance induction in thymus cells of younger and older mice. After thymectomy, there was a much more profound interference of a low dose of cyclophosphamide with tolerance induction. Results were discussed in terms of an age-dependent decline of thymus progenitors and of peripheral progenitors of suppressor cells.

Summary A strain survey has been undertaken to examine polymorphism of age‐dependent changes in antigen elimination, immune responsiveness and tolerance inducibility. In young mice of tested strains, other than C57BL/6J, aggregate‐freed mouse Ig was eliminated faster than was the xenogeneic Ig (RGG); C57BL/6J was the only exception. The rate of elimination increased with age in all strains, tested. Antibody formation to RGG decreased in 3 (BALB/cJ, 129/J, DBA/2J) out of the eleven strains tested at 5‐7 and 31‐40 weeks of age; there was a considerable strain variation in the extent of this decrease. Age‐dependent resistance to tolerance induction was observed in a fraction of animals of H‐2 haplotypes, H‐2k and H‐2d.