Mechanisms of clonal abortion tolerogenesis. IV. Verification of the hypothesis in living mice
Kay, T.W.; Pike, B.L.; Nossal, G.J.
Journal of Immunology 124(4): 1579-1584
1980
ISSN/ISBN: 0022-1767 PMID: 6154074 Document Number: 155686
Immunologic tolerance to the hapten fluorescein (FLU) can be induced in vivo by much lower molar concentrations of FLU-human .gamma.-globulin (FLU4-HGG) in newborn mice than in adult mice. Euthymic or congenitally athymic mice were given a single i.p. injection of FLU4-HGG. At various intervals thereafter, some mice were given a challenge injection of FLU-polymerized flagellin (FLU-POL) and killed 3-4 days later for a determination of the numbers of FLU-specific directly hemolytic plaque-forming cells (PFC) in their spleens, constituting an in vivo read-out of B [bone marrow-derived] cell tolerance. Other mice were killed after injection of the tolerogen without further challenge, and the capacity of their splenic B cells to form clones of anti-FLU PFC was assessed by a limiting dilution microculture technique, i.e., tolerance was read out in vitro as a diminution in the number of anti-FLU PFC precursors. Serum concentrations of tolerogen were estimated by trace labeling. Tolerance became progressively more profound over the 1st wk after injection of FLU-HGG. The degree of tolerance was routinely tested 7 days after injection of tolerogen. Four noteworthy findings emerged. With both assay systems a higher antigen concentration was required to achieve a given degree of tolerance in adult than in newborn mice. No significant differences were noted between normal and athymic nu/nu mice. Higher concentrations of antigen were required to achieve tolerance as measured by the in vitro clone enumeration assay. With the in vitro read-out, which was considered to give a more accurate picture of functional assessment of responsiveness by measuring reduction in the numbers of individual clonable B cells, the difference in susceptibility between adult and newborn cells was greater than with in vivo assessment of the tolerant state. The serum concentrations operative during tolerance induction were not substantially different from those previously shown to induce clonal abortion of immature B cells in vitro. For example, with the in vitro readout, 50% tolerance was achieved in newborn mice with tolerogen concentrations falling progressively from 0.2 .mu.g/ml (1.2 .times. 10-9 M) to 0.05 .mu.g/ml (3 .times. 10-10 M), compared with 0.08 .mu.g/ml (5 .times. 10-10 M) previously found to be tolerogenic in vitro. The requisite concentrations for equivalent effects on adult cells were 230-fold higher. The results were discussed within the framework of the clonal abortion theory of B lymphocyte tolerance, which they strongly support.