REDUCED LIFE-SPAN OF ANERGIC SELF-REACTIVE B-CELLS IN A DOUBLE-TRANSGENIC MODEL

The life span of anergic self-reactive B cells was determined by 5-bromo-2'-deoxyuridine (BrdU) loading of tolerant double-transgenic (Dbl-Tg) mice produced by mating hen egg lysozyme (HEL)-transgenic mice with the corresponding immunoglobulin-transgenic (Ig-Tg) mice, the B cells of which express anti-HEL IgM and IgD. B cells from Dbl-Tg mice, despite being exposed to soluble antigen throughout their development, are not deleted, but persist in an anergic state. As a prelude to studying the life span of these anergic B cells, BrdU was administered to nontransgenic mice; B cells from the bone marrow, spleen, and lymph nodes displayed distinct kinetic profiles based on reciprocal expression of the B220 isoform of CD45 and heat-stable antigen (HSA). Thus, immature B220(lo)/HSA(hi) B cells incorporated BrdU rapidly suggesting recent generation from dividing precursors, whereas uptake by B cells expressing the mature B220(hi)/HSA(lo) phenotype was significantly slower, consistent with a longer life span. Such gating allowed analysis to be directed at the stable mature B cell population in transgenic mice. Comparison of BrdU uptake in Ig- and Dbl-Tg mice indicated that B cells from Dbl-Tg mice were renewed at a much higher rate (50% renewal times of 0.64 vs. 3.4 wk for total B cells, and 1.2 vs. 5.0 wk for mature B200(hi)/HSA(lo) cells from Dbl- and Ig-transgenic mice, respectively). This difference was even more marked when analysis in Dbl-Tg mice was restricted to HEL-binding cells, which had a 50% renewal time of 3-4 d compared with 4-5 wk for non-HEL-binding B cells. While the proportion of B cells in cell cycle, and the rate of entry of newly generated B cells into the spleen of Ig- and Dbl-Tg mice, were similar, B cell numbers were reduced in Dbl-Tg mice. It was therefore concluded that anergic B cells have a markedly decreased life span in the periphery. According to studies in radiation chimeras produced by reconstituting HEL-transgenic recipients expressing different serum levels of antigen with Ig-Tg bone marrow, the reduced life span of anergic B cells was associated with the anergic state per se, the serum concentration of HEL being important only in attaining the critical threshold necessary for tolerance induction. B cells rendered tolerant by exposure to soluble self-antigen therefore survive for a relatively short period in an anergic state once they have reached peripheral lymphoid tissue and fail to enter the long-lived compartment. These findings have implications for the maintenance of self-tolerance and induction of autoimmunity in both B cell and T cell repertoires and suggest that the distinction between B cell anergy and deletion is more relative than absolute.