Engagement of the Antigen-Receptor on Immature Murine B Lymphocytes Results in Death by Apoptosis

The Journal of Immunology 1995, 154: 4404 ­ 4413

By Amanda Norvell, Laura Mandik and John B. Monroe

Work by Goodnow and others has shown that when immature B cells develop in an environment containing a "self"-antigen, the B cells are continually deleted from the repertoire. This paper by Monroe demonstrates that the deletion of immature B cells is mediated by apoptosis.

The authors have established an in vitro cell culture system to look at early immature mouse B cells that express surface IgM (sIgM). These B cells are treated with an anti-IgM antibody to cross link sIgM as a model of "self"-antigen binding. While the cells remain intact, many lose large amounts of DNA as measured by flow cytometry, suggesting apoptosis is occurring.

When B cells enriched from the bone marrow are placed in culture, they express surface IgM antibodies (sIgM). Following treatment with anti-IgM, about 20% new apoptotic cells are detected, a significant jump. An analysis determined that these were immature B cells. The effect was rapid and could be detected six hours post cross linking. The number of measurable apoptotic cells continued to increase in over the next 24 hours (the length of the experiment).

Apoptosis was not seen when more mature B cells from the spleen were treated. Thus, the presence of sIgM on the membrane was not sufficient to enable apoptosis to be triggered, the cells needed to be immature B cells. The authors also treated adult bone marrow B cells that not only express sIgM but also IgD. These B cells are likely to be mature memory cells that have returned to bone marrow and would not readily undergo apoptosis or be deleted. When treated with anti-IgM, no reduction in DNA was seen.

The authors also asked if the induction of B cell apoptosis required the synthesis of new proteins. They found that when cells were cultured in the presence of the protein synthesis inhibitor, cyclohexamide, apoptosis was blocked.