Definition of a novel cellular constituent of the bone marrow that regulates the response of immature B cells to B cell antigen receptor engagement.

Previously we defined a Thy1(dull) bone marrow-derived cell population that regulated fate decisions by immature B cells after Ag receptor signaling. The microenvironmental signals provided by this cell population were shown to redirect the B cell Ag receptor -induced apoptotic response of immature B cells toward continued recombination-activating gene (RAG) expression and secondary light chain recombination (receptor editing). Neither the identity of the cell responsible for this activity nor its role in immature B cell development in vivo were addressed by these previous studies. Here we show that this protective microenvironmental niche is defined by the presence of a novel Thy1(dull), DX5(pos) cell that can be found in close association with immature B cells in vivo. Depletion of this cell eliminates the anti-apoptotic effect of bone marrow in vitro and leads to a significant decrease in the number and frequency of bone marrow immature B cells in vivo. We propose that, just as the bone marrow environment is essential for the survival and progression of pro-B and pre-B cells through their respective developmental checkpoints, this cellular niche regulates the progression of immature stage B cells through negative selection.

Negative selection of immature B cells by receptor editing or deletion is determined by site of antigen encounter.

Immature B cells that encounter self-antigen are eliminated from the immune repertoire by negative selection. Negative selection has been proposed to take place by two distinct mechanisms: deletion by apoptosis or alteration of the antigen receptor specificity by receptor editing. While convincing evidence exists for each, the two models are inherently contradictory. In this paper, we propose a resolution to this contradiction by demonstrating that the site of first antigen encounter dictates which mechanism of negative selection is utilized. We demonstrate that the bone marrow microenvironment provides signals that block antigen-induced deletion and promote RAG reinduction. In the periphery, the absence of these signals allows the immature B cell to default to apoptosis as a result of BCR engagement.

B cell receptor-induced apoptosis in primary transitional murine B cells: signaling requirements and modulation by T cell help.

Self-reactive immature B cells may be eliminated in the bone marrow (BM) after B cell receptor (BCR) engagement in a process known as negative selection. Immature B cells emigrating from the BM, the so-called transitional cells, remain sensitive to negative selection and are likely to be important targets of tolerance towards peripheral antigens. Transitional cells are deleted through apoptosis after BCR cross-linking in vitro. Using anti-Ig as a surrogate antigen, we determined the signaling requirements for the induction of apoptosis in transitional cells. Treatment with anti-Ig for only 20 min causes most cells to be apoptotic 16 h later. Furthermore, apoptosis of transitional cells is induced with low doses of anti-Ig while mature cell proliferation requires extended culture at 30-fold higher concentrations. For both populations of B cells, total surface Ig expression is equivalent, therefore indicating that the threshold of BCR signaling required to elicit these responses is different. T cell help can modulate B cell tolerance. However, specific help may not be available when apoptosis is triggered by a peripheral antigen. The opportunity to reverse apoptosis of transitional cells is surprisingly long. Even 8 h after anti-Ig treatment, IL-4 or anti-CD40 antibody can block apoptosis. The upper time limit of protection is concurrent with irreversibility of apoptosis as measured by DNA fragmentation. These findings indicate that B cell negative selection is more easily triggered than activation, and that the induction of apoptosis and its reversal by T cell help can be events that occur in distinct microenvironments.