It is well established that immature B cells upon BCR cross-linking

undergo apoptosis. To analyze whether receptor editing could rescue immature B cells from apoptosis, we sorted κ-LC+ λ-LC– CD19+ CD93+ CD23– BAFF-R– (referred to as CD23– BAFF-R–) and κ-LC+ λ-LC– CD19+ CD93+ CD23– BAFF-R+ (referred to as CD23– BAFF-R+) cells and cultured them for 36 h in the presence of anti-κ. FACS analysis on propidium iodide (PI) negative cells revealed that 21.8 and 11.3% of the CD23– selleck BAFF-R–, respectively, CD23– BAFF-R+ expressed a λ-LC (Fig. 3C). To determine the extent of apoptosis in these cultures, we stained the cells with Annexin V and PI. Of the CD23– BAFF-R– cells around 30% were non-apoptotic (Annexin V and PI negative), around 32% were pro-apoptotic (Annexin V positive and PI negative) INCB024360 datasheet and around 35% were apoptotic (Annexin V and PI positive; Fig. 3C).

Of the CD23– BAFF-R+ cells around 12% were non-apoptotic (Annexin V and PI negative), around 15% were pro-apoptotic (Annexin V positive and PI negative) and around 66% were apoptotic (Annexin V and PI positive; Fig. 3C). Thus, the vast majority of κ-LC CD23– BAFF-R– and BAFF-R+ cells undergo apoptosis upon BCR cross-linking by anti-κ. However, the vast majority of κ+ CD23– BAFF-R– and BAFF-R+ cells that upon BCR cross-linking by anti-κ underwent receptor editing and now expressed λ-LC were Annexin V and PI negative (Fig. 3C, CD23– BAFF-R–, CD23– BAFF-R+).

Thus, also in this system immature B cells can be rescued from negative selection by receptor editing. It has been indicated that negative selection of B cells can also occur in the spleen, though the contribution next of this process to the mature B-cell pool is still a matter of debate. Moreover, it is unclear whether also transitional splenic B cells can undergo receptor editing. We, therefore, purified transitional type-1, type-2/3 and follicular B cells from the spleen by cell sorting and analyzed them for LC editing. Thus, κ-LC+ λ-LC– CD19+ CD93+ CD23– CD21– transitional type-1, κ-LC+ λ-LC– CD19+ CD93+ CD23+ CD21+ transitional type-2/3, κ-LC+ λ-LC– CD19+ CD93– CD23+ CD21+ follicular B cells were incubated either in the presence or absence of an anti-κ-LC+ antibody. Minimal spontaneous LC editing could only be observed for the most immature splenic subset, namely T1 cells, where only about 1.3% showed LC editing (Fig. 4A). Incubation with an anti-κ-LC+-antibody increased the occurrence of LC editing within this subset to about 4.4% (Fig. 4B). No signs of editing could be detected for the other two splenic B-cell subsets, independently of the stimulation (Fig. 4A and B). In fact, RAG-2 expression was induced upon anti-κ-LC+ stimulation only within the T1 subset (Fig. 4A and B), suggesting that no further BCR rearrangements are possible beyond this stage of development.