Table 3 Pre-BCR selection and N-additions in B-2 cells

The development of B cells in the BM occurs in two main sequential steps of immunoglobulin gene rearrangement. VDJ recombination occurs first in the heavy-chain locus (Ig_H) at the early pro-B cell stage by first recombining the heavy chain diversity (D) to the joining (J) gene segment. Later, the variable (V) to DJ segment is rearranged at the late pro-B cell stage. The resultant Ig_H pairs with a surrogate light chain (SLC) which is composed by VpreB and lambda5 proteins, forming the pre BCR. At this moment, successful signaling from the pre-BCR is required for the proliferative burst of the pre-B cell and subsequent rearrangement of the light chain (Ig_K) V to J. The cytokine IL-7 is key in directing the sequential ordering of these recombination events. Through IL-7R and its downstream signaling STAT5, IL-7 actively inhibits Igk rearrangement [39] (Fig. 1G). In B-2 cells, the pre-BCR is an additional mechanism of negative selection for self-reactive Ig_H chains, since the SLC pairs poorly with autoreactive IgH [40]. During B-2 cell lymphopoiesis in the adult BM, any B cell that generates self-reactive BCR will undergo a process of negative selection which culminates in further V(D)J recombination or, ultimately, cell death by apoptosis [42]

V(D)J recombination requires DNA strand breaks. Before the DNA ends are rejoined, non-templated (N)-nucleotides are added by the enzyme terminal deoxynucleotidyl transferase (TdT) [43]. TdT is an unusual DNA polymerase that catalyzes the template-independent addition of random nucleotides being expressed in the pro-B cell stage. However, TdT is not expressed during the fetal stage in mice, thus, little to no N-addition is found in FL-derived B cells [44], which is the case of B-1 cells. In humans, TdT is expressed during the fetal phase, and both fetal and adult human B cells produce Ig with N-additions [45]. However, it has been demonstrated that the number of N-additions and CDR3-H3 length in B cells from preterm and term infants are shorter than those in adults [46]