Repertoire-based selection into the marginal zone compartment during B cell development.

Marginal zone (MZ) B cells resemble fetally derived B1 B cells in their innate-like rapid responses to bacterial pathogens, but the basis for this is unknown. We report that the MZ is enriched in "fetal-type" B cell receptors lacking N regions (N(-)). Mixed bone marrow (BM) chimeras, made with adult terminal deoxynucleotidyl transferase (TdT)(+/+) and TdT(-/-) donor cells, demonstrate preferential repertoire-based selection of N(-) B cells into the MZ. Reconstitution of irradiated mice with adult TdT(+/+) BM reveals that the MZ can replenish N(-) B cells in adult life via repertoire-based selection and suggest the possibility of a TdT-deficient precursor population in the adult BM. The mixed chimera data also suggest repertoire-based bifurcations into distinct BM and splenic maturation pathways, with mature "recirculating" BM B cells showing a very strong preference for N(+) complementarity-determining region (CDR) 3 compared with follicular B cells. Because the T1 and MZ compartments are both the most enriched for N(-) H-CDR3, we propose a novel direct T1-->MZ pathway and identify a potential T1-MZ precursor intermediate. We demonstrate progressive but discontinuous repertoire-based selection throughout B cell development supporting multiple branchpoints and pathways in B cell development. Multiple differentiation routes leading to MZ development may contribute to the reported functional heterogeneity of the MZ compartment.

Paucity of V-D-D-J rearrangements and VH replacement events in lupus prone and nonautoimmune TdT-/- and TdT+/+ mice.

CDR3 regions containing two D segments, or containing the footprints of V(H) replacement events, have been reported in both mice and humans. However, the 12-23 bp rule for V(D)J recombination predicts that D-D rearrangements, which would occur between 2 recombination signal sequences (RSSs) with 12-bp spacers, should be extremely disfavored, and the cryptic RSS used for V(H) replacement is very inefficient. We have previously shown that newborn mice, which lack TdT due to the late onset of its expression, do not contain any CDR3 with D-D rearrangements. In the present study, we test our hypothesis that most D-D rearrangements are due to fortuitous matching of the second apparent D segment by TdT-introduced N nucleotides. We analyzed 518 sequences from adult MRL/lpr- and C57BL/6 TdT-deficient B cell precursors and found only two examples of CDR3 with D-D rearrangements and one example of a potential V(H) replacement event. We examined rearrangements from pre-B cells, marginal zone B cells, and follicular B cells from mice congenic for the Lbw5 (Sle3/5) lupus susceptibility loci and from other strains of mice and found very few examples of CDR3 with D-D rearrangements. We assayed B progenitor cells, and cells enriched for receptor editing, for DNA breaks at the "cryptic heptamer" but such breaks were rare. We conclude that many examples of apparent D-D rearrangements in the mouse are likely due to N additions that fortuitously match short stretches of D genes and that D-D rearrangements and V(H) replacement are rare occurrences in the mouse.