B cell biology : the role of Nbs1, KRC and [lamda]5

Antigen receptor variability is mostly generated by the V(D)J recombination
mechanism. During V(D)J recombination hairpinned DNA ends are generated as
intermediate reaction products, which have to be opened before religation can take
place. One complex, which had been suggested to constitute the hairpin opening
activity, was the Mre11/Rad50/Nbs1 (MRN) complex. This complex was known to be
involved in DNA repair and hypothesized to participate in the non homologous end
joining (NHEJ) pathway, the repair mechanism involved in V(D)J recombination.
Additionally, it was shown that Mre11, as part of the ternary MRN complex,
displayed hairpin opening activity in vitro, altogether making (Paull and Gellert,
1999) it conceivable that the complex is involved in V(D)J recombination.
To test whether or not the MRN complex, and accordingly Nbs1, is involved in
hairpin opening at coding ends, we quantitatively and qualitatively analyzed V(D)J
recombination with a cellular recombination assay in cells of healthy controls and
cells of Nijmegen breakage syndrome (NBS) patients, the latter carrying a mutated
Nbs1 gene. No obvious difference was detected, neither in recombination frequencies
nor in the quality of signal joint or coding joint formation. Endogenous CDR3 regions
of the Igκ and the Igλ chain loci of lymphocytes of NBS patients also showed no
differences compared to healthy controls. These results led to the conclusion that
Nbs1, and as a consequence the MRN complex, is not directly involved in the
processing of DNA ends during V(D)J recombination.
KRC, for kappa binding and RSS recognition component, was identified based on its
ability to bind to the RSS (Wu et al., 1993). This RSS binding specificity was shown
to be regulated by phosphorylation and to decrease upon RAG expression. Together
with its mainly lymphoid expression pattern, it made it conceivable that KRC
participates either in the V(D)J recombination mechanism itself or in the regulation of
it. Additionally, KRC had been shown to be involved as a modulator in TNF-α
signaling, suggesting an additional function for KRC in lymphocyte function and
survival.
To investigate the role of KRC in lymphocytes the KRC locus was targeted in order to
analyze the effects of KRC deficiency on V(D)J recombination and lymphoid cells in
general. Heterozygous mice were obtained but no homozygous mice were born due to
very early embryonic lethality caused by the introduced mutation. Closer
characterization of the targeted locus revealed that the integration of the targeting
construct into the locus had ocurred homologously only with one homology arm,
whereas the other arm had integrated randomly. Additionally, a second integration
into an intronic region of the KRC locus was detected. Whether the early embryonic
lethality observed in our line of mice was due to KRC deficiency, based on a possible
involvement of KRC in early zygotic/embryonic signaling, or rather due to the
additional integration event, remains to be determined.
During B cell development the IgH chain locus is the first locus where V(D)J
recombination takes place. The resulting μH chain is subsequently tested for pairing
with the surrogate light (SL) chain and for expression on the cell surface, as part of
the preBCR. Deficiency in λ5, one component of the SL, partially blocks early B cell
development at the preB-I to preB-II stage transition, resulting in B lymphopenia
(Kitamura et al., 1992). The rate of generation of B cells and also the size of the B cell
compartment itself apparently influence, amongst other factors, both the composition
and the size of peripheral B cell compartments. B lymphopenic situations, like in the
case of λ5-/- mice, seem to favour the development of B cell types, considered to be
more innate, like marginal zone B cells (MZB). λ5 deficient mice had not been
analyzed in this respect, so that we decided to characterize the mature B cell
compartments in λ5 deficient mice.
Analysis of the peritoneal B cell compartment of λ5-/- mice showed that conventional
B2 B cells were hardly detectable and the B1b B cell population was reduced,
whereas the B1a B cells were present in normal numbers. The splenic B cell
compartment of λ5 deficient animals was diminished, mainly due to the reduction of
follicular B (FOB) cells. In contrast, MZB cells were present in normal numbers in
λ5-/- mice. BrdU labeling kinetics revealed a slower turnover of FOB as well as MZB
cells in λ5 deficient animals, probably due to lowered influx of newly generated B
cells. Functionally, λ5-/- mice were able to mount both primary and secondary thymusdependent
as well as thymus-independent responses, albeit mostly at reduced levels.
Additionally, we tested the competitive reconstitution ability of λ5-/- cells in mixed
bone marrow chimeras between wild type and λ5-/- bone marrow cells. The results are
preliminary, but suggest that λ5 deficient bone marrow can reconstitute to a limited
extent, even in competition with wild type cells. Surprisingly, λ5-/- deficient cells were
enriched in the MZB compartment, implicating that they have an intrinsic propensity
to develop into MZB cells. In summary, the data show that in λ5 deficient mice B
cells preferentially develop into more innate-like B cell types.