Molecular mechanisms of antibody diversification in human B-cells

Sammanfattning: B-cells undergo several somatic rearrangement/mutational processes during development to diversify their immunoglobulin (Ig) receptors. First, the antigen binding Ig variable (V) region is assembled through V(D)J recombination. Later, B-cells undergo class switch recombination (CSR), which exchanges the constant region and thus Ig isotype and immune effector function. In addition, point mutations are introduced into the Ig V regions, to increase the affinity between the Ig and antigen, by somatic hypermutation (SHM). These processes rely on numerous DNA damage response (DDR) and DNA repair proteins, such as those from the classical non-homologous end-joining (c-NHEJ) pathway. The aim of this thesis was to elucidate the functions of some of these proteins during antibody diversification in human cells. In Paper I, cells from patients with Cornelia de Lange syndrome (CdLS), with mutations in the gene encoding the cohesin loader NIPBL, were studied. The analysis of CSR junctions from these patients revealed an altered repair pattern. It was characterized by reduced direct end-joining and increased microhomology (MH) usage, indicating a shift from c-NHEJ to alternative end-joining (A-EJ). A role for NIPBL in promoting NHEJ was shown to be conserved in yeast. Furthermore, the early recruitment of the DDR factor 53BP1 to DSBs was impaired in the NIPBL-deficient cells, suggesting a mechanism for the involvement of NIPBL/cohesin in NHEJ. In Paper II, the Ig heavy (H) chain and T cell receptor (TCR) β chain V region repertoires were analyzed in CdLS patients by high through-put sequencing methods. The cells from the CdLS patients showed reduced lymphocyte receptor diversity and a skewed V gene usage, which seemed dependent on the location of the genes at the IgH and TCRβ loci. Furthermore, the frequency of SHMs in the IgH V regions was reduced. These results might imply a role for cohesin in the regulation of SHM, as well as in processes ensuring that V genes throughout the Ig locus are used during V(D)J recombination. In Paper III, the CSR junctions were analyzed in individuals deficient in the tumor suppressor BRCA1. The CSR junctions showed several aberrations, including increased MH usage, elevated frequency of intra-switch deletions and unusual insertions containing inversions. Alterations were also observed at the CSR-junctions from patients with defects in the BRCA1-associated proteins BRIP1, BRCA2, CtIP and RNF168. Thus, it seems as BRCA1 together with its interaction partners play an important role in promoting the c-NHEJ pathway during CSR. In Paper IV, CSR junctions were examined in the two only patients described with DNAPKcs-deficiency to date. Furthermore, CSR-junctions from DNA-PKcs-deficient mice and the Ramos B-cell line, transfected with a switch plasmid and treated with DNA-PKcs inhibitor, were studied. The role of DNA-PKcs in CSR has been unclear, but the analysis of CSR junctions from all DNA-PKcs-deficient models revealed a shift to A-EJ, suggesting that CSR, indeed, is affected in DNA-PKcs-deficient cells.