DNA-PKcs Is Involved in Ig Class Switch Recombination in Human B Cells.

Nonhomologous end-joining (NHEJ) is one of the major DNA double-strand break repair pathways in mammalian cells and is required for both V(D)J recombination and class switch recombination (CSR), two Ig gene-diversification processes occurring during B cell development. DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) is a component of the classical NHEJ machinery and has a critical function during V(D)J recombination. However, its role in CSR has been controversial. In this study, we examined the pattern of recombination junctions from in vivo-switched B cells from two DNA-PKcs-deficient patients. One of them harbored mutations that did not affect DNA-PKcs kinase activity but caused impaired Artemis activation; the second patient had mutations resulting in diminished DNA-PKcs protein expression and kinase activity. These results were compared with those from DNA-PKcs-deficient mouse B cells. A shift toward the microhomology-based alternative end-joining at the recombination junctions was observed in both human and mouse B cells, suggesting that the classical NHEJ pathway is impaired during CSR when DNA-PKcs is defective. Furthermore, cells from the second patient showed additional or more severe alterations in CSR and/or NHEJ, which may suggest that DNA-PKcs and/or its kinase activity have additional, Artemis-independent functions during these processes.

Cernunnos influences human immunoglobulin class switch recombination and may be associated with B cell lymphomagenesis.

Cernunnos is involved in the nonhomologous end-joining (NHEJ) process during DNA double-strand break (DSB) repair. Here, we studied immunoglobulin (Ig) class switch recombination (CSR), a physiological process which relies on proper repair of the DSBs, in B cells from Cernunnos-deficient patients. The pattern of in vivo generated CSR junctions is altered in these cells, with unusually long microhomologies and a lack of direct end-joining. The CSR junctions from Cernunnos-deficient patients largely resemble those from patients lacking DNA ligase IV, Artemis, or ATM, suggesting that these factors are involved in the same end-joining pathway during CSR. By screening 269 mature B cell lymphoma biopsies, we also identified a somatic missense Cernunnos mutation in a diffuse large B cell lymphoma sample. This mutation has a dominant-negative effect on joining of a subset of DNA ends in an in vitro NHEJ assay. Translocations involving both Ig heavy chain loci and clonal-like, dynamic IgA switching activities were observed in this tumor. Collectively, our results suggest a link between defects in the Cernunnos-dependent NHEJ pathway and aberrant CSR or switch translocations during the development of B cell malignancies.

Rhesus macaque inhibitory and activating KIR3D interact with Mamu-A-encoded ligands.

Specific interactions between killer cell Ig-like receptors (KIRs) and MHC class I ligands have not been described in rhesus macaques despite their importance in biomedical research. Using KIR-Fc fusion proteins, we detected specific interactions for three inhibitory KIRs (3DLW03, 3DL05, 3DL11) and one activating KIR (3DS05). As ligands we identified Macaca mulatta MHC (Mamu)-A1- and Mamu-A3-encoded allotypes, among them Mamu-A1*001:01, which is well known for association with slow progression to AIDS in the rhesus macaque experimental SIV infection model. Interactions with Mamu-B or Mamu-I molecules were not found. KIR3DLW03 and KIR3DL05 differ in their binding sites to their shared ligand Mamu-A1*001:01, with 3DLW03 depending on presence of the α1 domain, whereas 3DL05 depends on both the α1 and α2 domains. Fine-mapping studies revealed that binding of KIR3DLW03 is influenced by presence of the complete Bw4 epitope (positions 77, 80-83), whereas that of KIR3DL05 is mainly influenced by amino acid position 77 of Bw4 and positions 80-83 of Bw6. Our findings allowed the successful prediction of a further ligand of KIR3DL05, Mamu-A1*002:01. These functional differences of rhesus macaque KIR3DL molecules are in line with the known genetic diversification of lineage II KIRs in macaques.

Erratum to: rhesus macaque MHC class I molecules show differential subcellular localizations.

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding alpha1 and alpha2 domains, suggesting failure of peptide binding is responsible for retaining 'intracellular' Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.

Characterization of rhesus macaque KIR genotypes and haplotypes.

Certain combinations of the killer immunoglobulin-like receptors (KIR) and major histocompatibility complex class I ligands in humans predispose carriers to a variety of diseases, requiring sophisticated genotyping of the highly polymorphic and diverse KIR and HLA genes. Particularly, KIR genotyping is challenging due to polymorphisms (allelic substitutions), genomic diversity (presence/absence of genes), and frequent duplications. Rhesus macaques are often used as important animal models of human diseases such as, e.g. AIDS. However, typing of rhesus macaque KIR genes has not been described so far. In this study, we report the identification of additional novel rhesus macaque KIR cDNA sequences and a sequence-specific KIR genotyping assay. From a cohort of four rhesus macaque families with a total of 70 individuals, we identified 25 distinct KIR genotypes. Segregation analyses of KIR genes and of two polymorphic microsatellite markers allowed the identification of 21 distinct KIR haplotypes in these families, with five to 11 segregating KIR genes per haplotype. Our analyses confirmed and extended knowledge on differential gene KIR gene content in macaques and indicate that rhesus macaque and human KIR haplotypes show a comparable level of diversity and complexity.

Rhesus macaque MHC class I molecules show differential subcellular localizations.

The MHC class I gene family of rhesus macaques is characterised by considerable gene duplications. While a HLA-C-orthologous gene is absent, the Mamu-A and in particular the Mamu-B genes have expanded, giving rise to plastic haplotypes with differential gene content. Although some of the rhesus macaque MHC class I genes are known to be associated with susceptibility/resistance to infectious diseases, the functional significance of duplicated Mamu-A and Mamu-B genes and the expression pattern of their encoded proteins are largely unknown. Here, we present data of the subcellular localization of AcGFP-tagged Mamu-A and Mamu-B molecules. We found strong cell surface and low intracellular expression for Mamu-A1, Mamu-A2 and Mamu-A3-encoded molecules as well as for Mamu-B*01704, Mamu-B*02101, Mamu-B*04801, Mamu-B*06002 and Mamu-B*13401. In contrast, weak cell surface and strong intracellular expression was seen for Mamu-A4*1403, Mamu-B*01202, Mamu-B*02804, Mamu-B*03002, Mamu-B*05704, Mamu-I*010201 and Mamu-I*0121. The different expression patterns were assigned to the antigen-binding alpha1 and alpha2 domains, suggesting failure of peptide binding is responsible for retaining 'intracellular' Mamu class I molecules in the endoplasmic reticulum. These findings indicate a diverse functional role of the duplicated rhesus macaque MHC class I genes.

A novel system of polymorphic and diverse NK cell receptors in primates.

There are two main classes of natural killer (NK) cell receptors in mammals, the killer cell immunoglobulin-like receptors (KIR) and the structurally unrelated killer cell lectin-like receptors (KLR). While KIR represent the most diverse group of NK receptors in all primates studied to date, including humans, apes, and Old and New World monkeys, KLR represent the functional equivalent in rodents. Here, we report a first digression from this rule in lemurs, where the KLR (CD94/NKG2) rather than KIR constitute the most diverse group of NK cell receptors. We demonstrate that natural selection contributed to such diversification in lemurs and particularly targeted KLR residues interacting with the peptide presented by MHC class I ligands. We further show that lemurs lack a strict ortholog or functional equivalent of MHC-E, the ligands of non-polymorphic KLR in "higher" primates. Our data support the existence of a hitherto unknown system of polymorphic and diverse NK cell receptors in primates and of combinatorial diversity as a novel mechanism to increase NK cell receptor repertoire.

Genotyping and segregation analyses indicate the presence of only two functional MIC genes in rhesus macaques.

MIC molecules are stress-inducible ligands of the activating receptor NKG2D, which is expressed on natural killer cells and subsets of T lymphocytes. In rhesus macaques (Macaca mulatta), three different MIC sequences (MIC1, MIC2, MIC3) have been described that are closely related to but, according to phylogenetic analysis, do not represent orthologues of the human MICA and MICB genes. Although a single haplotype of the rhesus macaque Mhc (Mamu) has been completely sequenced, it remained unknown so far whether these three sequences are derived from two or three Mamu-MIC genes. We genotyped a cohort of 115 rhesus macaque individuals for the presence of MIC1, MIC2, and MIC3 sequences and analysed the segregation in families. All individuals were positive for MIC2, whereas only 66.1 and 80.9 % were positive for MIC1 and MIC3, respectively. MIC1 and MIC3 sequences segregated in offspring, indicating that they behave as alleles. Thus, we conclude that two MIC genes are present in the rhesus macaque Mhc, which we propose to designate as Mamu-MICA (MIC1 and MIC3) and Mamu-MICB (MIC2). "MIC1" and "MIC3" are regarded as divergent allelic lineages of the Mamu-MICA gene. Mamu-MIC genotyping of DNA of a cohort of 68 experimentally simian immunodeficiency virus (SIV)-infected rhesus macaques revealed no significant association of either of the two Mamu-MICA allelic lineages with differences in progression to AIDS-like symptoms.