Jean-Claude Weill

A hyperconversion mechanism generates the chicken light chain preimmune repertoire

The chicken immunoglobulin light chain repertoire has been shown to be entirely derived from a single V lambda 1-J rearranged combination. The complete coding information of the lambda locus was determined: it comprises 25 V-hybridizing elements, all of which are pseudogenes, clustered in both orientations within 19 kb of DNA, starting 2.4 kb upstream of the V lambda 1 gene. Sequences of somatically rearranged V lambda 1 genes from embryonic and posthatching bursal cells show that diversification of light chain sequences occurs during ontogeny by a segmental gene conversion mechanism which takes place at a frequency of 0.05-0.1 per cell generation between the pseudogene pool and the unique rearranged functional V gene.

Multiple layers of B cell memory with different effector functions

Memory B cells are at the center of longstanding controversies regarding the presence of antigen for their survival and their re-engagement in germinal centers after secondary challenge. Using a new mouse model of memory B cell labeling dependent on the cytidine deaminase AID, we show that after immunization with a particulate antigen, B cell memory appeared in several subsets, comprising clusters of immunoglobulin M–positive (IgM+) and IgG1+ B cells in germinal center–like structures that persisted up to 8 months after immunization, as well as IgM+ and IgG1+ B cells with a memory phenotype outside of B cell follicles. After challenge, the IgG subset differentiated into plasmocytes, whereas the IgM subset reinitiated a germinal center reaction. This model, in which B cell memory appears in several layers with different functions, reconciles previous conflicting propositions.

Hypermutation generating the sheep immunoglobulin repertoire is an antigen-independent process

Somatic hypermutation of light chain V genes during development of B cells in sheep ileal Peyers patches was studied in three experimental conditions: in sterile fragments of the ileum surgically isolated from the gut during fetal life, in germ-free sheep, and in animals thymectomized during early fetal life. The somatic mutation pattern was found identical to control tissues in all three experiments. The same age-dependent amount of mutations, a higher than theoretical R/S ratio in complementarity-determining regions (CDRs), and a similar clustering of mutations in CDRs were observed. The mechanism, as estimated from the silent mutation pattern, appears to target mutations to CDRs; moreover, the major V lambda genes have a specific codon usage with a high purine content at the first two bases of the codons and a low content at the third position, which, together with a specific targeting of mutations to purines, favors replacement mutations in CDRs.

Human Marginal Zone B Cells

Human marginal zone (MZ) B cells are, in a sense, a new entity. Although they share many properties with their mouse counterpart, they also display striking differences, such as the capacity to recirculate and the presence of somatic mutations in their B cell receptor. These differences are the reason they are often not considered a separate, rodent-like B cell lineage, but rather are considered IgM memory B cells. We review here our present knowledge concerning this subset and the arguments in favor of the proposition that humans have evolved for their MZ B cell compartment a separate B cell population that develops and diversifies its Ig receptor during ontogeny outside T-dependent or T-independent immune responses.

A single rearrangement event generates most of the chicken immunoglobulin light chain diversity.

The chicken immunoglobulin lambda locus contains a single C lambda gene with a unique J lambda element, 1.9 kb upstream. The same V lambda gene (V lambda 1) is rearranged in most cells of the Bursa of Fabricius. This V lambda 1 gene is located, in germ-line configuration, 1.7 kb upstream from J lambda and in the same transcriptional orientation. Eight to twelve variable genes of the same set are found adjacent to the V lambda 1 gene, indicating that V-gene amplification did occur. Three of these genes were sequenced and proved to be pseudogenes, one of them having an inverted polarity. Data suggesting extensive somatic diversification of the V lambda 1 sequence are reported, including the possible use of nonfunctional V elements in a somatic gene-conversion-like process.

Induction of somatic hypermutation in immunoglobulin genes is dependent on DNA polymerase iota

Somatic hypermutation of immunoglobulin genes is a unique, targeted, adaptive process. While B cells are engaged in germinal centres in T-dependent responses, single base substitutions are introduced in the expressed Vh/Vl genes to allow the selection of mutants with a higher affinity for the immunizing antigen. Almost every possible DNA transaction has been proposed to explain this process, but each of these models includes an error-prone DNA synthesis step that introduces the mutations. The Y family of DNA polymerases—pol η, pol ι, pol κ and rev1—are specialized for copying DNA lesions and have high rates of error when copying a normal DNA template. By performing gene inactivation in a Burkitts lymphoma cell line inducible for hypermutation, we show here that somatic hypermutation is dependent on DNA polymerase iota.

Contribution of DNA polymerase η to immunoglobulin gene hypermutation in the mouse

The mutation pattern of immunoglobulin genes was studied in mice deficient for DNA polymerase η, a translesional polymerase whose inactivation is responsible for the xeroderma pigmentosum variant (XP-V) syndrome in humans. Mutations show an 85% G/C biased pattern, similar to that reported for XP-V patients. Breeding these mice with animals harboring the stop codon mutation of the 129/Olain background in their DNA polymerase ι gene did not alter this pattern further. Although this G/C biased mutation profile resembles that of mice deficient in the MSH2 or MSH6 components of the mismatch repair complex, the residual A/T mutagenesis of polη-deficient mice differs markedly. This suggests that, in the absence of polη, the MSH2–MSH6 complex is able to recruit another DNA polymerase that is more accurate at copying A/T bases, possibly polκ, to assume its function in hypermutation.

AID-dependent somatic hypermutation occurs as a DNA single-strand event in the BL2 cell line

Immunoglobulin (Ig) gene hypermutation can be induced in the BL2 Burkitts lymphoma cell line by IgM cross-linking and coculture with normal or transformed T helper clones. We describe here a T cell–independent in vitro induction assay, by which hypermutation is induced in BL2 cells through simultaneous aggregation of three surface receptors: IgM, CD19 and CD21. The mutations arise as a post-transcriptional event within 90 min. They are stably introduced in the G1 phase of the cell cycle, occurring in one of the two variable gene DNA strands, and eventually become fixed by replication in one of the daughter cells. Inactivation of AID (activation-induced cytidine deaminase) by homologous recombination in BL2 cells completely inhibits the process, thus validating this induction procedure as a model for the in vivo mechanism.

Mismatch Repair Deficiency Interferes with the Accumulation of Mutations in Chronically Stimulated B Cells and Not with the Hypermutation Process

Primary responses to the hapten phenyloxazolone and chronic responses to environmental antigens occurring in Peyers patches were analyzed in two different mismatch repair-deficient backgrounds. Paradoxically, whereas primary responses were found normal in MSH2- and only slightly diminished in PMS2-deficient mice, mutations in Peyers patch B cells from both k.o. animals were reduced three times, the subset of Peyers patch B cells with highly mutated sequences being specifically missing in the mismatch repair-deficient context. Strikingly, germinal center B cells from Peyers patches of k.o. animals showed microsatellite instability at an unprecedented level. We thus propose that the amount of DNA damages generated prevents these cells from recycling in germinal centers and that mismatch repair deficiency is only the indirect cause of the lower mutation incidence observed.

FORMATION OF THE CHICKEN B-CELL REPERTOIRE: ONTOGENESIS, REGULATION OF IG GENE REARRANGEMENT, AND DIVERSIFICATION BY GENE CONVERSION

Publisher Summary The chapter focuses on the actual knowledge of the formation of the B-cell repertoire, including data on early B-cell commitment and the regulation of rearrangement obtained with chicken substrates in transgenic mice. The chicken B-cell immune system is attractive because of its apparent simplicity. During B-cell expansion in the bursa, gene conversion generates a diversified B-cell repertoire. Rabbits use gene conversion to generate B-cell diversity, and this process may well occur during a short period of development in gut-associated lymphoid tissues (GALT). Sheep, and probably ruminants in general, use gut-associated lymphoid tissues (GALT) that are only present during early development to generate their B cell repertoire. In chickens, sheep, and rabbits, immunoglobulin (Ig) gene rearrangement is not the key event for Ig diversity: The post rearrangement diversification processes taking place during an early phase of B-cell amplification generate the B-cell repertoire in these three species. The molecular mechanisms differ, however, among them: in the chicken, gene conversion diversifies a unique rearranged gene at both heavy- and light-chain loci by recombination with a pool of pseudo-gene elements; in the sheep, several functional light-chain V genes undergo extensive modification by untemplated somatic mutations; and for the rabbit heavy chain, a major rearranged gene undergoes gene conversion with possibly extensive somatic mutation of the D region. The organization of Ig genes in the chicken and the generation of the chicken B-Cell repertoire by gene conversion are also discussed in the chapter.