Do developing B cells need antigen?

Just as potentially useful T cells are positively selected by MHC–peptide complexes in the thymus, it has been proposed that self or commensal bacterial epitopes might select B cell populations with the capacity to recognize polysaccharide or protein structures on pathogens. Recent studies indicate that the repertoire of B cells entering the periphery is not shaped by specific stimuli, but that mature B cell subsets may be under different selective pressures

Les cellules B de la zone marginale chez l'homme (un lignage NOTCH 2 dépendant)

Les lymphocytes B de la zone marginale (MZB) dérivent d une voie de différenciation indépendante du centre germinatif chez la souris, mais cette question reste controversée chez l homme. Chez la souris, la différenciation de ces cellules s effectue dans la rate en utilisant la voie Notch2. Un précurseur lymphoïde murin a été identifié, et sa différenciation en MZB par des signaux Notch2 a été démontrée in vitro. Nous avons cherché un tel précurseur dans le compartiment B IgD+ CD27- splénique chez l homme. Sur la base de sa capacité à acquérir in vitro les marqueurs des cellules MZB en réponse à un signal donné par le ligand de Notch, Delta Like One (Dll1), nous avons identifié dans la rate humaine deux populations de cellules B potentiellement précurseurs. La première présente un taux de mutations somatiques se rapprochant du taux des MZB et semble engagée dans une voie de différenciation plus mature qui reste à préciser ; la deuxième très peu mutée parait être un précurseur très précoce des MZB. In vitro cette population acquière un programme qui la rapproche des MZB, ce qui est confirmé par l induction de gènes que nous avons identifié comme spécifiquement exprimés par les MZB (comme le facteur de transcription SOX7) comparés aux cellules B mémoires. Cette population est majoritairement présente durant l enfance puis diminue fortement. Nous avons aussi identifié, dans la rate de jeunes enfants, des cellules non-lymphoïdes exprimant DLL1 à la périphérie de la zone marginale. Pour confirmer ces résultats, nous avons analysés le sang de trois patients présentant une mutation génétique invalidant un des deux allèles du gène NOTCH2 (Syndrome d Alagille). Contrairement aux contrôles sains, ces patients présentent un taux de cellules MZB dans le sang 2 à 3 fois diminué en comparaison du taux de cellules B mémoires. Ce phénotype rappelle celui des souris haploinsuffisantes pour Notch2, ce qui semble bien confirmer que, chez l homme comme chez la souris, la population zone marginale représente un lignage cellulaire spécifique dont la mise en place est indépendante du centre germinatif.Marginal zone B cell (MZB) is a specific B cell lineage in mice. The existence of such a lineage in human remains controversial. In mice, MZB differentiation takes place in the spleen and is under the control of a Notch2 signal. A marginal zone B cell precursor (MZP) has been identified in mice and its in vitro differentiation into MZB cell requires a Notch2 signaling. We have looked for such a precursor among IgD+ CD27- splenic B cell compartment in human. Based on its ability to acquire MZB cell markers in vitro after a Notch signaling, provided by the Notch ligand Delta Like One (Dll1), we identified in human spleen two B cell subsets that could be putative precursors. One shows a mutational rate close to the rate found in MZB and seems already engaged in an unidentified mature differentiation stage. The second population appears almost unmutated and seems to be a very early precursor for MZB. In vitro, this population acquires a transcriptional program resembling the MZB program. This is confirmed by the induction of genes specifically expressed by MZB (like SOX7) compared to B cell memory. Putative MZP is found in a higher proportion in children compared to adults and we also identified in children spleen non-lymphoid cells expressing DLL1 at the marginal zone border. To confirm all these results, we analyzed blood samples of three patients that are mutated in one of the two NOTCH2 alleles and suffering from the Alagille syndrome. Contrarily to healthy controls, the 3 Alagille patients showed a 2 to 3 fold decrease in MZB level compared to memory B cell level. Interestingly this phenotype is similar to mice showing a Notch2 haploinsufficiency, thus confirming that MZB population is, as in mice, a specific B cell lineage in human.PARIS5-Bibliotheque electronique (751069902) / SudocSudocFranceF

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

DNA Polymerase η Is Involved in Hypermutation Occurring during Immunoglobulin Class Switch Recombination

Base substitutions, deletions, and duplications are observed at the immunoglobulin locus in DNA sequences involved in class switch recombination (CSR). These mutations are dependent upon activation-induced cytidine deaminase (AID) and present all the characteristics of the ones observed during V gene somatic hypermutation, implying that they could be generated by the same mutational complex. It has been proposed, based on the V gene mutation pattern of patients with the cancer-prone xeroderma pigmentosum variant (XP-V) syndrome who are deficient in DNA polymerase η (pol η), that this enzyme could be responsible for a large part of the mutations occurring on A/T bases. Here we show, by analyzing switched memory B cells from two XP-V patients, that pol η is also an A/T mutator during CSR, in both the switch region of tandem repeats as well as upstream of it, thus suggesting that the same error-prone translesional polymerases are involved, together with AID, in both processes

Somatic diversification in the absence of antigen-driven responses is the hallmark of the IgM+IgD+CD27+ B cell repertoire in infants

T cell–dependent immune responses develop soon after birth, whereas it takes 2 yr for humans to develop T cell–independent responses. We used this dissociation to analyze the repertoire diversification of IgM+IgD+CD27+ B cells (also known as “IgM memory” B cells), comparing these cells with switched B cells in children <2 yr of age, with the aim of determining whether these two subsets are developmentally related. We show that the repertoire of IgM+IgD+CD27+ B cells in the spleen and blood displays no sign of antigen-driven activation and expansion on H-CDR3 spectratyping, despite the many antigenic challenges provided by childhood vaccinations. This repertoire differed markedly from those of switched B cells and splenic germinal center B cells, even at the early stage of differentiation associated with μ heavy chain expression. These data provide evidence for the developmental diversification of IgM+IgD+CD27+ B cells, at least in very young children, outside of T cell–dependent and –independent immune responses

Visualizing antibody affinity maturation in germinal centers

open12siAntibodies somatically mutate to attain high affinity in germinal centers (GCs). There, competition between B cell clones and among somatic mutants of each clone drives an increase in average affinity across the population. The extent to which higher-affinity cells eliminating competitors restricts clonal diversity is unknown. By combining multiphoton microscopy and sequencing, we show that tens to hundreds of distinct B cell clones seed each GC and that GCs lose clonal diversity at widely disparate rates. Furthermore, efficient affinity maturation can occur in the absence of homogenizing selection, ensuring that many clones can mature in parallel within the same GC. Our findings have implications for development of vaccines in which antibodies with nonimmunodominant specificities must be elicited, as is the case for HIV-1 and influenza.openTas J.M.J.; Mesin L.; Pasqual G.; Targ S.; Jacobsen J.T.; Mano Y.M.; Chen C.S.; Weill J.-C.; Reynaud C.-A.; Browne E.P.; Meyer-Hermann M.; Victora G.D.Tas, J. M. J.; Mesin, L.; Pasqual, G.; Targ, S.; Jacobsen, J. T.; Mano, Y. M.; Chen, C. S.; Weill, J. -C.; Reynaud, C. -A.; Browne, E. P.; Meyer-Hermann, M.; Victora, G. D

B cell intrinsic and extrinsic factors impacting memory recall responses to SRBC challenge

MBCs (MBCs) generated in T-dependent immune responses can persist for a lifetime and rapidly react upon secondary antigen exposure to differentiate into plasma cells (PCs) and/or to improve the affinity of their BCR through new rounds of hypermutation in germinal centers (GCs). The fate of a MBC in secondary immune reactions appears to depend upon multiple parameters, whose understanding is mandatory for the design of efficient vaccine strategies. We followed the behavior of MBCs in recall responses to SRBCs using an inducible AID fate mapping mouse model in which B cells engaged in a germinal center (GC) response are irreversibly labeled upon simultaneous tamoxifen ingestion and immunization. We used different schemes of mouse immunization and tamoxifen feeding in adoptive-transfer experiments of total splenic B cells into congenic mice that have been pre-immunized or not, to assess the contribution of the different effector subsets in a physiological competitive context. We were able to show that naive B cells can differentiate into GC B cells with kinetics similar to MBCs in the presence of previously activated T follicular helper (TFH) cells and a primed microenvironment. We also showed that MBCs are recruited into secondary GCs, together with naive B cells. In contrast, PC differentiation, which dominated secondary MBC responses, was not dependent upon a previous TFH activation. We observed that the presence of persisting germinal centers and circulating antibody levels are key factors determining the germinal center versus plasma cell fate in a recall response. Notably, disruption of persistent germinal center structures by a lymphotoxin beta-receptor fusion protein or a longer timing between the prime and the boost, which correlated with reduced antigen-specific immunoglobulin levels in serum, were two conditions with an opposite impact, respectively inhibiting or promoting a GC fate for MBCs. Altogether, these studies highlight the complexity of recall responses, whose outcome varies according to immunization contexts