IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors

Tandem Substitutions in Somatic Hypermutation

Upon antigen recognition, activation-induced cytosine deaminase initiates affinity maturation of the B-cell receptor by somatic hypermutation (SHM) through error-prone DNA repair pathways. SHM typically creates single nucleotide substitutions, but tandem substitutions may also occur. We investigated incidence and sequence context of tandem substitutions by massive parallel sequencing of V(D)J repertoires in healthy human donors. Mutation patterns were congruent with SHM-derived single nucleotide mutations, delineating initiation of the tandem substitution by AID. Tandem substitutions comprised 5,7% of AID-induced mutations. The majority of tandem substitutions represents single nucleotide juxtalocations of directly adjacent sequences. These observations were confirmed in an independent cohort of healthy donors. We propose a model where tandem substitutions are predominantly generated by translesion synthesis across an apyramidinic site that is typically created by UNG. During replication, apyrimidinic sites transiently adapt an extruded configuration, causing skipping of the extruded base. Consequent strand decontraction leads to the juxtalocation, after which exonucleases repair the apyramidinic site and any directly adjacent mismatched base pairs. The mismatch repair pathway appears to account for the remainder of tandem substitutions. Tandem substitutions may enhance affinity maturation and expedite the adaptive immune response by overcoming amino acid codon degeneracies or mutating two adjacent amino acid residues simultaneously

N-Glycosylation Site Analysis of Citrullinated Antigen-Specific B-Cell Receptors Indicates Alternative Selection Pathways During Autoreactive B-Cell Development

Many autoimmune diseases are hallmarked by autoreactive B and plasma cell responses that are directly or indirectly involved in disease pathogenesis. These B-cell responses show large variability between diseases, both in terms of the secreted autoantibody repertoire and the dynamics and characteristics of the underlying B-cell responses. Hence, different mechanisms have been proposed to explain the emergence of autoreactive B cells in an otherwise self-tolerant immune system. Notably, most mechanistic insights have been obtained from murine studies using models harboring genetic modifications of B and T cells. Given recent technological advances that have rendered autoreactive human B cells accessible for analysis, we here discuss the phenomenon of extensive N-glycosylation of the B-cell receptor (BCR) variable domain of a prototypic human autoreactive B-cell response and its potential role in the generation of autoimmunity. Anti-citrullinated protein antibodies (ACPA) hallmark the most disease-specific autoimmune response in Rheumatoid Arthritis (RA). Remarkably, ACPA-IgG are heavily N-glycosylated in the variable domain due to somatic mutations that generate abundant N-glycosylation consensus sequences. These sites, obtained from full-length BCR sequences of ACPA-expressing B cells from 12 ACPA-positive RA patients, were here analyzed in detail. Sites that required a single nucleotide mutation to be generated were defined as single somatic hypermutation (s-SHM) sites, whereas sites requiring multiple mutations were defined as m-SHM sites. IgG sequences of 12 healthy donors were used as control. Computational modeling of the germinal center reaction (CLONE algorithm) was used with the germline counterparts of ACPA-IgG heavy chain (HC) sequences to simulate the germinal center response. Our analyses revealed an abundance of N-glycosylation sites in ACPA-IgG HC that frequently required multiple mutations and predominated in specific positions. Based on these data, and taking into account recent insights into the dynamics of the ACPA-response during disease development, we here discuss the hypothesis that N-glycosylation sites in ACPA-IgG variable domains could lead to alternative, possibly antibody affinity-independent selection forces. Presumably, this occurs during germinal center responses allowing these B cells to escape from putative tolerance checkpoints, thereby driving autoreactive B cell development in the pathogenesis of RA

MuSK myasthenia gravis monoclonal antibodies: Valency dictates pathogenicity

ObjectiveTo isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level.MethodsWe generated recombinant MuSK antibodies from patient-derived clonal MuSK-specific B cells and produced monovalent Fab fragments from them. Both the antibodies and Fab fragments were tested for their effects on neural agrin-induced MuSK phosphorylation and acetylcholine receptor (AChR) clustering in myotube cultures.ResultsThe isolated MuSK monoclonal antibody sequences included IgG1, IgG3, and IgG4 that had undergone high levels of affinity maturation, consistent with antigenic selection. We confirmed their specificity for the MuSK Ig-like 1 domain and binding to neuromuscular junctions. Monovalent MuSK Fab, mimicking functionally monovalent MuSK MG patient Fab-arm exchanged serum IgG4, abolished agrin-induced MuSK phosphorylation and AChR clustering. Surprisingly, bivalent monospecific MuSK antibodies instead activated MuSK phosphorylation and partially induced AChR clustering, independent of agrin.ConclusionsPatient-derived MuSK antibodies can act either as MuSK agonist or MuSK antagonist, depending on the number of MuSK binding sites. Functional monovalency, induced by Fab-arm exchange in patient serum, makes MuSK IgG4 antibodies pathogenic