Antigen-independent, autonomous B cell receptor signaling drives activated B cell DLBCL.

Diffuse large B cell lymphoma of activated B cell type (ABC-DLBCL), a major cell-of-origin DLBCL subtype, is characterized by chronic active B cell receptor (BCR) signaling and NF-κB activation, which can be explained by activating mutations of the BCR signaling cascade in a minority of cases. We demonstrate that autonomous BCR signaling, akin to its essential pathogenetic role in chronic lymphocytic leukemia (CLL), can explain chronic active BCR signaling in ABC-DLBCL. 13 of 18 tested DLBCL-derived BCR, including 12 cases selected for expression of IgM, induced spontaneous calcium flux and increased phosphorylation of the BCR signaling cascade in murine triple knockout pre-B cells without antigenic stimulation or external BCR crosslinking. Autonomous BCR signaling was associated with IgM isotype, dependent on somatic BCR mutations and individual HCDR3 sequences, and largely restricted to non-GCB DLBCL. Autonomous BCR signaling represents a novel immunological oncogenic driver mechanism in DLBCL originating from individual BCR sequences and adds a new dimension to currently proposed genetics- and transcriptomics-based DLBCL classifications.

Autonomous B-cell receptor signaling and genetic aberrations in chronic lymphocytic leukemia-phenotype monoclonal B lymphocytosis in siblings of patients with chronic lymphocytic leukemia.

Clonal expansion of CD5-expressing B cells, commonly designated as monoclonal B lymphocytosis (MBL), is a precursor condition for chronic lymphocytic leukemia (CLL). The mechanisms driving subclinical MBL B-cell expansion and progression to CLL, occurring in approximately 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for the pathogenesis of CLL. The objectives of this study were functional characterization of the BCR of MBL in siblings of CLL patients and a comparison of genetic variants in MBL-CLL sibling pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per microliter (median: 37/µL) in 34 of 191 (17.8%) siblings of CLL patients. Clonal BCR isolated from highly purified CLL-phenotype cells induced robust calcium mobilization in BCR-deficient murine pre-B cells in the absence of external antigen and without experimental crosslinking. This autonomous BCR signal was less intense than the signal originating from the CLL BCR of their CLL siblings. According to genotyping by single nucleotide polymorphism array, whole exome, and targeted panel sequencing, CLL risk alleles were found with high and similar prevalence in CLL patients and MBL siblings, respectively. Likewise, the prevalence of recurrent CLL-associated genetic variants was similar between CLL and matched MBL samples. However, copy number variations and small variants were frequently subclonal in MBL cells, suggesting their acquisition during subclinical clonal expansion. These findings support a stepwise model of CLL pathogenesis, in which autonomous BCR signaling leads to a non-malignant (oligo)clonal expansion of CD5+ B cells, followed by malignant progression to CLL after acquisition of pathogenic genetic variants.

A Comparison of Immunoglobulin Variable Region N-Linked Glycosylation in Healthy Donors, Autoimmune Disease and Lymphoma.

N-linked glycans play an important role in immunity. Although the role of N-linked glycans in the Fragment crystallizable (Fc) region of immunoglobulins has been thoroughly described, the function of N-linked glycans present in Ig-variable domains is only just being appreciated. Most of the N-linked glycans harbored by immunoglobulin variable domain are of the complex biantennary type and are found as a result of the presence of N-linked glycosylation that most often have been introduced by somatic hypermutation. Furthermore, these glycans are ubiquitously present on autoantibodies observed in some autoimmune diseases as well as certain B-cell lymphomas. For example, variable domain glycans are abundantly found by anti-citrullinated protein antibodies (ACPA) in rheumatoid arthritis (RA) as well as by the B-cell receptors of follicular lymphoma (FL). In FL, variable domain glycans are postulated to convey a selective advantage through interaction with lectins and/or microbiota, whereas the contribution of variable domain glycans on autoantibodies is not known. To aid the understanding how these seemingly comparable phenomena contribute to a variety of deranged B-responses in such different diseases this study summarizes the characteristics of ACPA and other auto-antibodies with FL and healthy donor immunoglobulins, to identify the commonalities and differences between variable domain glycans in autoimmune and malignant settings. Our finding indicate intriguing differences in variable domain glycan distribution, frequency and glycan composition in different conditions. These findings underline that variable domain glycosylation is a heterogeneous process that may lead to a number of pathogenic outcomes. Based on the current body of knowledge, we postulate three disease groups with distinct variable domain glycosylation patterns, which might correspond with distinct underlying pathogenic processes.

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.

Acquired N-Linked Glycosylation Motifs in B-Cell Receptors of Primary Cutaneous B-Cell Lymphoma and the Normal B-Cell Repertoire.

Primary cutaneous follicle center lymphoma (PCFCL) is a rare mature B-cell lymphoma with an unknown etiology. PCFCL resembles follicular lymphoma (FL) by cytomorphologic and microarchitectural criteria. FL B cells are selected for N-linked glycosylation motifs in their B-cell receptors (BCRs) that are acquired during continuous somatic hypermutation. The stimulation of mannosylated BCR by lectins on the tumor microenvironment is therefore a candidate driver in FL pathogenesis. We investigated whether the same mechanism could play a role in PCFCL pathogenesis. Full-length functional variable, diversity, and joining gene sequences of 18 PCFCL and 8 primary cutaneous diffuse large B-cell lymphoma, leg-type were identified by unbiased Anchoring Reverse Transcription of Immunoglobulin Sequences and Amplification by Nested PCR and BCR reconstruction from RNA sequencing data. Low BCR variation demonstrated negligible ongoing somatic hypermutation in PCFCL and primary cutaneous diffuse large B-cell lymphoma, leg-type, and indicated that the PCFCL microarchitecture does not act as a functional germinal center. Similar to FL but in contrast to primary cutaneous diffuse large B-cell lymphoma, leg-type, BCR genes of 15 PCFCLs (83%) had acquired N-linked glycosylation motifs. These motifs were located at the BCR positions converted to N-linked glycosylation motifs in normal B-cell repertoires with low prevalence but mostly at different positions than those found in FL. The cutaneous localization of PCFCL might suggest a role for lectins from commensal skin bacteria in PCFCL lymphomagenesis.

MuSK myasthenia gravis monoclonal antibodies: Valency dictates pathogenicity.

Objective: To isolate and characterize muscle-specific kinase (MuSK) monoclonal antibodies from patients with MuSK myasthenia gravis (MG) on a genetic and functional level. Methods: We 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. Results: The 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. Conclusions: Patient-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.