AID Overlapping and Polη Hotspots Are Key Features of Evolutionary Variation Within the Human Antibody Heavy Chain (IGHV) Genes

© Copyright © 2020 Tang, Bagnara, Chiorazzi, Scharff and MacCarthy. Somatic hypermutation (SHM) of the immunoglobulin variable (IgV) loci is a key process in antibody affinity maturation. The enzyme activation-induced deaminase (AID), initiates SHM by creating C → U mismatches on single-stranded DNA (ssDNA). AID has preferential hotspot motif targets in the context of WRC/GYW (W = A/T, R = A/G, Y = C/T) and particularly at WGCW overlapping hotspots where hotspots appear opposite each other on both strands. Subsequent recruitment of the low-fidelity DNA repair enzyme, Polymerase eta (Polη), during mismatch repair, creates additional mutations at WA/TW sites. Although there are more than 50 functional immunoglobulin heavy chain variable (IGHV) segments in humans, the fundamental differences between these genes and their ability to respond to all possible foreign antigens is still poorly understood. To better understand this, we generated profiles of WGCW hotspots in each of the human IGHV genes and found the expected high frequency in complementarity determining regions (CDRs) that encode the antigen binding sites but also an unexpectedly high frequency of WGCW in certain framework (FW) sub-regions. Principal Components Analysis (PCA) of these overlapping AID hotspot profiles revealed that one major difference between IGHV families is the presence or absence of WGCW in a sub-region of FW3 sometimes referred to as “CDR4.” Further differences between members of each family (e.g., IGHV1) are primarily determined by their WGCW densities in CDR1. We previously suggested that the co-localization of AID overlapping and Polη hotspots was associated with high mutability of certain IGHV sub-regions, such as the CDRs. To evaluate the importance of this feature, we extended the WGCW profiles, combining them with local densities of Polη (WA) hotspots, thus describing the co-localization of both types of hotspots across all IGHV genes. We also verified that co-localization is associated with higher mutability. PCA of the co-localization profiles showed CDR1 and CDR2 as being the main contributors to variance among IGHV genes, consistent with the importance of these sub-regions in antigen binding. Our results suggest that AID overlapping (WGCW) hotspots alone or in conjunction with Polη (WA/TW) hotspots are key features of evolutionary variation between IGHV genes

Recognition of antigen-specific B-cell receptors from chronic lymphocytic leukemia patients by synthetic antigen surrogates

In patients with chronic lymphocytic leukemia (CLL), a single neoplastic antigen-specific B cell accumulates and overgrows other B cells, leading to immune deficiency. CLL is often treated with drugs that ablate all B cells, leading to further weakening of humoral immunity, and a more focused therapeutic strategy capable of targeting only the pathogenic B cells would represent a significant advance. One approach to this would be to develop synthetic surrogates of the CLL antigens allowing differentiation of the CLL cells and healthy B cells in a patient. Here, we describe nonpeptidic molecules capable of targeting antigen-specific B cell receptors with good affinity and selectivity using a combinatorial library screen. We demonstrate that our hit compounds act as synthetic antigen surrogates and recognize CLL cells and not healthy B cells. Additionally, we argue that the technology we developed can be used to identify other classes of antigen surrogates

Excessive antigen reactivity may underlie the clinical aggressiveness of chronic lymphocytic leukemia stereotyped subset #8

Subset #8 is a distinctive subset of patients with chronic lymphocytic leukemia (CLL) defined by the expression of stereotyped IGHV4-39/IGKV1(D)-39 B-cell receptors. Subset #8 patients experience aggressive disease and exhibit the highest risk for Richter transformation among all CLL. In order to obtain biological insight into this behavior, we profiled the antigen reactivity and signaling capacity of subset #8 vs other clinically aggressive stereotyped subsets, namely subsets #1 and #2. Twenty-seven monoclonal antibodies (mAbs) from subsets #1, #2, and #8 CLL clones were prepared as recombinant human immunoglobulin G1 and used as primary antibodies in enzyme-linked immuno-sorbent assays against representatives of the major classes of established antigenic targets for CLL. Subset #8 CLL mAbs exhibited broad polyreactivity as they bound to all antigens tested, in clear contrast with the mAbs from the other subsets. Antigen challenge of primary CLL cells indicated that the promiscuous antigen-binding activity of subset #8 mAbs could lead to significant cell activation, again in contrast to the less responsive CLL cells from subsets #1 and #2. These features constitute a distinctive profile for CLL subset #8, supporting the existence of distinct mechanisms of aggressiveness in different immunogenetic subsets of CLL

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

© 2020 National Academy of Sciences. All rights reserved. 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 complementaritydetermining 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 ofmonoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors

Internal standard-based analysis of microarray data2—Analysis of functional associations between HVE-genes

In this work we apply the Internal Standard-based analytical approach that we described in an earlier communication and here we demonstrate experimental results on functional associations among the hypervariably-expressed genes (HVE-genes). Our working assumption was that those genetic components, which initiate the disease, involve HVE-genes for which the level of expression is undistinguishable among healthy individuals and individuals with pathology. We show that analysis of the functional associations of the HVE-genes is indeed suitable to revealing disease-specific differences. We show also that another possible exploit of HVE-genes for characterization of pathological alterations is by using multivariate classification methods. This in turn offers important clues on naturally occurring dynamic processes in the organism and is further used for dynamic discrimination of groups of compared samples. We conclude that our approach can uncover principally new collective differences that cannot be discerned by individual gene analysi