Clonotypic Features of Rearranged Immunoglobulin Genes Yield Personalized Biomarkers for Minimal Residual Disease Monitoring in Multiple Myeloma.

BACKGROUND: Due to improved treatment, more patients with multiple myeloma (MM) reach a state of minimal residual disease (MRD). Different strategies for MM MRD monitoring include flow cytometry, allele-specific oligonucleotide-quantitative PCR, next-generation sequencing, and mass spectrometry (MS). The last 3 methods rely on the presence and the stability of a unique immunoglobulin fingerprint derived from the clonal plasma cell population. For MS-MRD monitoring it is imperative that MS-compatible clonotypic M-protein peptides are identified. To support implementation of molecular MRD techniques, we studied the presence and stability of these clonotypic features in the CoMMpass database. METHODS: An analysis pipeline based on MiXCR and HIGH-VQUEST was constructed to identify clonal molecular fingerprints and their clonotypic peptides based on transcriptomic datasets. To determine the stability of the clonal fingerprints, we compared the clonal fingerprints during disease progression for each patient. RESULTS: The analysis pipeline to establish the clonal fingerprint and MS-suitable clonotypic peptides was successfully validated in MM cell lines. In a cohort of 609 patients with MM, we demonstrated that the most abundant clone harbored a unique clonal molecular fingerprint and that multiple unique clonotypic peptides compatible with MS measurements could be identified for all patients. Furthermore, the clonal immunoglobulin gene fingerprints of both the light and heavy chain remained stable during MM disease progression. CONCLUSIONS: Our data support the use of the clonal immunoglobulin gene fingerprints in patients with MM as a suitable MRD target for MS-MRD analyses.

Single-cell whole-genome sequencing reveals the functional landscape of somatic mutations in B lymphocytes across the human lifespan.

Accumulation of mutations in somatic cells has been implicated as a cause of aging since the 1950s. However, attempts to establish a causal relationship between somatic mutations and aging have been constrained by the lack of methods to directly identify mutational events in primary human tissues. Here we provide genome-wide mutation frequencies and spectra of human B lymphocytes from healthy individuals across the entire human lifespan using a highly accurate single-cell whole-genome sequencing method. We found that the number of somatic mutations increases from <500 per cell in newborns to >3,000 per cell in centenarians. We discovered mutational hotspot regions, some of which, as expected, were located at Ig genes associated with somatic hypermutation (SHM). B cell-specific mutation signatures associated with development, aging, or SHM were found. The SHM signature strongly correlated with the signature found in human B cell tumors, indicating that potential cancer-causing events are already present even in B cells of healthy individuals. We also identified multiple mutations in sequence features relevant to cellular function (i.e., transcribed genes and gene regulatory regions). Such mutations increased significantly during aging, but only at approximately one-half the rate of the genome average, indicating selection against mutations that impact B cell function. This full characterization of the landscape of somatic mutations in human B lymphocytes indicates that spontaneous somatic mutations accumulating with age can be deleterious and may contribute to both the increased risk for leukemia and the functional decline of B lymphocytes in the elderly.

Low frequency of broadly neutralizing HIV antibodies during chronic infection even in quaternary epitope targeting antibodies containing large numbers of somatic mutations.

Neutralizing antibodies (Abs) are thought to be a critical component of an appropriate HIV vaccine response. It has been proposed that Abs recognizing conformationally dependent quaternary epitopes on the HIV envelope (Env) trimer may be necessary to neutralize diverse HIV strains. A number of recently described broadly neutralizing monoclonal Abs (mAbs) recognize complex and quaternary epitopes. Generally, many such Abs exhibit extensive numbers of somatic mutations and unique structural characteristics. We sought to characterize the native antibody (Ab) response against circulating HIV focusing on such conformational responses, without a prior selection based on neutralization. Using a capture system based on VLPs incorporating cleaved envelope protein, we identified a selection of B cells that produce quaternary epitope targeting Abs (QtAbs). Similar to a number of broadly neutralizing Abs, the Ab genes encoding these QtAbs showed extensive numbers of somatic mutations. However, when expressed as recombinant molecules, these Abs failed to neutralize virus or mediate ADCVI activity. Molecular analysis showed unusually high numbers of mutations in the Ab heavy chain framework 3 region of the variable genes. The analysis suggests that large numbers of somatic mutations occur in Ab genes encoding HIV Abs in chronically infected individuals in a non-directed, stochastic, manner.

Ectopic restriction of DNA repair reveals that UNG2 excises AID-induced uracils predominantly or exclusively during G1 phase.

Immunoglobulin (Ig) affinity maturation requires the enzyme AID, which converts cytosines (C) in Ig genes into uracils (U). This alone produces C:G to T:A transition mutations. Processing of U:G base pairs via U N-glycosylase 2 (UNG2) or MutSα generates further point mutations, predominantly at G:C or A:T base pairs, respectively, but it is unclear why processing is mutagenic. We aimed to test whether the cell cycle phase of U processing determines fidelity. Accordingly, we ectopically restricted UNG2 activity in vivo to predefined cell cycle phases by fusing a UNG2 inhibitor peptide to cell cycle-regulated degradation motifs. We found that excision of AID-induced U by UNG2 occurs predominantly during G1 phase, inducing faithful repair, mutagenic processing, and class switching. Surprisingly, UNG2 does not appear to process U:G base pairs at all in Ig genes outside G1 phase.

Scattered regulatory regions of the chicken immunoglobulin-ß gene and two adjacent promoters of ubiquitously expressed genes interact with the immunoglobulin-ß promoter in DT40 cells.

Recent studies indicate that several transcription units assemble to form a 'transcription factory' where active transcription occurs in the nuclei. Previously, we generated chicken B-lymphocyte-derived DT40 cells lacking six transcriptional regulatory regions scattered in and around the immunoglobulin (Ig)-ß gene. The deletions caused a complete shut down of transcription and epigenetic regulation of the Ig-ß gene, demonstrating that the scattered regulatory regions cooperated in the transcriptional and epigenetic regulation of the gene. However, the in vivo 3-dimensional spatial relationships between the Ig-ß promoter and these six regulatory regions were not investigated. In this study, we used chromosome conformation capture (3C) technology and demonstrated that the Ig-ß promoter physically interacted with the scattered regulatory regions. We found that the Ig-ß promoter also interacted with two downstream promoters of ubiquitously expressed genes, rad motif 1 (RDM1) and Plekhm1, to form a transcription factory, but not with three ubiquitously expressed genes, BAF60b, p45/SUG, and RRMJ3, located upstream of the Ig-ß gene. In this factory, the chromatin from the three promoters and the scattered regulatory regions of the Ig-ß gene formed a complex structure with many chromatin loops.

Is there a role for antigen selection in mantle cell lymphoma? Immunogenetic support from a series of 807 cases.

We examined 807 productive IGHV-IGHD-IGHJ gene rearrangements from mantle cell lymphoma (MCL) cases, by far the largest series to date. The IGHV gene repertoire was remarkably biased, with IGHV3-21, IGHV4-34, IGHV1-8, and IGHV3-23 accounting for 46.3% of the cohort. Eighty-four of 807 (10.4%) cases, mainly using the IGHV3-21 and IGHV4-34 genes, were found to bear stereotyped heavy complementarity-determining region 3 (VH CDR3) sequences and were placed in 38 clusters. Notably, the MCL stereotypes were distinct from those reported for chronic lymphocytic leukemia. Based on somatic hypermutation (SHM) status, 238/807 sequences (29.5%) carried IGHV genes with 100% germ line identity; the remainder (569/807; 70.5%) exhibited different SHM impact, ranging from minimal (in most cases) to pronounced. Shared replacement mutations across the IGHV gene were identified for certain subgroups, especially those using IGHV3-21, IGHV1-8, and IGHV3-23. Comparison with other entities, in particular CLL, revealed that several of these mutations were "MCL-biased." In conclusion, MCL is characterized by a highly restricted immunoglobulin gene repertoire with stereotyped VH CDR3s and very precise SHM targeting, strongly implying a role for antigen-driven selection of the clonogenic progenitors. Hence, an antigen-driven origin of MCL could be envisaged, at least for subsets of cases.

Innate control of B cell responses.

Mature B cells generate protective immunity by undergoing immunoglobulin (Ig) class switching and somatic hypermutation, two Ig gene-diversifying processes that usually require cognate interactions with T cells that express CD40 ligand. This T cell-dependent pathway provides immunological memory but is relatively slow to occur. Thus, it must be integrated with a faster, T cell-independent pathway for B cell activation through CD40 ligand-like molecules that are released by innate immune cells in response to microbial products. Here, we discuss recent advances in our understanding of the interplay between the innate immune system and B cells, particularly at the mucosal interface. We also review the role of innate signals in the regulation of Ig diversification and production.

Recovering probabilities for nucleotide trimming processes for T cell receptor TRA and TRG V-J junctions analyzed with IMGT tools.

BACKGROUND: Nucleotides are trimmed from the ends of variable (V), diversity (D) and joining (J) genes during immunoglobulin (IG) and T cell receptor (TR) rearrangements in B cells and T cells of the immune system. This trimming is followed by addition of nucleotides at random, forming the N regions (N for nucleotides) of the V-J and V-D-J junctions. These processes are crucial for creating diversity in the immune response since the number of trimmed nucleotides and the number of added nucleotides vary in each B or T cell. IMGT sequence analysis tools, IMGT/V-QUEST and IMGT/JunctionAnalysis, are able to provide detailed and accurate analysis of the final observed junction nucleotide sequences (tool "output"). However, as trimmed nucleotides can potentially be replaced by identical N region nucleotides during the process, the observed "output" represents a biased estimate of the "true trimming process." RESULTS: A probabilistic approach based on an analysis of the standardized tool "output" is proposed to infer the probability distribution of the "true trimmming process" and to provide plausible biological hypotheses explaining this process. We collated a benchmark dataset of TR alpha (TRA) and TR gamma (TRG) V-J rearranged sequences and junctions analysed with IMGT/V-QUEST and IMGT/JunctionAnalysis, the nucleotide sequence analysis tools from IMGT, the international ImMunoGeneTics information system, http://imgt.cines.fr. The standardized description of the tool output is based on the IMGT-ONTOLOGY axioms and concepts. We propose a simple first-order model that attempts to transform the observed "output" probability distribution into an estimate closer to the "true trimming process" probability distribution. We use this estimate to test the hypothesis that Poisson processes are involved in trimming. This hypothesis was not rejected at standard confidence levels for three of the four trimming processes: TRAV, TRAJ and TRGV. CONCLUSION: By using trimming of rearranged TR genes as a benchmark, we show that a probabilistic approach, applied to IMGT standardized tool "outputs" opens the way to plausible hypotheses on the events involved in the "true trimming process" and eventually to an exact quantification of trimming itself. With increasing high-throughput of standardized immunogenetics data, similar probabilistic approaches will improve understanding of processes so far only characterized by the "output" of standardized tools.

Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes.

Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.

Clonal expansion of IgA-positive plasma cells and axon-reactive antibodies in MS lesions.

Immunoglobulin A (IgA), the predominant immunoglobulin class in mucosal secretions, has been found in the cerebrospinal fluid of patients with multiple sclerosis (MS). In this study we examined the infiltration of clonally expanded IgA plasma cells in lesions of MS brains. Sequences of complementarity-determining region 3 of IgA variable heavy chain (V(H)) genes demonstrated the clonal expansion of IgA-bearing plasma cells in MS lesions. Somatic mutations and ongoing intra-clonal mutations occurred in their V(H) genes. Immunohistochemical study demonstrated infiltration of dimer and polymer IgA1- and A2-positive plasma cells in perivascular spaces, in the parenchyma of MS lesions, and in the adjacent white matter. Double immunofluorescence staining showed binding of IgA antibody on axons and walls of microvessels in the areas of chronic active and inactive demyelination. Bielshowsky's silver impregnation revealed axonal damage in these areas. These findings suggest that IgA in the CNS are localized on axons in lesions and may contribute to axonal damage in MS.

Patients with chronic lymphocytic leukemia with mutated VH genes presenting with Binet stage B or C form a subgroup with a poor outcome.

BACKGROUND AND OBJECTIVES: The immunoglobulin VH gene mutation status is a strong prognostic indicator in B-cell chronic lymphocytic leukemia (CLL), since unmutated VH genes are correlated with short survival. However, the traditional cut-off level dividing mutated and unmutated cases, i.e. more or less than 2% mutations, has been questioned and other cut-offs have been suggested. We investigated whether an alternative cut-off should be applied and the relation of mutational status to another prognostic marker, Binet staging. DESIGN AND METHODS: VH gene mutation status was assessed in 332 CLL cases by polymerase chain reaction amplification and nucleotide sequencing and was further correlated with overall survival using different VH mutation cut-offs (1-7%) and Binet stage. RESULTS: After testing different mutation borders, the 2% cut-off remained the best discriminative level for determining prognosis. Interestingly, prognostic stratification was improved by combining the information on VH gene mutation status with that of Binet stage: unmutated cases (all stages, n=151, mutated cases with stage A (n=77), and mutated cases with stage B or C (n=37) had a median survival of 82, 179 and 74 months, respectively. INTERPRETATION AND CONCLUSIONS: CLL cases displaying mutated VH genes with Binet stage B or C had a survival similar to that of unmutated cases and significantly shorter than that of mutated stage A CLL. Our result reveals clinical heterogeneity within the VH mutated CLL group by inclusion of Binet stage data, a finding which is of importance when considering surrogate marker(s) for VH mutation status.

Analysis of VH genes in marginal zone lymphoma reveals marked heterogeneity between splenic and nodal tumors and suggests the existence of clonal selection.

BACKGROUND AND OBJECTIVES: To clarify the relationship between splenic (SMZL) and nodal marginal zone (NMZL) lymphomas, we analyzed immunoglobulin variable heavy chain (VH) gene usage and mutation patterns in these tumors. DESIGN AND METHODS: VH genes were cloned and sequenced from 49 lymphoma samples (35 SMZL and 14 NMZL). RESULTS: A biased usage of VH gene was found with overrepresentation of VH1 in SMZL cases (13/35) and VH4 in NMZL cases (7/14). Evidence for antigen driven mutations was identified in 8 SMZL and 4 NMZL cases. Three cases out of 18 with clones analyzed from spleen and peripheral blood demonstrated intra-clonal diversity, with evidence of clonal selection in one case, indicating the possibility of antigen-driven clonal expansion. Eleven SMZL cases (31%) but only 2 NMZL (14%) cases were unmutated. No differences in clinical outcome and overall survival were found between the unmutated and mutated cases. INTERPRETATION AND CONCLUSIONS: The pattern of somatic mutation and the VH gene segment usage appear to differ between SMZL and NMZL, suggesting that these are distinct pathological entities. Moreover, a biased usage of certain sequences suggests that tumor cells in SMZL may be subjected to antigen selection.

In vitro and in vivo studies on the generation of the primary T-cell receptor repertoire.

The primary T-cell receptor repertoire is generated by somatic rearrangement of discontinuous gene segments. The shape of the combinatorial repertoire is stereotypical and, in part, evolutionarily conserved among mammals. Rearrangement is initiated by specific interactions between the recombinase and the recombination signals (RSs) that flank the gene segments. Conserved sequence variations in the RS, which modulate its interactions with the recombinase, appear to be a major factor in shaping the primary repertoire. In vitro, biochemical studies have revealed distinct steps in these complex recombinase-RS interactions that may determine the final frequency of gene segment rearrangement. These studies offer a plausible model to explain gene segment selection, but new, more physiological approaches will have to be developed to verify and refine the mechanism by which the recombinase targets the RS in its endogenous chromosomal context in vivo.

Antisense intergenic transcription in V(D)J recombination.

Antigen receptor genes undergo variable, diversity and joining (V(D)J) recombination, which requires ordered large-scale chromatin remodeling. Here we show that antisense transcription, both genic and intergenic, occurs extensively in the V region of the immunoglobulin heavy chain locus. RNA fluorescence in situ hybridization demonstrates antisense transcription is strictly developmentally regulated and is initiated during the transition from DJ(H) to VDJ(H) recombination and terminates concomitantly with VDJ(H) recombination. Our data show antisense transcription is specific to the V region and suggest transcripts extend across several genes. We propose that antisense transcription remodels the V region to facilitate V(H)-to-DJ(H) recombination. These findings have wider implications for V(D)J recombination of other antigen receptor loci and developmental regulation of multigene loci.

Ganglion cell axon pathfinding in the retina and optic nerve.

The eye is a highly specialized structure that gathers and converts light information into neuronal signals. These signals are relayed along axons of retinal ganglion cells (RGCs) to visual centers in the brain for processing. In this review, we discuss the pathfinding tasks RGC axons face during development and the molecular mechanisms known to be involved. The data at hand support the presence of multiple axon guidance mechanisms concentrically organized around the optic nerve head, each of which appears to involve both growth-promoting and growth-inhibitory guidance molecules. Together, these strategies ensure proper optic nerve formation and establish the anatomical pathway for faithful transmission of information between the retina and the brain.

Pax5 activates immunoglobulin heavy chain V to DJ rearrangement in transgenic thymocytes.

Mice deficient for the B cell-restricted transcription factor Pax5 show a defect in the VH to DJH rearrangement step of immunoglobulin heavy chain gene assembly even though the expression of the V(D)J recombinase is not diminished in Pax5-/- pro-B cells. To investigate whether Pax5 is limiting for VH to DJH rearrangement, we generated transgenic mice which express Pax5 in developing thymocytes. We show that enforced expression of Pax5 in thymocytes results in a partial block in T cell development due to defective pre-TCR signaling in beta-selection. Moreover, our results demonstrate that expression of Pax5 in early thymocytes is sufficient to induce VH to DJH rearrangements in CD4+CD8+ T cells and lead us to suggest that Pax5 may play a direct role in the lineage-specific regulation of immunoglobulin heavy chain gene rearrangement.