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 app. 1% of affected individuals, are unknown. An autonomously signaling B-cell receptor (BCR) is essential for CLL pathogenesis. The objective of this study was functional characterization of the BCR of MBL in siblings of CLL patients and comparison of genetic variants in MBL-CLL siblings pairs. Screening of peripheral blood by flow cytometry detected 0.2-480 clonal CLL-phenotype cells per μL (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 cross-linking. This autonomous BCR signal was less intense than the signal originating from CLL BCR of their CLL sibling. 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. 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 CLL pathogenetic model wherein 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.

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DOI: https://doi.org/10.3324/haematol.2022.282542

The ongoing COVID-19 pandemic is arguably one of the most challenging health crises in modern times. The development of effective strategies to control the spread of SARS-CoV-2 were major goals for governments and policy makers. Mathematical modeling and machine learning emerged as potent tools to guide and optimize the different control measures. This review briefly summarizes the SARS-CoV-2 pandemic evolution during the first 3 years. It details the main public health challenges focusing on the contribution of mathematical modeling to design and guide government action plans and spread mitigation interventions of SARS-CoV-2. Next describes the application of machine learning methods in a series of study cases, including COVID-19 clinical diagnosis, the analysis of epidemiological variables, and drug discovery by protein engineering techniques. Lastly, it explores the use of machine learning tools for investigating long COVID, by identifying patterns and relationships of symptoms, predicting risk indicators, and enabling early evaluation of COVID-19 sequelae.

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DOI: https://doi.org/10.3389/fpubh.2023.1140353

Genomic surveillance of infectious diseases allows monitoring circulating and emerging variants and quantifying their epidemic potential. However, due to the high costs associated with genomic sequencing, only a limited number of samples can be analysed. Thus, it is critical to understand how sampling impacts the information generated. Here, we combine a compartmental model for the spread of COVID-19 (distinguishing several SARS-CoV-2 variants) with different sampling strategies to assess their impact on genomic surveillance. In particular, we compare adaptive sampling, i.e., dynamically reallocating resources between screening at points of entry and inside communities, and constant sampling, i.e., assigning fixed resources to the two locations. We show that adaptive sampling uncovers new variants up to five weeks earlier than constant sampling, significantly reducing detection delays and estimation errors. This advantage is most prominent at low sequencing rates. Although increasing the sequencing rate has a similar effect, the marginal benefits of doing so may not always justify the associated costs. Consequently, it is convenient for countries with comparatively few resources to operate at lower sequencing rates, thereby profiting the most from adaptive sampling. Finally, our methodology can be readily adapted to study undersampling in other dynamical systems.

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DOI: https://doi.org/10.1016/j.chaos.2022.113093

Clinical and molecular heterogeneity are hallmarks of chronic lymphocytic leukemia (CLL), a neoplasm characterized by accumulation of mature and clonal long-lived CD5 + B-lymphocytes. Mutational status of the IgHV gene of leukemic clones is a powerful prognostic tool in CLL, and it is well established that unmutated CLLs (U-CLLs) have worse evolution than mutated cases. Nevertheless, progression and treatment requirement of patients can evolve independently from the mutational status. Microenvironment signaling or epigenetic changes partially explain this different behavior. Thus, we think that detailed characterization of the miRNAs landscape from patients with different clinical evolution could facilitate the understanding of this heterogeneity. Since miRNAs are key players in leukemia pathogenesis and evolution, we aim to better characterize different CLL behaviors by comparing the miRNome of clinically progressive U-CLLs vs. stable U-CLLs. Our data show up-regulation of miR-26b-5p, miR-106b-5p, and miR-142-5p in progressive cases and indicate a key role for miR-26b-5p during CLL progression. Specifically, up-regulation of miR-26b-5p in CLL cells blocks TGF-β/SMAD pathway by down-modulation of SMAD-4, resulting in lower expression of p21−Cip1 kinase inhibitor and higher expression of c-Myc oncogene. This work describes a new molecular mechanism linking CLL progression with TGF-β modulation and proposes an alternative strategy to explore in CLL therapy.

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DOI: https://doi.org/10.3390/cancers14071676

SARS-CoV-2 variant Lambda was dominant in several South American countries, including Chile. To ascertain the efficacy of local vaccination efforts, we used pseudotyped viruses to characterize the neutralization capacity of antibodies elicited by CoronaVac (n = 53) and BNT162b2 (n = 56) in healthcare workers from Clínica Santa María and the Faculty of Medicine at Universidad de Chile, as well as in convalescent plasma from individuals infected during the first wave visiting the Hospital Clínico at Pontificia Universidad Católica (n = 30). We observed that BNT162b2 elicits higher neutralizing antibody titres than CoronaVac, with differences ranging from 7.4-fold for the ancestral spike (Wuhan-Hu-1) to 8.2-fold for the Lambda spike and 13-fold for the Delta spike. Compared with the ancestral virus, neutralization against D614G, Alpha, Gamma, Lambda and Delta variants was reduced by between 0.93- and 4.22-fold for CoronaVac, 1.04- and 2.38-fold for BNT162b2, and 1.26- and 2.67-fold for convalescent plasma. Comparative analyses among the spike structures of the different variants suggest that mutations in the spike protein from the Lambda variant, including the 246–252 deletion in an antigenic supersite at the N-terminal domain loop and L452Q/F490S within the receptor-binding domain, may account for immune escape. Interestingly, analyses using pseudotyped and whole viruses showed increased entry rates into HEK293T-ACE2 cells, but reduced replication rates in Vero-E6 cells for the Lambda variant when compared with the Alpha, Gamma and Delta variants. Our data show that inactivated virus and messenger RNA vaccines elicit different levels of neutralizing antibodies with different potency to neutralize SARS-CoV-2 variants, including the variant of interest Lambda.

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DOI: https://doi.org/10.1038/s41564-022-01092-1

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

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DOI: https://doi.org/10.3389/fimmu.2021.807015

Immune responses at the boundary between the host and the world beyond are complex and mucosal tissue homeostasis relies on them. Obstructive sleep apnea (OSA) is a syndrome suffered by children with hypertrophied tonsils. We have previously demonstrated that these tonsils present a defective regulatory B cell (Breg) compartment. Here, we extend those findings by uncovering the crucial role of resident pro-inflammatory B and T cells in sustaining tonsillar hypertrophy and hyperplasia by producing TNFα and IL17, respectively, in ex vivo cultures. Additionally, we detected prominent levels of expression of CD1d by tonsillar stratified as well as reticular epithelium, which have not previously been reported. Furthermore, we evidenced the hypertrophy of germinal centers (GC) and the general hyperplasia of B lymphocytes within the tissue and the lumen of the crypts. Of note, such B cells resulted mainly (IgG/IgM)+ cells, with some IgA+ cells located marginally in the follicles. Finally, by combining bacterial culture from the tonsillar core and subsequent identification of the respective isolates, we determined the most prevalent species within the cohort of OSA patients. Although the isolated species are considered normal oropharyngeal commensals in children, we confirmed their capacity to breach the epithelial barrier. Our work sheds light on the pathological mechanism underlying OSA, highlighting the relevance taken by the host immune system when defining infection versus colonization, and opening alternatives of treatment.

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DOI: https://doi.org/10.3389/fimmu.2021.648064

Activation-induced deaminase (AID) is required for somatic hypermutation in immunoglobulin genes, but also induces off-target mutations. Follicular lymphoma (FL) and chronic lymphocytic leukemia (CLL), the most frequent types of indolent B-cell tumors, are exposed to AID activity during lymphomagenesis. We designed a workflow integrating de novo mutational signatures extraction and fitting of COSMIC (Catalogue Of Somatic Mutations In Cancer) signatures, with tridimensional chromatin conformation data (Hi-C). We applied the workflow to exome sequencing data from lymphoma samples. In 33 FL and 30 CLL samples, 42% and 34% of the contextual mutations could be traced to a known AID motif. We demonstrate that both CLL and FL share mutational processes dominated by spontaneous deamination, failures in DNA repair, and AID activity. The processes had equiproportional distribution across active and nonactive chromatin compartments in CLL. In contrast, canonical AID activity and failures in DNA repair pathways in FL were significantly higher within the active chromatin compartment. Analysis of DNA repair genes revealed a higher prevalence of base excision repair gene mutations (p = 0.02) in FL than CLL. These data indicate that AID activity drives the genetic landscapes of FL and CLL. However, the final result of AID-induced mutagenesis differs between these lymphomas depending on chromatin compartmentalization and mutations in DNA repair pathways.

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DOI: https://doi.org/10.3390/ijms222313015

The emergence of SARS-CoV-2 variants, as observed with the D614G spike protein mutant, and more recently with B.1.1.7 (501Y.V1), B.1.351 (501Y.V2) and B.1.1.28.1 (P.1) lineages, represent a continuous threat and might lead to strains of higher infectivity and/or virulence. We report on the occurrence of a SARS-CoV-2 haplotype with nine mutations including D614G/T307I double-mutation of the spike. This variant expanded and completely replaced previous lineages within a short period in the subantarctic Magallanes Region, southern Chile. The rapid lineage shift was accompanied by a significant increase of cases, resulting in one of the highest incidence rates worldwide. Comparative coarse-grained molecular dynamic simulations indicated that T307I and D614G belong to a previously unrecognized dynamic domain, interfering with the mobility of the receptor binding domain of the spike. The T307 mutation showed a synergistic effect with the D614G. Continuous surveillance of new mutations and molecular analyses of such variations are important tools to understand the molecular mechanisms defining infectivity and virulence of current and future SARS-CoV-2 strains.

DOI Zenodo: 10.5281/zenodo.4730896
Material disponible en Zenodo

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DOI: https://doi.org/10.3390/v13050883

The enzyme activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin (Ig) genes, critical actions for an effective adaptive immune response. However, in addition to the benefits generated by its physiological roles, AID is an etiological factor for the development of human and murine leukemias and lymphomas. This review highlights the pathological role of AID and the consequences of its actions on the development, progression, and therapeutic refractoriness of chronic lymphocytic leukemia (CLL) as a model disease for mature lymphoid malignancies. First, we summarize pertinent aspects of the expression and function of AID in normal B lymphocytes. Then, we assess putative causes for AID expression in leukemic cells emphasizing the role of an activated microenvironment. Thirdly, we discuss the role of AID in lymphomagenesis, in light of recent data obtained by NGS analyses on the genomic landscape of leukemia and lymphomas, concentrating on the frequency of AID signatures in these cancers and correlating previously described tumor-gene drivers with the presence of AID off-target mutations. Finally, we discuss how these changes could affect tumor suppressor and proto-oncogene targets and how they could be associated with disease progression. Collectively, we hope that these sections will help to better understand the complex paradox between the physiological role of AID in adaptive immunity and its potential causative activity in B-cell malignancies.

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DOI: https://doi.org/10.3389/fonc.2021.634383

Most cancers become more dangerous by the outgrowth of malignant subclones with additional DNA mutations that favor proliferation or survival. Using chronic lymphocytic leukemia (CLL), a disease exemplary of this process, and a model for neoplasms in general, we created transgenic mice overexpressing the enzyme, activation-induced deaminase (AID), whose normal function is to induce DNA mutations in B lymphocytes. AID allows normal B lymphocytes to develop more effective immunoglobulin (Ig)-mediated immunity, but also is able to mutate non-Ig genes, predisposing to cancer. In chronic lymphocytic leukemia (CLL), AID expression correlates with poor prognosis suggesting a role for this enzyme in disease progression. Nevertheless, direct experimental evidence identifying the specific genes that are mutated by AID and indicating that those genes are associated with disease progression is not available. To address this point, we overexpressed Aicda in a murine model of CLL (Eμ-TCL1). Analyses of TCL1/AID mice demonstrate a role for AID in disease kinetics, CLL-cell proliferation, and the development of cancer-related target mutations with canonical AID signatures in non-Igs genes. Notably, our mouse models can accumulate mutations in the same genes that are mutated in human cancers. Moreover, some of these mutations occur at homologous positions, leading to identical or chemically-similar amino acid substitutions as in human CLL and lymphoma. Together, these findings support a direct link between aberrant AID activity and CLL driver mutations that are then selected for their oncogenic effects, whereby AID promotes aggressiveness in CLL and other B-cell neoplasms.

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DOI: https://doi.org/10.1182/blood.2020008654

The use of convalescent plasma (CP) to treat COVID-19 has shown promising results; however, its effectiveness remains uncertain. The purpose of this study was to determine the safety and mortality of CP among patients hospitalized with COVID-19. Study Design and Methods: This multicenter, open-label, uncontrolled clinical trial is currently being conducted at nine hospitals in Chile. Patients hospitalized due to COVID-19 who were still within 14 days since symptom onset were classified into four groups: Patients with cancer and severe COVID-19. Patients with cancer and non-severe COVID-19. Patients with severe COVID-19 and patients with non-severe COVID-19 only. The intervention involved two 200-cc. CP transfusions with anti-SARS-CoV-2 IgG titers ≥ 1:320 collected from COVID-19-recovered donors. Results: 192 patients hospitalized for COVID-19 received CP transfusions. At the first transfusion, 90.6% fulfilled the criteria for severity, and 41.1% required mechanical ventilation. 11.5% of the patients had cancer. Overall 7-day and 30-day mortality since the first CP transfusion was 5.7% and 16.1% respectively. There were no differences at either time point in mortality between the four groups. Patients on mechanical ventilation when receiving CP had higher mortality rates than those who were not (22.8% vs. 11.5%; p = 0.037). Overall 30-day mortality was higher in patients over 65 than in younger patients (p = 0.019). Severe adverse events were reported in four patients (2.1%) with an overall transfusion-related lung injury rate of 1.56%. No CP-related deaths occurred. Discussion: CP is safe when used in patients with COVID-19 even when also presenting severity criteria or risk factors. Our mortality rate is comparable to reports from larger studies. Controlled clinical trials are required to determine efficacy.

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DOI: https://doi.org/10.1101/2020.11.30.20218560

Predicting the effect of mutations in proteins is one of the most critical challenges in protein engineering; by knowing the effect a substitution of one (or several) residues in the protein’s sequence has on its overall properties, could design a variant with a desirable function. New strategies and methodologies to create predictive models are continually being developed. However, those that claim to be general often do not reach adequate performance, and those that aim to a particular task improve their predictive performance at the cost of the method’s generality. Moreover, these approaches typically require a particular decision to encode the amino acidic sequence, without an explicit methodological agreement in such endeavor. To address these issues, in this work, we applied clustering, embedding, and dimensionality reduction techniques to the AAIndex database to select meaningful combinations of physicochemical properties for the encoding stage. We then used the chosen set of properties to obtain several encodings of the same sequence, to subsequently apply the Fast Fourier Transform (FFT) on them. We perform an exploratory stage of Machine-Learning models in the frequency space, using different algorithms and hyperparameters. Finally, we select the best performing predictive models in each set of properties and create an assembled model. We extensively tested the proposed methodology on different datasets and demonstrated that the generated assembled model achieved notably better performance metrics than those models based on a single encoding and, in most cases, better than those previously reported. The proposed method is available as a Python library for non-commercial use under the GNU General Public License (GPLv3) license.

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The purpose of genome sequencing is to determine the DNA sequence of a given organism. Current sequencing technologies can be classified by the type of output data. Whereas Nanopore technology generates long reads with high error rates, short read technologies – such as Illumina sequencing – generate shorter reads but with low error rate. Since de novo genome assembly of sequencing reads is defined as a NP-hard problem, it remains as one of the major challenges for defining reference genomes of different species. This paper aims to improve the quality of reads obtained through Oxford Nanopore Technologies (ONT). We developed an algorithm to associate the reads obtained from Illumina with the ones obtained with Nanopore. Low accuracy ONT reads were corrected with the high quality Illumina reads to achieve an improved sequencing data. The inclusion of this algorithm as a preprocessing step resulted in improved coverage, contig length, and mismatch rate when performing de novo genome assembly of a bacterial genome with well known tools.

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DOI: https://doi.org/10.1007/978-3-030-58814-4_63

Understanding the mechanisms of interaction between antigen-antibody systems is one of the most relevant problems in the field of Immunology, since, supported by computational tools and data mining techniques, it would be possible to implement new antibody design tools. As a first approach and taking advantage of microarray techniques, a biological pattern recognition pipeline is presented from multiple ProtoArray chips, based on data mining techniques and bioinformatic analysis. This new approach, proposes 3 key stages in data processing: cleaning, statistical-frequency clustering of antigens and characterization by bioinformatic methods. This methodology was tested in a set of 370,000 interactions (45 antibodies, 8,000 antigens), achieving an effective filtering and grouping, in addition, a group obtained is characterized, in order to test the proposed bioinformatic methods. It is expected that this new method allows the identification of patterns in a simple and efficient way from multidimensional sets generated by protein microarrays.

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DOI: 10.1109/SCCC49216.2019.8966421

The immunoglobulin receptor represents a central molecule in acquired immunity. The complete set of immunoglobulins present in an individual is known as immunological repertoire. The identification of this repertoire is particularly relevant in immunology and cancer research and diagnostics. In a seminal work we provided a proof of concept of the novel ARTISAN-PCR amplification method, we adapted this technology for sequencing using Nanopore technology. This approach may represent a faster, more portable and cost-effective alternative to current methods. In this study we present the pipeline for the analysis of immunological repertoires obtained by this approach. This paper shows the performance of immune repertoires sequenced by Nanopore technology, using measures of error, coverage and gene usage identification.

In the bioinformatic methodology used in this study, first, Albacore Base calling software, was used to translate the electrical signal of Nanopore to DNA bases. Subsequently, the sequons, introduced during amplification, were aligned using bl2seq from Blast. Finally, selected reads were mapped using IMGT/HighV-QUEST and IgBlast.

Our results demonstrate the feasibility of immune repertoire sequencing by Nanopore technology, obtaining higher depth than PacBio sequencing and better coverage than pair-end based technologies. However, the high rate of systematic errors indicates the need of improvements in the analysis pipeline, sequencing chemistry and/or molecular amplification.

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DOI: 10.1007/978-3-030-17938-0_27

IgE-mediated allergic disease represents an increasing health problem. Although numerous studies have investigated IgE sequences in allergic patients, little information is available on the healthy IgE repertoire. IgM, IgG, IgA, and IgE transcripts from peripheral blood B cells of five healthy, non-atopic individuals were amplified by unbiased, template-switching, isotype-specific PCR. Complete VDJ regions were sequenced to near-exhaustion on the PacBio platform. Sequences were analyzed for clonal relationships, degree of somatic hypermutation, IGHV gene usage, evidence of antigenic selection, and N-linked glycosylation motifs. IgE repertoires appeared to be highly oligoclonal with preferential usage of certain IGHV genes compared to the other isotypes. IgE sequences carried more somatic mutations than IgM, yet fewer than IgG and IgA. Many IgE sequences contained N-linked glycosylation motifs. IgE sequences had no clonal relationship with the other isotypes. The IgE repertoire in healthy individuals is derived from relatively few clonal expansions without apparent relations to immune reactions that give rise to IgG or IgA. The mutational burden of normal IgE suggests an origin through direct class-switching from the IgM repertoire with little evidence of antigenic drive, and hence presumably low affinity for specific antigens. These findings are compatible with a primary function of the healthy IgE repertoire to occupy Fcε receptors for competitive protection against mast cell degranulation induced by allergen-specific, high-affinity IgE. This background knowledge may help to elucidate pathogenic mechanisms in allergic disease and to design improved desensitization strategies.

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DOI: 10.3389/fimmu.2019.01543

Lipoprotein lipase (LPL) mRNA expression in chronic lymphocytic leukaemia (CLL) is associated with an unmutated immunoglobulin profile and poor clinical outcome. We evaluated the subcellular localization of LPL protein in CLL cells that did or did not express LPL mRNA. Our results show that LPL protein is differently located in CLL cells depending on whether it is incorporated from the extracellular medium in mutated CLL or generated de novo by leukaemic cells of unmutated patients. The specific quantification of endogenous LPL protein correlates with mRNA expression levels and mutational IGHV status, suggesting LPL protein as a possible reliable prognostic marker in CLL.

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DOI: https://doi.org/10.1111/bjh.15427

The comprehension of unconventional immune functions of tonsillar B cells, their role in tolerance induction and protective immune responses, is crucial to unveil the dynamic interactions of the upper aero digestive tract with polymicrobial commensal flora and pathogens, in health and disease. Here, we describe the kinetics of IL10 intracellular expression and compare it with that of cytokines known to be produced by tonsillar B cells. Additionally, we detected a relevant proportion of IL17-expressing tonsillar B cells, which has not previously been reported. We immunophenotyped tonsillar IL10-expressing B cells (B10) and observed IL10 production in activated B cells at every developmental stage. Finally, we identified a relationship between decreased B10 percentages, increased proportion of the germinal centre (GC) population and hypertrophied tonsils (HT). Our findings provide greater insight into the role of B10 in GC reactions and characterized their involvement in the pathogenesis of tonsillar dysfunction

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DOI: https://doi.org/10.1038/s41598-017-09689-x

Patients with leukemia who receive a T cell–depleted allogeneic stem cell graft followed by postponed donor lymphocyte infusion (DLI) can experience graft-versus-leukemia (GVL) reactivity, with a lower risk of graft-versus-host disease (GVHD). Here, we have investigated the magnitude, diversity, and specificity of alloreactive CD8 T cells in patients who developed GVL reactivity after DLI in the absence or presence of GVHD. We observed a lower magnitude and diversity of CD8 T cells for minor histocompatibility antigens (MiHAs) in patients with selective GVL reactivity without GVHD. Furthermore, we demonstrated that MiHA-specific T cell clones from patients with selective GVL reactivity showed lower reactivity against nonhematopoietic cells, even when pretreated with inflammatory cytokines. Expression analysis of MiHA-encoding genes showed that similar types of antigens were recognized in both patient groups, but in patients who developed GVHD, T cell reactivity was skewed to target broadly expressed MiHAs. As an inflammatory environment can render nonhematopoietic cells susceptible to T cell recognition, prevention of such circumstances favors induction of selective GVL reactivity without development of GVHD.

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DOI: 10.1172/JCI86175

Follicular lymphoma (FL) is the second most common indolent B-cell lymphoma (1). FL is characterized by a heterogeneous clinical evolution and remains incurable despite recent progress. The accepted founding event of FL pathogenesis is the acquisition of the t(14;18)(q32;q21) during primary VDJ recombination in the bone marrow (2). This chromosomal translocation juxtaposes the antiapoptotic proto-oncogene BCL2 to the immunoglobulin heavy chain locus during VDJ recombination (3). The resulting dysregulation of BCL2 expression provides a survival advantage through activation of antiapoptotic programs. The consequences of this early genetic event become manifest much later during B-cell maturation, when FL cells clonally expand and adopt malignant growth in germinal centers (GC) (2). The precise mechanisms involved in the initiation of malignant growth of apoptosisresistant t(14:18)-positive B-cells remain unclear.

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DOI: Marcelo A. Navarrete, Pablo Oppezzo

B cells recognize specific antigens by their membrane‐bound B‐cell receptor (BCR). Functional BCR genes are assembled in pre‐B cells by recombination of the variable (V), diversity (D) and joining (J) genes [V(D)J recombination]. When B cells participate in germinal centre reactions, non‐templated point mutations are introduced into BCR genes by somatic hypermutation (SHM) (Rajewsky, 1996). V(D)J recombination and SHM create virtually unlimited BCR repertoires.

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DOI: 10.1111/bjh.14180

Follicular lymphoma (FL) has a paradigmatic stepwise pathogenesis with the balanced translocation (14;18)(q32;q21) as the founding event (Kridel et al, 2012). Most healthy individuals harbour t(14;18)‐positive premalignant FL‐like cells (FLLC) (Roulland et al, 2011). FLLC have a memory phenotype and have often undergone immunoglobulin class switch recombination (CSR) on the translocated IGH allele. Frank FL develops only after repetitive entry of FLLC into germinal centres (GC) and multiple GC passages (Sungalee et al, 2014). Paradoxically, FLLC fail to switch the functional IGH allele, suggesting dependence on IgM signalling at this stage. In contrast, many diagnosed FL cases have undergone CSR of their B‐cell receptor (BCR) (Sachen et al, 2012; Scherer et al, 2015). Therefore, class‐switched FL cells have apparently lost the characteristic dependency of FLLC on IgM signalling for recruitment into the GC. In addition, expression of activation‐induced deaminase (AID) seems to be dissociated from somatic hypermutation (SHM) and aberrant SHM in class‐switched FL (Scherer et al, 2015).

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DOI: 10.1111/bjh.13901

In B-cells, activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) and class switch recombination (CSR) of immunoglobulin genes. AID introduces mutations in immunoglobulin variable regions (IGV) during B-cell receptor affinity maturation, but may also introduce aberrant mutations into non-immunoglobulin genes, most commonly BCL6. Follicular lymphoma (FL) B-cells constitutively express AID and undergo CSR, SHM and aberrant SHM. We have studied AID expression, the presence of SHM mutations, CSR, and aberrant SHM in BCL6 in a cohort of 75 FL patients. Whereas IgM-expressing (non-switched) FL were characterized by an expected positive correlation between AID and IGV and BCL6 mutations, isotype-switched FL showed dissociation between AID expression and aberrant SHM, and inverse correlation between SHM and AID expression. Our results unveil two manifest biological subgroups of FL and indicate that the specific dissociation between AID and SHM after isotype switch may correlate with the clinical outcome of this heterogeneous disease.

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DOI: https://doi.org/10.3109/10428194.2015.1037758

Antigen receptors of B cells (BCR) exhibit a virtually unlimited repertoire, created by V(D)J recombination through combinatorial and junctional diversity of genetic elements in B-precursor cells. B cells further increase the antigen affinity of their BCR through somatic hypermutation (SHM). VDJ recombination and SHM create unique and novel peptide sequences that are not encoded in germline (GL) and may, therefore, act as neoantigens for the adaptive immune system. Naturally occurring BCR-directed immunity could, therefore, influence expansion of individual B-cell clones. We have previously found evidence for HLA class I-dependent, BCR-directed immunosurveillance in follicular lymphoma (FL).1 We here report lack of evidence for this mechanism in other indolent B-cell lymphomas and an apparent dependence of possible immunosurveillance on the high SHM rate in FL.

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DOI: 10.3324/haematol.2014.111252

Discovering functionalities for unknown enzymes has been one of the most common bioinformatics tasks. Functional annotation methods based on phylogenetic properties have been the gold standard in every genome annotation process. However, these methods only succeed if the minimum requirements for expressing similarity or homology are met. Alternatively, machine learning and deep learning methods have proven helpful in this problem, developing functional classification systems in various bioinformatics tasks. Nevertheless, there needs to be a clear strategy for elaborating predictive models and how amino acid sequences should be represented. In this work, we address the problem of functional classification of enzyme sequences (EC number) via machine learning methods, exploring various alternatives for training predictive models and numerical representation methods. The results show that the best performances are achieved by applying representations based on pre-trained models. However, there needs to be a clear strategy to train models. Therefore, when exploring several alternatives, it is observed that the methods based on CNN architectures proposed in this work present a more outstanding facility for learning and pattern extraction in complex systems, achieving performances above 97% and with error rates lower than 0.05 of binary cross entropy. Finally, we discuss the strategies explored and analyze future work to develop integrated methods for functional classification and the discovery of new enzymes to support current bioinformatics tools.

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DOI: https://doi.org/10.1007/978-3-031-34953-9_24

Predicting the affinity between two proteins is one of the most relevant challenges in bioinformatics and one of the most useful for biotechnological and pharmaceutical applications. Current prediction methods use the structural information of the interaction complexes. However, predicting the structure of proteins requires enormous computational costs. Machine learning methods emerge as an alternative to this bioinformatics challenge. There are predictive methods for protein affinity based on structural information. However, for linear information, there are no development guidelines for elaborating predictive models, being necessary to explore several alternatives for processing and developing predictive models. This work explores different options for building predictive protein interaction models via deep learning architectures and classical machine learning algorithms, evaluating numerical representation methods and transformation techniques to represent structural complexes using linear information. Six types of predictive tasks related to the affinity and mutational variant evaluations and their effect on the interaction complex were explored. We show that classical machine learning and convolutional network-based methods perform better than graph convolutional network methods for studying mutational variants. In contrast, graph-based methods perform better on affinity problems or association constants, using only the linear information of the protein sequences. Finally, we show an illustrative use case, expose how to use the developed models, discuss the limitations of the explored methods and comment on future development strategies for improving the studied processes.

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DOI: https://doi.org/10.1007/978-3-031-36805-9_16
Lugar del congreso: Atenas, Grecia.

Somatic mutations associated with distinctive types of cancer can be globally classified by the trinucleotide context of single nucleotide variants into mutational signatures. Distinct signatures can be attributed to underlying causative mechanisms such as UV irradiation or smoking. B-cell lymphomas have only been analyzed globally and carry a signature characterized by APOBEC activity (Alexandrov, 2014).

The two most common indolent B-cell neoplasms, chronic lymphocytic leukemia (CLL) and follicular lymphoma (FL), are characterized by distinctive pathogenic processes, but mutagenesis caused by the APOBEC enzyme activation-induced cytosine deaminase (AID) may be shared by both malignancies. AID deaminates deoxycytidine to deoxyuridine, which is processed by DNA repair enzymes into a larger spectrum of point mutations and also DNA double-strand breaks. AID activity and subsequent DNA repair create two different mutation signatures: the canonical signature characterized by C>T/G transitions at RCY motifs and the non-canonical defined by A>C transversions at WAN motifs. More recently a third AID signature characterized by C>T transitions at RCG motifs has been associated with AID-mediated CpG-methylation dependent mutagenesis.

We here investigate the hypothesis that AID mutational signatures contribute to the pathogenesis of FL, a lymphoma characterized by constitutional expression of AID, but also of CLL since high AID expression levels may account for a more aggressive disease, and a subset of CLL B-cells with AID expression display dissociation between class switch recombination and somatic hypermutation.

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DOI: https://doi.org/10.1182/blood.V130.Suppl_1.2741.2741
Lugar del congreso: Atlanta, GA. USA: Blood; 2017

Activation-induced cytidine deaminase (AID) initiates somatic hypermutation (SHM) and class switch recombination (CSR) of the immunoglobulin (Ig) genes,which are critically important for an effective immune response development. In addition, AID seems to contribute to B cell tolerance in mice and humans eliminating developing autoreactive B cells. As a trade-off of the beneficial roles of AID physiological activity, this enzyme can also contribute to cellular transformation and tumor progression through its off-target mutagenic activity. Mounting evidences indicate a role for AID in disease progression and poor prognosis of chronic lymphoid neoplasms. Since these leukemias are not immediately derived from germinal center B cells, normal AID regulation might not be fully functional. Specifically, in CLL we have shown that AID isanomalously expressed in the peripheral blood of patients with Unmutated (Um) VH genes, correlating with active CSR and poor clinical outcome (Oppezzo et al., Blood 2005). In addition, others and us have described that AID expression is mainly restricted to distinct proliferative subsets within the leukemic clone(Albesiano et al., Blood, 2003; Palacios et al., Blood, 2010 and Patten et al., Blood, 2012). Despite the fact that AID expression has been associated with adverse cytogenetics and worse outcome (Leuenberger, M., XVII INTERNATIONAL WORKSHOP ON CHRONIC LYMPHOCYTIC LEUKEMIA 2017 207 Downloaded by [University of Florida] at 09:55 17 December 2017 Mod Pathol., 2010) definitive experimental demonstration that AID contributes to CLL progression is missing. To obtain insight into this issue we analyzed mutation load and signatures in Um patients in which anomalous expression of AID in peripheral blood was sustainedthroughout disease evolution. We compared the mutational profile by Whole Exome Sequencing(WES) of purified CD19þ cells extracted from 5 progressivepatients expressing AID (Um/AIDpos) andfrom 5 progressive patients in which AID was notdetected (Um/AIDneg). Samples were obtained atdiagnosis and at second time point after the first treatment(AFT). Analysis was performed according toGATK-best practice guidelines, variant discovery wasperformed by Varscan algorithm. Unsupervised AIDmutational profile was assessed by the Bayesian nonnegativematrix factorization algorithm as previouslydescribed (Alexandrov et. al., Nature, 2013), with thisstrategy we identified non-canonical AID signatures(nc-AID), i.e. A>C mutations at WA motifs in sensestrand and T>G mutation at TW motifs in antisensestrand. For supervised analysis of canonical AID signatures(c-AID) we quantified C to T mutations at WRCYmotifs in the sense strand or T to C variants in YGRWmotifs in antisense strand.We found that the total number of somatic mutationsis significantly increased in Um/AIDpos compared withUm/AIDneg patients independently of the diseasetime of the analyzed sample. A higher frequency ofsomatic mutations were observed in Um/AIDpos samplesat debut of the disease compared with Um/AIDpos samples AFT. We next analyzed mutation signaturesin the different groups: Um/AIDpos at diagnosis;Um/AIDpos AFT; Um/AIDneg at diagnosis and Um/AIDneg AFT. Our results show no significant differencesobserved between the groups concerning c-AIDmutations. However, an increased and significant differencewas found in the group Um/AIDpos comparedwith Um/AIDneg. Interestingly, a higher frequency ofnc-AID mutations was observed in Um/AIDpos groupat diagnosis time compared with Um/AIDpos AFT,which might suggest selection for samples with lowerAID levels and/or mutation loads. In addition, theunsupervised analysis identified the presence otherpreviously described signatures (Alexandrov et. al.,Nature, 2013), such as Aging, and APOBEC in all samples.We also analyzed AID-catalyzed mutations thatwere found at the debut of the disease and maintainedduring disease evolution, as well as those thatappeared only in the AFT samples. Presently, mutationsin selected genes are being confirmed in a largercohort. We will discuss these results.AID expression could be specially problematic inchronic diseases, where even a small but continuouslevel of AID activity can lead to selectable geneticmutations over time, giving rise to more aggressivetumors and treatment resistance. Our results supportthis notion and describes for the first time the mutationalprofile of Um and progressive patients withanomalous AID expression through their evolution.Under the light of the hypothesis that AID expressionis a hallmark of an activated microenvironment resultingin disease progression our results will allow us toidentify AID mutated genes which could be implicatedin CLL tumor progression.This work was supported by ANII. ProyectoFMV_1_2014_1_104397 and CSIC-Udelar. Programa deIþD 2014.

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DOI: 10.1080/10428194.2017.1377942
Lugar del congreso: XVII International Workshop on Chronic Lymphocytic Leukemia. New York; 2017

T cells recognizing solid tumors and Hodgkin’s lymphoma can be released from anergy by immune checkpoint inhibitors. Its therapeutic success seems to be associated with the abundance of neoepitopes within the tumor mutanome. In neoplastic B cells, VDJ recombination and somatic hypermutation (SHM) generate unique immunoglobulin (Ig) peptide sequences that predictably contribute to the lymphoma mutanome. Efficient presentation of a neoepitope by an individual’s HLA complex is an essential requirement for neoepitope-directed T-cell immunity. Presentation of Ig-derived peptides in HLA class I has hitherto been demonstrated only indirectly, and there is as yet no direct evidence for presentation of Ig-derived neoepitopes of primary human lymphoma cells.

We analyzed HLA-presented Ig-derived peptides in 9 clonal B-cell populations: 3 chronic lymphocytic leukemias (CLL), 1 hairy cell leukemia, 1 follicular lymphoma (FL), 3 EBV-transformed lymphoblastoid cell lines, and the U299 myeloma cell line. Full-length B-cell receptor (BCR) VDJ and VJ sequences were obtained by unbiased ARTISAN PCR and Pacific Biosciences next generation sequencing. 1067 unique Ig-derived nonamers were predicted to bind the HLA class I alleles expressed by the respective B-cell clone by the NetCTLpan bioinformatics tool. 650 candidate epitopes (60.9%) were derived from constant (C) regions; 417 (39.1%) from variable (V) regions. 146 predicted peptides from V regions (35.0%) contained at least one amino acid change due to SHM or at least one amino acid from CDR3 and were therefore considered potential neoepitopes.

To investigate experimentally which epitopes are processed intracellularly and presented by HLA class I, HLA class I-peptide complexes were immunoaffinity purified by the monoclonal antibody W6.23 and analyzed by liquid chromatography and tandem mass spectrometry. In total, 53,663 unique peptides (8-15mers) were identified by Uniprot matching with a Mascot Ion Score of >20. HLA ligandome sizes of individual B-cell populations ranged from 600 to 14,091 unique peptides per case. Within any HLA ligandome, 6 to 81 peptides were annotated as BCR-derived epitopes if they matched the individual BCR sequences. Of the total of 276 eluted BCR peptides, 203 (73.5%) where derived from C regions and 73 (26.5%) from V regions. 25 eluted peptides (range 0-10 per case) were derived from CDR3 regions or contained SHM-induced amino acid changes, thus fulfilling the definition of neoepitopes. Neoepitopes were detected in all cases with more than 109 cells as input material. Up to date, 4 neoepitopes have been synthesized and their mass spectra confirmed. Confirmation of all remaining neoepitopes is ongoing. No BCR-derived neoepitopes could be detected in an IGHV-unmutated CLL and the FL. These two cases had the lowest number of input cells and small overall ligandome sizes of only 600 and 1015 unique UniProt-matched peptides, respectively.

We demonstrate for the first time that primary neoplastic B cells process and present BCR-derived neoepitopes in the context of HLA class I. Despite their origin from only two polypeptides, BCR epitopes and neoepitopes represent app. 0.5% and 0.05% of the total HLA class I ligandome, respectively. The challenging identification of BCR neoepitopes was possible by unbiased identification of BCR transcripts combined with next generation sequencing and targeted search for HLA class I-bound peptides. Matching fragmentation patterns of native and synthetic peptides suggest high specificity of this strategy. With respect to sensitivity, the ability to identify low frequency peptides appears to be strongly dependent on the amount of input cells.

Our data close a gap in the mechanism underlying the evidence that highly immunogenic formulations of an idiotype vaccine are able to induce MHC class I-restricted cytotoxic T-cell responses. While phase III trials of idiotype vaccination aiming to induce anti-Ig antibodies failed to demonstrate convincing prolongation of clinical remissions achieved by chemotherapy, our data lend support for exploring idiotype-specific T-cell immunity against B-cell lymphomas. With recent advances in peptide synthesis, adjuvant formulations, and the availability of check point inhibitors to surpass regulatory activity, active immunotherapy targeting the lymphoma idiotype may regain appeal as truly personalized immune therapy.

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DOI: https://doi.org/10.1182/blood.V128.22.914.914

Background Primary mediastinal large B-cell lymphoma (PM LBCL) is putatively derived from thymic B cells. PM LBCL is clinically and morphologically distinct from diffuse large B-cell lymphoma (DLBCL), but shares certain phenotypic characteristics with nodular sclerosing classical Hodgkin’s lymphoma. The expression and function of an intact B-cell receptor (BCR) in PM LBCL is controversial. In addition, the pathological mechanisms that drive lymphomagenesis in PM DLBCL are largely unknown. Here, we investigate the expression of functional BCR in PM LBCL and explore the hypothesis that autonomous signaling of the BCR as observed in CLL (Dühren-von Minden, Nature 2012) plays a key role in PM DLBCL pathogenesis. Methods The PM LBCL cell lines Farage, MedB-1, Karpas 1106P, and U2940 were tested in vitro by comparing calcium flux with and without inhibition of BCR signaling by blockade of Syk with the tyrosine kinase inhibitor R406. BCR transcripts were identified from archived fresh-frozen biopsies from two histologically confirmed PM LBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching (Koning, BJH 2016). Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. To test for autonomous BCR signaling, murine triple KO (TKO) cells were transduced with functional BCR of PM LBCL (Dühren-von Minden, Nature 2012). TKO cells lack the rag2 and lambda5 genes and are therefore developmentally arrested at the pre-B-cell stage. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65 variant. When transduced with a functional BCR, autonomous or antigen-induced BCR signaling can be measured upon induction with tamoxifen as calcium flux by flow cytometry or as robust cellular proliferation. Results All four PM-LBCL lines had higher calcium flux in the absence of R406, suggesting autonomous BCR signaling activity without antigenic stimulation or artificial BCR crosslinking. Functional BCR transcripts could be readily identified in both primary PM LBCL biopsies by ARTISAN PCR. In accordance with our hypothesis, TKO cells transduced with the BCR of both primary PM LBCL cases showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. To test whether this autonomous BCR signal would represent a druggable therapeutic target, the PM DLBCL cell lines were cultured in the presence of increasing doses of the second generation, highly BTK-specific kinase inhibitor acalabrutinib. The ABC DLBCL cell lines OCI-Ly10, TMD8, and U2932, and the GCB cell lines Karpas 422, OCI-Ly7, and OCI-Ly19 were tested in parallel as controls. Cell viability decreased in all cell lines with increasing concentrations of acalabrutinib (Figure). Consistent with their known dependency on active BCR signalling (Young, PNAS 2015; Koning, ASH 2016), ABC DLBCL cell lines were very sensitive to acalabrutinib. As suggested by the experimental evidence for autonomous BCR signalling, PM LBCL cell lines also responded to acalabrutinib, albeit to a lesser degree than ABC DLBCL. Growth inhibition of GCB DLBCL cell lines generally required excessively high doses of acalabrutinib, consistent with the notion that this DLBCL subtype depends on tonic BCR signaling only. Conclusion Consistent with our hypothesis, our data demonstrate expression of an intact BCR with autonomous signaling activity in all investigated cases of PM LBCL. These results point to an oncogenic role of structurally normal BCR in PM LBCL akin to CLL. Similarly to CLL and ABC DLBCL, PM DLBCL cell lines respond to BTK inhibitors, albeit less strongly than the ABC DLBCL cell lines tested. The results identify BCR signaling as a novel therapeutic target in PM LBCL and suggest the initiation of clinical trials with BTK inhibitors in PM LBCL. Figure Figure. Disclosures No relevant conflicts of interest to declare.

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DOI: DOI: 10.1182/blood.V128.22.4171.4171

The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In app. 10% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CARD11. Recent evidence suggests that autoantigen recognition by the BCR sustains the viability of ABC DLBCL in vitro (Young, PNAS 2015), although the functional relationship to CARD11 mutations remains unresolved. Antigen-independent, constitutively active BCR signaling is a fundamental oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We therefore explored the hypothesis that autonomous BCR signaling akin to CLL could be operative in ABC DLBCL and would represent an alternative but functionally complementary oncogenic mechanism to activating CARD11 mutations in ABC DLBCL lymphomagenesis.

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DOI: https://doi.org/10.1182/blood.V128.22.778.778

The activated B-cell (ABC) type of diffuse large B-cell lymphoma (DLBCL) is clinically more aggressive than the germinal center (GCB) type. The gene expression profile of ABC DLBCL resembles that of mature B cells upon stimulation via their B-cell receptor (BCR). In up to 30% of ABC DLBCL cases, this signature can be explained by gain-of-function mutations in CD79A, CD79B, or CARD11. Antigen-independent, constitutively active (BCR) signaling is a general oncogenic mechanism of CLL (Dühren-von Minden, Nature 2012). We hypothesized that autonomous BCR signaling akin to CLL may operate in ABC DLBCL and could explain the characteristic ABC gene expression signature.

BCR transcripts were identified from fresh-frozen biopsies from 12 histologically confirmed, IgM-expressing DLBCL by ARTISAN PCR, a novel anchored RT-PCR for unbiased amplification of BCR transcripts facilitated by template switching. Clonal full-length BCR sequences were identified by PacBio next-generation sequencing. Triple KO (TKO) cells were transduced with functional DLBCL BCR. TKO cells are arrested at the pre-B-cell stage due to lack of the rag2 and lambda5 genes. In addition, TKO cells have their wild-type SLP65 adaptor replaced with a tamoxifen-dependent SLP65. When transduced with expression constructs encoding a functional BCR, autonomous or antigen-induced BCR signalling can be measured as calcium flux upon induction with tamoxifen.

TKO cells transduced with seven of the twelve DLBCL BCR (58%) showed robust calcium flux and proliferation upon activation of SLP65 by tamoxifen without additional BCR crosslinking. By histomorphology and immunohistochemistry for CD10, PAX5, MUM1, and Bcl-6, these cases were classified as non-GCB-type DLBCL, including one primary CNS DLBCL and two primary mediastinal (PM-)DLBCL. In accordance with our hypothesis, BCR from three DLBCL classified as GCB lacked an autonomous BCR signal. The remaining two negative cases were non-GCB-type and are currently being analyzed for activating CD79/CARD11 mutations.

To further explore the detection of autonomous BCR signaling in DLBCL, PM-DLBCL and ABC DLBCL cell lines were tested in vitro by comparing calcium flux with and without blockade of Syk signaling by the tyrosine kinase inhibitor R406. All four PM-DLBCL and two of five ABC DLBCL cell lines had higher calcium flux in the absence of R406, indicating autonomous signaling activity without antigenic stimulation or artificial BCR crosslinking.

In summary, we demonstrate autonomous BCR activity in a substantial fraction (72%) of non-GCB DLBCL (including PM-DLBCL). These results point to an oncogenic role of structurally normal BCR in non-GCB DLBCL akin to CLL. Autonomous BCR signaling is a candidate alternative mechanism to the previously described activating mutations of components of the BCR signaling cascade to induce the characteristic gene expression signature of ABC DLBCL. Formal delineation of autonomous BCR signalling to activating mutations in the BCR signalling cascade is ongoing.

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DOI: 10.1158/1538-7445.AM2016-LB-012