Purpose-Built Immunoinformatics for BcR IG/TR Repertoire Data Analysis.

The study of antigen receptor gene repertoires using next-generation sequencing (NGS) technologies has disclosed an unprecedented depth of complexity, requiring novel computational and analytical solutions. Several bioinformatics workflows have been developed to this end, including the T-cell receptor/immunoglobulin profiler (TRIP), a web application implemented in R shiny, specifically designed for the purposes of comprehensive repertoire analysis, which is the focus of this chapter. TRIP has the potential to perform robust immunoprofiling analysis through the extraction and processing of the IMGT/HighV-Quest output, via a series of functions, ensuring the analysis of high-quality, biologically relevant data through a multilevel process of data filtering. Subsequently, it provides in-depth analysis of antigen receptor gene rearrangements, including (a) clonality assessment; (b) extraction of variable (V), diversity (D), and joining (J) gene repertoires; (c) CDR3 characterization at both the nucleotide and amino acid level; and (d) somatic hypermutation analysis, in the case of immunoglobulin gene rearrangements. Relevant to mention, TRIP enables a high level of customization through the integration of various options in key aspects of the analysis, such as clonotype definition and computation, hence allowing for flexibility without compromising on accuracy.

Understanding Monoclonal B Cell Lymphocytosis: An Interplay of Genetic and Microenvironmental Factors.

The term monoclonal B-cell lymphocytosis (MBL) describes the presence of a clonal B cell population with a count of less than 5 × 109/L and no symptoms or signs of disease. Based on the B cell count, MBL is further classified into 2 distinct subtypes: 'low-count' and 'high-count' MBL. High-count MBL shares a series of biological and clinical features with chronic lymphocytic leukemia (CLL), at least of the indolent type, and evolves to CLL requiring treatment at a rate of 1-2% per year, whereas 'low-count' MBL seems to be distinct, likely representing an immunological rather than a pre-malignant condition. That notwithstanding, both subtypes of MBL can carry 'CLL-specific' genomic aberrations such as cytogenetic abnormalities and gene mutations, yet to a much lesser extent compared to CLL. These findings suggest that such aberrations are mostly relevant for disease progression rather than disease onset, indirectly pointing to microenvironmental drive as a key contributor to the emergence of MBL. Understanding microenvironmental interactions is therefore anticipated to elucidate MBL ontogeny and, most importantly, the relationship between MBL and CLL.

TRIP - T cell receptor/immunoglobulin profiler.

BACKGROUND: Antigen receptors are characterized by an extreme diversity of specificities, which poses major computational and analytical challenges, particularly in the era of high-throughput immunoprofiling by next generation sequencing (NGS). The T cell Receptor/Immunoglobulin Profiler (TRIP) tool offers the opportunity for an in-depth analysis based on the processing of the output files of the IMGT/HighV-Quest tool, a standard in NGS immunoprofiling, through a number of interoperable modules. These provide detailed information about antigen receptor gene rearrangements, including variable (V), diversity (D) and joining (J) gene usage, CDR3 amino acid and nucleotide composition and clonality of both T cell receptors (TR) and B cell receptor immunoglobulins (BcR IG), and characteristics of the somatic hypermutation within the BcR IG genes. TRIP is a web application implemented in R shiny. RESULTS: Two sets of experiments have been performed in order to evaluate the efficiency and performance of the TRIP tool. The first used a number of synthetic datasets, ranging from 250k to 1M sequences, and established the linear response time of the tool (about 6 h for 1M sequences processed through the entire BcR IG data pipeline). The reproducibility of the tool was tested comparing the results produced by the main TRIP workflow with the results from a previous pipeline used on the Galaxy platform. As expected, no significant differences were noted between the two tools; although the preselection process seems to be stricter within the TRIP pipeline, about 0.1% more rearrangements were filtered out, with no impact on the final results. CONCLUSIONS: TRIP is a software framework that provides analytical services on antigen receptor gene sequence data. It is accurate and contains functions for data wrangling, cleaning, analysis and visualization, enabling the user to build a pipeline tailored to their needs. TRIP is publicly available at https://bio.tools/TRIP_-_T-cell_Receptor_Immunoglobulin_Profiler .

T-Cell Dynamics in Chronic Lymphocytic Leukemia under Different Treatment Modalities.

PURPOSE: Using next-generation sequencing (NGS), we recently documented T-cell oligoclonality in treatment-naïve chronic lymphocytic leukemia (CLL), with evidence indicating T-cell selection by restricted antigens. EXPERIMENTAL DESIGN: Here, we sought to comprehensively assess T-cell repertoire changes during treatment in relation to (i) treatment type [fludarabine-cyclophosphamide-rituximab (FCR) versus ibrutinib (IB) versus rituximab-idelalisib (R-ID)], and (ii) clinical response, by combining NGS immunoprofiling, flow cytometry, and functional bioassays. RESULTS: T-cell clonality significantly increased at (i) 3 months in the FCR and R-ID treatment groups, and (ii) over deepening clinical response in the R-ID group, with a similar trend detected in the IB group. Notably, in constrast to FCR that induced T-cell repertoire reconstitution, B-cell receptor signaling inhibitors (BcRi) preserved pretreatment clones. Extensive comparisons both within CLL as well as against T-cell receptor sequence databases showed little similarity with other entities, but instead revealed major clonotypes shared exclusively by patients with CLL, alluding to selection by conserved CLL-associated antigens. We then evaluated the functional effect of treatments on T cells and found that (i) R-ID upregulated the expression of activation markers in effector memory T cells, and (ii) both BcRi improved antitumor T-cell immune synapse formation, in marked contrast to FCR. CONCLUSIONS: Taken together, our NGS immunoprofiling data suggest that BcRi retain T-cell clones that may have developed against CLL-associated antigens. Phenotypic and immune synapse bioassays support a concurrent restoration of functionality, mostly evident for R-ID, arguably contributing to clinical response.

Disease-biased and shared characteristics of the immunoglobulin gene repertoires in marginal zone B cell lymphoproliferations.

The B cell receptor immunoglobulin (Ig) gene repertoires of marginal zone (MZ) lymphoproliferations were analyzed in order to obtain insight into their ontogenetic relationships. Our cohort included cases with MZ lymphomas (n = 488), i.e. splenic (SMZL), nodal (NMZL) and extranodal (ENMZL), as well as provisional entities (n = 76), according to the WHO classification. The most striking Ig gene repertoire skewing was observed in SMZL. However, restrictions were also identified in all other MZ lymphomas studied, particularly ENMZL, with significantly different Ig gene distributions depending on the primary site of involvement. Cross-entity comparisons of the MZ Ig sequence dataset with a large dataset of Ig sequences (MZ-related or not; n = 65 837) revealed four major clusters of cases sharing homologous ('public') heavy variable complementarity-determining region 3. These clusters included rearrangements from SMZL, ENMZL (gastric, salivary gland, ocular adnexa), chronic lymphocytic leukemia, but also rheumatoid factors and non-malignant splenic MZ cells. In conclusion, different MZ lymphomas display biased immunogenetic signatures indicating distinct antigen exposure histories. The existence of rare public stereotypes raises the intriguing possibility that common, pathogen-triggered, immune-mediated mechanisms may result in diverse B lymphoproliferations due to targeting versatile progenitor B cells and/or operating in particular microenvironments. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

B Cell Anergy Modulated by TLR1/2 and the miR-17∼92 Cluster Underlies the Indolent Clinical Course of Chronic Lymphocytic Leukemia Stereotyped Subset #4.

Chronic lymphocytic leukemia (CLL) patients assigned to stereotyped subset #4 (mutated IGHV4-34/IGKV2-30 BCR Ig) display a particularly indolent disease course. Immunogenetic studies of the clonotypic BCR Ig of CLL subset #4 suggested a resemblance with B cells rendered anergic through chronic autoantigenic stimulation. In this article, we provide experimental evidence that subset #4 CLL cells show low IgG levels, constitutive ERK1/2 activation, and fail to either release intracellular Ca(2+) or activate MAPK signaling after BCR cross-linking, thus displaying a signature of B cell anergy at both biochemical and functional levels. Interestingly, TLR1/2 triggering restored BCR functionality, likely breaching the anergic state, and this was accompanied by induction of the miR-17∼92 cluster, whose members target critical BCR-associated molecules, including MAPKs. In conclusion, we demonstrate BCR anergy in CLL subset #4 and implicate TLR signaling and the miR-17∼92 cluster in the regulation of the anergic state. This detailed signaling profiling of subset #4 has implications for advanced understanding of the complex regulation of intracellular signaling pathways in CLL, currently a major therapeutic target of the disease.