A Pan-Cancer Analysis of Tumor-Infiltrating B Cell Repertoires

Tumor-infiltrating B cells can play an important role in anti-tumor responses but their presence is not well understood. In this study, we extracted the B cell receptor repertoires from 9522 tumor and adjacent non-tumor samples across 28 tumor types in the Cancer Genome Atlas project and performed diversity and network analysis. We identified differences in diversity and network statistics across tumor types and subtypes and observed a trend towards increased clonality in primary tumors compared to adjacent non-tumor tissues. We also found significant associations between the repertoire features and mutation load, tumor stage, and age. Our V-gene usage analysis identified similar V-gene usage patterns in colorectal and endometrial cancers. Lastly, we evaluated the prognostic value of the repertoire features and identified significant associations with survival in seven tumor types. This study warrants further research into better understanding the role of tumor-infiltrating B cells across a wide range of tumor types.

Trav15-dv6 family Tcrd rearrangements diversify the Tcra repertoire

The Tcra repertoire is generated by multiple rounds of Vα-Jα rearrangement. However, Tcrd recombination precedes Tcra recombination within the complex Tcra-Tcrd locus. Here, by ablating Tcrd recombination, we report that Tcrd rearrangement broadens primary Vα use to diversify the Tcra repertoire in mice. We reveal that use of Trav15-dv6 family V gene segments in Tcrd recombination imparts diversity in the Tcra repertoire by instigating use of central and distal Vα segments. Moreover, disruption of the regions containing these genes and their cis-regulatory elements identifies the Trav15-dv6 family as being responsible for driving central and distal Vα recombinations beyond their roles as substrates for Tcrd recombination. Our study demonstrates an indispensable role for Tcrd recombination in general, and the Trav15-dv6 family in particular, in the generation of a combinatorially diverse Tcra repertoire.

Fully Automated Workflows Quantify and Report Key T-Cell and B-Cell Receptor Biomarkers Relevant to Immuno-Oncology and Heme-Oncology Research

Background T-cell and B-cell repertoire analysis is used in oncology research, to understand the etiology of complex disease phenotypes, for the identification of biomarkers predictive of disease burden, outcome, and response to treatment, and for research in diagnosis and recurrence monitoring. Key predictors include secondary and tertiary repertoire features not reported by existing sequencing software solutions. For example, due to ongoing somatic hypermutation in mature B-cell receptors, the underlying sequence of a given clone can accumulate base differences and appear as several distinct clones with smaller frequencies, thereby hampering the ability of analysis software to detect its presence as a single dominant clone with the highest frequency. This has particularly detrimental implications for research in disorders such as follicular lymphoma and may require clonal lineage analysis for proper mitigation. Therefore, to aid the downstream analytics of biomarker identification and the study of complex disease, we developed fully automated analysis solutions that directly compute and report several key features (clonal lineage, amongst several others described below) pertinent to this area of research. Results We developed the Oncomine™ TCR Beta-SR, TCR Gamma-SR, BCR IGH-SR and BCR IGKL-SR workflows on Ion Reporter™ to characterize T-cell (β, γ chains) and B-cell (heavy and light (κ, δ) chains) repertoires. These workflows generate output tables and visualizations for primary repertoire features such as detected clones (viz., unique rearrangements in the receptor DNA sequence), their frequencies, as well as their somatic hypermutation levels in the case of B-cells (Figure 1a & 1b) for clonality assessment and rare clone detection. The software also quantifies and reports several secondary and tertiary repertoire features in a sample, such as clonal diversity, evenness of the clonal population, and B-cell lineage groupings useful in identifying related sub-clones. It includes spectratyping format plots to simultaneously assess the above features as a function of v-gene usage and CDR3 length combinations (Figure 1c & 1d), thereby providing users a complete snapshot of the repertoire, and also the capability to quickly determine CDR3 lengths and V-gene usage of highly expanded or mutated clones. A separate CDR3 lengths histogram is included, as well as a heatmap that depicts the distributions/intensity of Variable-Joining gene combinations (Figure 1e & 1f). Furthermore, the TCR workflows also report (i) convergence frequencies (fraction of clones with different nucleotide sequences, but identical amino acid sequences), and (ii) haplotype grouping for an analyzed sample, based on V-gene allele genotyping and clustering (Figure 1g). In addition, the long read Oncomine™ BCR IGH-LR workflow uniquely reports the isotype class for every detected clone, and includes a visualization of total reads, clones and lineages in the sample represented by isotype (Figure 1h). Conclusion The Oncomine™ immune repertoire workflows for T-cell and B-cell receptor sequencing were designed to be of high utility in distinct areas of malignancy research, and we expect them to greatly simplify complex downstream analyses. The unique capabilities of the workflows to automatically report secondary and tertiary repertoire features such as (i) clonal lineages for improved dominant clone detection in blood cancers, (ii) TCR clone convergence for prediction of response to immune checkpoint inhibitors [1,2], (iii) TCR haplotype grouping for evaluation of risk factors for autoimmunity and immune-related adverse events [3], and (iv) isotype classification in BCRs for studying pan-cancer immune evasion mechanisms, demonstrate the clear advantages of using these automated workflows over other existing solutions. For research use only. References 1) Looney TJ et al. (2020) TCR Convergence in Individuals Treated With Immune Checkpoint Inhibition for Cancer. Front. Immunol. 10:2985. 2) Naidus et al. (2021) Early changes in the circulating T cells are associated with clinical outcomes after PD-L1 blockade by durvalumab in advanced NSCLC patients. Cancer Immunology, Immunotherapy 70:2095-2102 3) Looney TJ et al. (2019) Haplotype Analysis of the T-Cell Receptor Beta (TCRB) Locus by Long-amplicon TCRB Repertoire Sequencing. Journal of Immunotherapy and Precision Oncology. 2 (4): 137-143. Figure 1 Figure 1. Disclosures Sarda: Thermo Fisher Scientific: Current Employment. Lowman: Thermo Fisher Scientific: Current Employment. Toro: Thermo Fisher Scientific: Current Employment. Pickle: Thermo Fisher Scientific: Current Employment. Looney: Thermo Fisher Scientific: Ended employment in the past 24 months; Singular Genomics: Current Employment. Hyland: Thermo Fisher Scientific: Current Employment.

A pan-cancer analysis of tumor-infiltrating B cell repertoires

Tumor-infiltrating B cells can play an important role in anti-tumor responses but their presence is not well understood. In this study, we extracted the B cell receptor repertoires from 9522 tumor and adjacent non-tumor samples across 28 tumor types in the Cancer Genome Atlas (TCGA) project and performed diversity and network analysis. We identified differences in diversity and network statistics across tumor types and subtypes and we observed a trend towards increased clonality in primary tumor samples compared to adjacent non-tumor tissues. We also found significant associations between the repertoire features and mutation load, tumor stage, and age. Our V-gene usage analysis identified similar V-gene usage patterns in colorectal and endometrial cancers. Lastly, we evaluated the prognostic value of the repertoire features and identified significant associations with survival in seven tumor types. This study warrants further research into better understanding the role of tumor-infiltrating B cells across a wide range of tumor types.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

A comparison of the IgM and IgT repertoires reveals the structure of B cell communities in rainbow trout is a consequence of mechanistic and evolutionary forces

In rainbow trout (Oncorhynchus mykiss), three classes of antibodies have been identified: IgM, IgD, and IgT, which differ in their abundance and effector functions. Though the process of VDJ recombination that creates these antibodies is often described as a stochastic process, recent findings in mammals suggest that there are biases in antibody construction. Because class switching is absent in teleosts, studying the IgM and IgT communities of rainbow trout provides the opportunity to see how evolution has differentially shaped the IgM and IgT repertoires. Even though it has not yet been demonstrated that biases exist in variable region construction for trout immunoglobulins, it seems reasonable that both natural and artificial selection have driven preferential gene-segment usage and pairing biases in rainbow trout. In this study, we sequenced the heavy chain variable regions of membrane IgM and IgT from multiple fish and predicted that given the more generalist role of the abundant IgM there would be less pressure to bias V gene segment usage and DJ pairings; rather, natural selection would have favored diversity and the ability to respond to a plethora of pathogens. Conversely, as IgT is substantially less abundant in the serum than IgM and specialized in its function, there would be selective pressure to make the most out of a little, thus favoring biased V segment usage and preferential DJ pairings. In support of our hypotheses, for IgT, over 70% of DJ pairs were biased and over 60% of antibodies were constructed with just two V gene segments. These biases were not prevalent in the IgM repertoire, where only 4% of DJ pairs were biased and no single V gene segment was utilized in more than 10% of antibodies. We found that these biases have profound influences in the richness and evenness of the repertoires, with the IgM repertoire investing more equitably in nearly double the number of VDJ combinations compared with IgT.

The TR locus annotation and characteristics of Rhinolophus ferrumequinum

Rhinolophus ferrumequinumin vertebrate could be divided into αβ or γδ, which are encoded by TRA/D, TRG and TRB loci respectively. TCRs play a central role in mammal cellular immunity, which are functionally produced by rearrangement of V gene, D gene, J gene and C gene in the loci. Bat is the only mammal with flying ability, and is considered as the main host of zoonotic virus, which occupies an important position in the field of public health. At present, little is known about the composition of bat TRs gene. According to the whole genome sequencing results of the Rhinolophus ferrumequinum, and referring to the TR/IG annotation rules formulated by IMGT. We make a complete annotation on the TRA/D, TRG and TRB loci of the Rhinolophus ferrumequinum. A total of 128 V segments, 3 D segments, 85 J segments and 6 C segments were annotated, in addition to compared with the known mammalian, the characteristics of the TRs locus and germline genes of the Rhinolophus ferrumequinum were analyzed.

The TR locus annotation and characteristics of Rhinolophus ferrumequinum

T cell antigen receptors (TCRs) in vertebrate could be divided into αβ or γδ, which are encoded by TRA/D, TRG and TRB loci respectively. TCRs play a central role in mammal cellular immunity, which are functionally produced by rearrangement of V gene, D gene, J gene and C gene in the loci. Bat is the only mammal with flying ability, and is considered as the main host of zoonotic virus, which occupies an important position in the field of public health. At present, little is known about the composition of bat TRs gene. According to the whole genome sequencing results of the Rhinolophus ferrumequinum, and referring to the TR/IG annotation rules formulated by IMGT. We make a complete annotation on the TRA/D, TRG and TRB loci of the Rhinolophus ferrumequinum. A total of 128 V segments, 3 D segments, 85 J segments and 6 C segments were annotated, in addition to compared with the known mammalian, the characteristics of the TRs locus and germline genes of the Rhinolophus ferrumequinum were analyzed.