Comprehensive Profiling of T- and B-Cell Receptor Repertoires Demonstrates Impaired Developmental and Activation of Adaptive Immunity in DOCK8 Deficiency.

Recent successes in tumour immunotherapies have highlighted the importance of tumour immunity. However, most previous studies to date have focused on T-cell immune response, although B cells are key players in the core immune network and are associated with T-cell immune response. Based on our previous study delineating T-cell receptor (TCR) repertoire in seven patients with oesophageal squamous cell carcinoma (ESCC), this study profiled the B-cell receptor (BCR) repertoire of multiple tumour regions, adjacent normal tissue, and blood from the same seven patients to reveal the characteristics of B-cell immunity and the relationship to TCR repertoire in ESCC patients. We found that intratumour BCR repertoire was significantly more oligoclonal than matched adjacent normal tissue or peripheral blood and, moreover, clonal amplification of B cells in multiple tumour regions was significantly heterogeneous, although clonal amplification of the TCR repertoire across different tissue compartments and regions of the same tumour was similar. However, both BCR and TCR repertoires in the tumour microenvironment were distinct from those in adjacent normal tissues and blood, and thus represented a group of B and T cells that were spatially confined to the tumour microenvironment and could react to tumour antigens. Additionally, B- and T-cell clones varying between different tumour regions showed intratumour heterogeneity of B- and T-cell immune response. Thus, multiple tumour biopsies could be essential to comprehensively delineate the adaptive immune response to an individual ESCC. These findings expand our understanding of adaptive anti-tumour immunity and shed more light on ESCC immunotherapy. This study provides insights into the intratumour heterogeneity of the BCR repertoire as well as the difference and relationship between the BCR and TCR repertoire in ESCC, expanding our understanding of adaptive anti-tumour immunity and ESCC immunotherapy. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Introduction: Autoimmune hepatitis (AIH) is a chronic liver disease characterized by inflammation resulting from the body’s immune system attacking its own liver cells. T-cell receptor (TCR) and B-cell receptor (BCR) repertoires are crucial mediators of autoimmune diseases, with the cause of B-lineage dysregulation remaining elusive. Objective: This study aims to comprehensively describe the changes in TCR and BCR repertoires in the peripheral blood and liver of patients with AIH. Methods: High-throughput sequencing and multiplex polymerase chain reaction amplification were applied to explore the TCR and BCR gene repertoire characteristics in 60 patients with AIH and control groups. Results: The findings revealed a significant reduction in the diversity of BCR heavy-chain (BCR-H) repertoire and the length of BCR-H complementarity-determining region 3 (CDR3) in the peripheral blood of AIH patients. Interestingly, the pre-selection BCR-H CDR3s in AIH patients were abnormally short, suggesting irregularities in the early stages of B-cell development and repertoire formation in the bone marrow. Furthermore, compared with controls, peripheral blood samples from AIH patients showed reduced diversity in the TCR β-chain (TCR-β) repertoire, longer CDR3 lengths, biased TCR-β variable gene usage, and extensive TCR-β sharing. The analysis revealed a comparable distribution of clone diversity between liver tissue samples from AIH and control groups, potentially attributable to the architectural characteristics of AIH liver T and B cells, which are marked by the presence of multiple antigen-responsive T and B cells rather than a single reactive T and B lymphocyte clone. Conclusion: This study reveals distinct alterations in the TCR and BCR repertoires among patients with AIH, potentially offering novel insights for its prevention and diagnosis.

Although B cells and T cells are integral players of the adaptive immune system and act in co-dependent ways to orchestrate immune responses, existing methods to study the immune repertoire have largely focused on separate analyses of B cell receptor (BCR) and T cell receptor (TCR) repertoires. Based on our hypothesis that the shared history of immune exposures and the shared cellular machinery for recombination result in similarities between BCR and TCR repertoires in an individual, we examine any commonalities and interrelationships between BCR and TCR repertoires. We find that the BCR and TCR repertoires have covarying clonal architecture and diversity, and that the pattern of correlations appears to be altered in immune-mediated diseases. Furthermore, hierarchical clustering of public B and T cell clonotypes in both health and disease based on correlation of clonal proportion revealed distinct clusters of B and T cell clonotypes that exhibit increased sequence similarity, share motifs, and have distinct amino acid characteristics. Our findings point to common principles governing memory formation, recombination, and clonal expansion to antigens in B and T cells within an individual. A significant proportion of public BCR and TCR repertoire can be clustered into nonoverlapping and correlated clusters, suggesting a novel way of grouping B and T cell clonotypes.

Introduction: Pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA), and aplastic anemia (AA) are immune-mediated diseases that affect mainly erythrocytes or erythroid progenitor cells. This study aimed to investigate changes related to autoimmunity in B-cell receptor (BCR) and T-cell receptor (TCR) repertoires in patients with these diseases. Methods: Patients with primary PRCA, AIHA, and AA and normal controls (NCs) were recruited. Peripheral blood was collected, and BCR and TCR repertoires were sequenced by next-generation immunosequencing. Results: Ten patients with PRCA, 10 with AIHA, 10 with AA, and 7 NCs were ultimately enrolled. According to the broad repertoire metric analysis, only the TCR repertoire of the AA group was more diverse than that of the NC group (p < 0.05). Regarding BCR and TCR repertoires, the PRCA, AIHA, and AA groups had uniform gene characteristics. The preferential gene of immunoglobulin heavy chains in PRCA patients was correlated with memory cell and red blood cell antigen recognition; genes expressed in the AIHA group were associated with the secretion of autoantibodies, whereas AA patients had more genes related to neutralizing antibodies. For T-cell receptor β (TRB) chains, PRCA patients had skewed use of genes associated with T-cell dysregulation and hyperinflammation, whereas AIHA and AA patients had similar genes as patients with other autoimmune diseases, which correlated with abnormal antigen recognition. PRCA and AA patients had specific TRBV-J gene combinations. For the BCR light chains, PRCA and AIHA patients tended to use more κ chains, whereas AA patients tended to use more λ chains. Regarding the TCRα chains, patients with each of the three diseases expressed more genes related to hypersensitivity reactions (p < 0.05). Compared to the NC group, the PRCA and AIHA groups had greater BCR somatic hypermutation (SHM). The length of CDR3 was similar, but the hydrophobicity differed among the different disease groups. Different motifs were found in BCRs and TCRs of the three diseases. Compared to NCs, the PRCA, AIHA, and AA groups showed a considerable lack of physiological T-cell clusters, and some disease-specific T-cell clusters were found in each disease group. Conclusion: PRCA, AIHA, and AA patients had different BCR and TCR repertoire characteristics in terms of genes and gene combinations, hydrophobicity and CDR3 motifs, and T-cell clustering, which might contribute to autoimmune antigen recognition. The abnormalities were mainly T-cell related for PRCA patients, B-cell related for AIHA patients and both for AA patients.

Lung cancer is one of the greatest threats to human health, and is initially detected and attacked by the immune system through tumor-reactive T cells. The aim of this study was to determine the basic characteristics and clinical significance of the peripheral blood T-cell receptor (TCR) repertoire in patients with advanced lung cancer. To comprehensively profile the TCR repertoire, high-throughput sequencing was used to identify hypervariable rearrangements of complementarity determining region 3 (CDR3) of the TCR β chain in peripheral blood samples from 64 advanced lung cancer patients and 31 healthy controls. We found that the TCR repertoire differed substantially between lung cancer patients and healthy controls in terms of CDR3 clonotype, diversity, V/J segment usage, and sequence. Specifically, baseline diversity correlated with several clinical characteristics, and high diversity reflected a better immune status. Dynamic detection of the TCR repertoire during anticancer treatment was useful for prognosis. Both increased diversity and high overlap rate between the pre- and post-treatment TCR repertoires indicated clinical benefit. Combination of the diversity and overlap rate was used to categorize patients into immune improved or immune worsened groups and demonstrated enhanced prognostic significance. In conclusion, TCR repertoire analysis served as a useful indicator of disease development and prognosis in advanced lung cancer and may be utilized to direct future immunotherapy.

Intra-Tumoral CD8+ T-Cells in Follicular Lymphoma Contain Large Clonal Expansions That Are Amenable to Dual-Checkpoint Blockade

Exploration of B and T Cell Receptor Repertoires Reveals Distinct Mechanisms in Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and Aplastic Anemia

Methods for sequence and structural analysis of B and T cell receptor repertoires

Simple SummaryThe recent advancement in high-throughput sequencing has become indispensable for immune-genomics and profiling the T- and B-cell receptor repertoires. Immune repertoire sequencing (IR-seq) and whole transcriptome sequencing (WTS) can be implemented to investigate and quantitatively characterize the complex pattern of the CDR3 region. We conducted T-cell diversity analysis result comparisons of these sequencing methods and suggest an intuitive approach to discriminate reliable TCR sequences and clonotype patterns from capturing errors. Although bulk-RNA sequencing is commonly used for cancer analysis, we confirmed capturing highly enriched TCR transcripts with IR-seq is more reliable for accurate immune repertoire discovery, and singleton read filtering criteria should be applied to capture true clonotypes from error-prone sequencing data. The use of such well-established data and analytical methodologies can broaden understanding of antigen specificity in immunity and enabling efficient therapeutic antibody finding.Analysis of the T-cell receptor (TCR) repertoire is essential to characterize the extensive collections of T-cell populations with recognizing antigens in cancer research, and whole transcriptome sequencing (WTS) and immune repertoire sequencing (IR-seq) are commonly used for this measure. To date, no standard read filtering method for IR measurement has been presented. We assessed the diversity of the TCR repertoire results from the paired WTS and IR-seq data of 31 multiple myeloma (MM) patients. To invent an adequate read filtering strategy for IR analysis, we conducted comparisons with WTS results. First, our analyses for determining an optimal threshold for selecting clonotypes showed that the clonotypes supported by a single read largely affected the shared clonotypes and manifested distinct patterns of mapping qualities, unlike clonotypes with multiple reads. Second, although IR-seq could reflect a wider TCR region with a higher capture rate than WTS, an adequate comparison with the removal of unwanted bias from potential sequencing errors was possible only after applying our read filtering strategy. As a result, we suggest that TCR repertoire analysis be carried out through IR-seq to produce reliable and accurate results, along with the removal of single-read clonotypes, to conduct immune research in cancer using high-throughput sequencing.

Large-scale study of the properties of T-cell receptor (TCR) and B-cell receptor (BCR) repertoires through next-generation sequencing is providing excellent insights into the understanding of adaptive immune responses. Variable(Diversity)Joining [V(D)J] germline genes and alleles must be characterized in detail to facilitate repertoire analyses. However, most species do not have well-characterized TCR/BCR germline genes because of their high homology. Also, more germline alleles are required for humans and other species, which limits the capacity for studying immune repertoires. Herein, we developed “Immune Germline Prediction” (IMPre), a tool for predicting germline V/J genes and alleles using deep-sequencing data derived from TCR/BCR repertoires. We developed a new algorithm, “Seed_Clust,” for clustering, produced a multiway tree for assembly and optimized the sequence according to the characteristics of rearrangement. We trained IMPre on human samples of T-cell receptor beta (TRB) and immunoglobulin heavy chain and then tested it on additional human samples. Accuracy of 97.7, 100, 92.9, and 100% was obtained for TRBV, TRBJ, IGHV, and IGHJ, respectively. Analyses of subsampling performance for these samples showed IMPre to be robust using different data quantities. Subsequently, IMPre was tested on samples from rhesus monkeys and human long sequences: the highly accurate results demonstrated IMPre to be stable with animal and multiple data types. With rapid accumulation of high-throughput sequence data for TCR and BCR repertoires, IMPre can be applied broadly for obtaining novel genes and a large number of novel alleles. IMPre is available at https://github.com/zhangwei2015/IMPre.

The immunotherapy of cancer has revolutionized the treatment of oncologic cancer, but the patient response has been very heterogeneous and this has greatly raised the need to develop strong, minimally invasive, biomarkers that predict and monitor the reaction to the treatment. Although T-cell receptor (TCR) repertoire profiling has enjoyed much focus, recent studies have demonstrated that the variety of circulating B-cell receptor (BCR) repertoire is critical and understudied in determining antitumor immunity and immunotherapy response. With the current development of high-throughput immune repertoire sequencing, BCR diversity, clonality, somatic hypermutation, and lineage evolution can now be comprehensively characterized using a peripheral blood sample. The current article assesses the circulating BCR repertoire diversity as an immunotherapy responsiveness biomarker by synthesising the recent findings of immune checkpoint blockade, cancer vaccines, and combination immunotherapies biomarkers. We discuss methodological platforms of BCR sequencing, analytical diversity measures and computing methods of repertoire architecture. The existing data shows that the responders to immunotherapy tend to be characterized by either dynamic BCR repertoire remodelling, clonal amplification of tumour-surveillance B cells, and changes in indices of diversity throughout therapy. Moreover, peripheral BCR signatures have been linked to the treatment efficacy, immune-related adverse events, as well as long-term immune memory formation. We also comment on technical and biological confounders, which are sequencing depth, sampling time, tumor heterogeneity and immune compartmentalization. The problems of standardization and reproducibility are mitigated, and the novel methods of single-cell and multi-omics that increase interpretability are described. Lastly, we suggest a translational system of applying circulating BCR repertoire measures to foresee biomarker and clinical trial layout.

A commonly used immunoglobulin loci-transgenic mouse, the Kymouse, has B cells that go through complete development and have receptor repertoires that show key differences in encoding gene frequencies but structural similarity in their most variable regions.

A commonly used immunoglobulin loci-transgenic mouse, the Kymouse, has B cells that go through complete development and have receptor repertoires that show key differences in encoding gene frequencies but structural similarity in their most variable regions.

The liver is the primary site for colorectal metastases and hepatic resection often fails to cure these patients. Little is known about T-cell immune response in these patients or its changes after interleukin-2 (IL-2) infusion. Using polymerase chain reaction methodology, we investigated T-cell receptor (TCR) V alpha and V beta gene segment subfamily usage in tumor, hepatic tissue, and blood of six patients undergoing hepatic resection and, in addition, in six patients receiving preoperative adjuvant IL-2 therapy (randomized phase I trial). The objectives were (a) to analyze the TCR repertoire in patients undergoing hepatic resection (without IL-2), (b) to analyze the TCR repertoire in patients undergoing hepatic resection after IL-2 therapy, (c) to analyze the effects of preoperative IL-2 infusion on the tumor-infiltrating lymphocyte (TIL) TCR repertoire by comparing TCR V gene segment usage in IL-2-treated versus nontreated patients, and (d) to analyze the effect of IL-2 infusion on the peripheral blood mononuclear cell (PBMC) TCR repertoire by comparing TCR V gene segment expression in pre- versus posttreatment PBMC of IL-2-treated patients. With regard to the first objective, we observed an unrestricted use of V alpha and V beta gene segment subfamily specificities in tumor hepatic tissue and blood of patients undergoing hepatic resection. In addition, we found that some V alpha and V beta specificities were overexpressed in tumor compared with hepatic tissue and blood, suggesting that the corresponding T-cell subpopulations were expanded at the tumor site. In IL-2-treated patients, in whom the tumor and liver were heavily infiltrated by T lymphocytes, the TIL TCR repertoire was also highly diverse and roughly similar to that detected in hepatic tissue and blood, except for a few overexpressions. To analyze the effect of IL-2 on TIL, we next compared the data obtained in IL-2-treated versus nontreated patients. No significant difference between the two groups was observed. Finally, no major changes in the PBMC TCR repertoire were found after IL-2 infusion. Further studies are needed to identify discrete T-cell subpopulations in these patients that may participate in either tumor immune surveillance or active immunotherapy mechanisms triggered by systemic IL-2 infusion.

<h3>Background</h3> Intratumoral B-cells are associated with improved survival with ICB in sarcomas.¹ We investigated the dynamics of intratumoral and peripheral BCR repertoires and their association with survival in dedifferentiated liposarcoma...