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1
Harding, Taylor, Qidi Yang, Brittany Mineo, Jenna Malinauskas, Jason Perera, Karl Beutner, Denise Lau, and Aly Khan. "73 Characterization of tumor-infiltrating T-cell repertoire in human cancers." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A81. http://dx.doi.org/10.1136/jitc-2021-sitc2021.073.
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BackgroundTCR and BCR repertoire profiling is a promising technique that can provide a clinically useful window into the complex interactions between tumor cells and infiltrating lymphocytes. Despite recent advances in repertoire sequencing methods, the characterization of tumor-infiltrating T-cell repertoires has been limited to small sample sizes due to technical and material constraints. In this study, we constructed a large multidimensional database of repertoire data covering a diverse landscape of HLA genotypes and tumor neoantigens from routine clinical sequencing. We present a descriptive summary of repertoire profiles derived from tens of thousands of tumor samples from over fifty different cancer cohorts and characterize the associations between T-cell repertoires and various clinical and molecular features.MethodsTo enrich immune receptor transcripts detected by the Tempus RNA-sequencing workflow, hybrid capture probes tiling TCR and BCR genes were used. Repertoire profiling reads were aligned, assembled, and annotated against IMGT reference sequences. Repertoires are profiled as a component of Tempus|xT RNA sequencing and are summarized here for >25 thousand tumor samples from over 50 different cancer cohorts.ResultsWe demonstrate that the use of TCR/BCR hybrid capture probes is an effective method for enriching immune receptor transcripts in RNA-sequencing data without interfering with downstream transcriptomic analysis. These repertoires were profiled as part of a larger, multimodal DNA/RNA-sequencing pipeline that quantifies a variety of tumor clinical and molecular features. We explored the correlation between high-level repertoire metrics like richness (the number of unique receptor clonotypes in a given repertoire) and clonality/evenness (Shannon entropy) against both gene expression-based metrics (i.e. immune cell infiltration estimates, etc.) and mutational patterns (mutational burden and neoantigen load). Finally, we observed that the repertoire clonality of B-cell and T-cell driven cancers frequently exhibits clear monoclonal dominance for the tumor cells’ lymphoid receptors.ConclusionsTCR/BCR repertoire profiling can be incorporated into high-volume clinical RNA sequencing to generate a diverse multimodal dataset for studying the tumor-immune microenvironment. By creating a large-scale database of TCR/BCR repertoire profiles from a variety of tissue, HLA genotypes, and mutational contexts, we can better resolve the molecular and clinical correlates of cancer with host adaptive immunity.
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Gupta, Neetu, Christina Labib, Veronique Giudicelli, Safa Aouinti, Marie-Paul Lefranc, and Sofia Kossida. "Cytoskeletal control of the developing and mature B cell antigen receptor repertoire." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 151.13. http://dx.doi.org/10.4049/jimmunol.204.supp.151.13.
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Abstract The existence of diverse B cell antigen receptor (BCR) specificities is essential for development of protective antibody responses against a wide variety of pathogens. In developing B cells, the BCR repertoire is crafted through sequential rearrangements of heavy and light chain genes, and involves both combinatorial and junctional diversification catalyzed by recombinases and nucleases. Mature B cells undergo further diversification in peripheral lymphoid organs through isotype switching and somatic hypermutations in response to foreign antigens; although these events are not known to alter BCR antigen specificity. Cytoskeletal proteins modulate BCR signal strength and thereby antibody responses, but it is not known if they play a role in the generation of BCR diversity. Using next generation sequencing, and iRepertoire, Inc and ImMunoGeneTics (IMGT) software platforms we examined BCR repertoires in developing and mature B cells that lack the expression of Ezrin, a prominent cytoskeletal regulator of BCR organization, signaling and activation. We show that Ezrin deficiency impacts the generation of BCR repertoire diversity during B cell development, as well as the mature BCR repertoire. Our analysis provides a window into the major processes involved in diversifying the B cell repertoire in control and Ezrin-deficient B cells, including V gene usage, number, frequency and length of unique CDR3s, nucleotide additions and trimming, and shared and unique clonotypes. Our data suggest that the Ezrin cytoskeleton in B cells impacts humoral immunity not only through modulation of B cell activation upon foreign antigen encounter, but also through qualitative and quantitative modulation of the available BCR repertoire.
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Fraley, Elizabeth R., Santosh Khanal, Stephen H. Pierce, Cas A. LeMaster, Rebecca McLennan, Tomi Pastinen, and Todd Bradley. "Effects of Prior Infection with SARS-CoV-2 on B Cell Receptor Repertoire Response during Vaccination." Vaccines 10, no. 9 (September 6, 2022): 1477. http://dx.doi.org/10.3390/vaccines10091477.
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Understanding the B cell response to SARS-CoV-2 vaccines is a high priority. High-throughput sequencing of the B cell receptor (BCR) repertoire allows for dynamic characterization of B cell response. Here, we sequenced the BCR repertoire of individuals vaccinated by the Pfizer SARS-CoV-2 mRNA vaccine. We compared BCR repertoires of individuals with previous COVID-19 infection (seropositive) to individuals without previous infection (seronegative). We discovered that vaccine-induced expanded IgG clonotypes had shorter heavy-chain complementarity determining region 3 (HCDR3), and for seropositive individuals, these expanded clonotypes had higher somatic hypermutation (SHM) than seronegative individuals. We uncovered shared clonotypes present in multiple individuals, including 28 clonotypes present across all individuals. These 28 shared clonotypes had higher SHM and shorter HCDR3 lengths compared to the rest of the BCR repertoire. Shared clonotypes were present across both serotypes, indicating convergent evolution due to SARS-CoV-2 vaccination independent of prior viral exposure.
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Weng, Ruiqiang, Sudong Liu, Xiaodong Gu, and Zhixiong Zhong. "Clonal diversity of the B cell receptor repertoire in patients with coronary in-stent restenosis and type 2 diabetes." Open Life Sciences 16, no. 1 (January 1, 2021): 884–98. http://dx.doi.org/10.1515/biol-2021-0091.
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Abstract Type 2 diabetes mellitus (T2DM) is known as a risk factor for coronary in-stent restenosis (ISR) in patients with coronary artery disease (CAD). Evidence suggests that B cells play a functional role in the progression of atherosclerotic lesions. However, the B cell receptor (BCR) repertoire in patients with ISR remains unclear. This study aims to profile the BCR repertoire in patients with coronary ISR/T2DM. A total of 21 CAD patients with or without ISR/T2DM were enrolled. PBMCs were isolated and examined for BCR repertoire profiles using DNA-seq. Our results showed that the diversity of amino acid sequences in ISR DM patients was higher than that in ISR −DM patients. The frequencies of 21 V/J paired genes differed between ISR DM and −ISR DM patients, while frequencies of 5 V/J paired genes differed between ISR DM and ISR −DM. The −ISR −DM group presented the highest clonotype overlap rate, while ISR DM patients presented the lowest overlap rate. Our study presented the BCR repertoires in patients with ISR/T2DM. The data suggested different BCR signatures between patients with ISR and T2DM. Further analysis of BCR profiles would enhance understanding of ISR.
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Krishna, Chirag, and A. Ari Hakimi. "Rules of Engagement: The Lymphocyte Receptor Ecosystem in Renal Cell Carcinoma." Cancer Research 82, no. 5 (March 1, 2022): 764–65. http://dx.doi.org/10.1158/0008-5472.can-22-0146.
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Immune receptor repertoires provide insight into the clonal distribution of tumor-infiltrating lymphocytes, yet the clinical implications of T-cell receptor (TCR) and B-cell receptor (BCR) repertoire diversity in cancer are unclear. In this issue of Cancer Research, Ferral-Fairbanks and colleagues reveal the interplay between repertoire diversity, tumor molecular features, and clinical outcome in renal cell carcinoma (RCC). The authors show that aggressive tumors harbor increasingly diverse TCR and BCR repertoires and that both repertoires are altered by common RCC driver mutations. Moreover, the authors demonstrate that high TCR diversity is associated with improved overall survival. This study highlights the contribution of lymphocyte receptor dynamics to the emerging complexity of RCC antitumor immune responses. See related article by Ferral-Fairbanks et al., p. 929
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Shi, Xiaodong, Tihong Shao, Feifei Huo, Chenqing Zheng, Wanyu Li, and Zhenyu Jiang. "An analysis of abnormalities in the B cell receptor repertoire in patients with systemic sclerosis using high-throughput sequencing." PeerJ 8 (January 14, 2020): e8370. http://dx.doi.org/10.7717/peerj.8370.
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Systemic sclerosis is a chronic multisystem autoimmune disease that is associated with polyclonal B cell hyperreactivity. The CDR3 of BCRs is the major site of antigen recognition. Therefore, we analyzed the BCR repertoire of patients with SSc. The BCR repertoires in 12 subjects including eight SSc patients and four healthy controls were characterized by high-throughput sequencing, and bioinformatics analysis were studied. The average CDR3 length in the SSc group was significantly shorter. The SSc patient displayed more diverse BCR. Moreover, SSc patients with mild skin sclerosis, anti-Scl70, interstitial lung disease or female sex were more diversified. B cells from the SSc patients showed a differential V and J gene usage. SSc patients had distinct BCR repertoires.These findings reflected the differences of BCR repertoires between SSc patients and controls. The higher-usage genes for the BCR sequence might be potential biomarkers of B cell-targeted therapies or diagnosis for SSc.
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de Bourcy, Charles F. A., Cesar J. Lopez Angel, Christopher Vollmers, Cornelia L. Dekker, Mark M. Davis, and Stephen R. Quake. "Phylogenetic analysis of the human antibody repertoire reveals quantitative signatures of immune senescence and aging." Proceedings of the National Academy of Sciences 114, no. 5 (January 17, 2017): 1105–10. http://dx.doi.org/10.1073/pnas.1617959114.
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The elderly have reduced humoral immunity, as manifested by increased susceptibility to infections and impaired vaccine responses. To investigate the effects of aging on B-cell receptor (BCR) repertoire evolution during an immunological challenge, we used a phylogenetic distance metric to analyze Ig heavy-chain transcript sequences in both young and elderly individuals before and after influenza vaccination. We determined that BCR repertoires become increasingly specialized over a span of decades, but less plastic. In 50% of the elderly individuals, a large space in the repertoire was occupied by a small number of recall lineages that did not decline during vaccine response and contained hypermutated IgD+B cells. Relative to their younger counterparts, older subjects demonstrated a contracted naive repertoire and diminished intralineage diversification, signifying a reduced substrate for mounting novel responses and decreased fine-tuning of BCR specificities by somatic hypermutation. Furthermore, a larger proportion of the repertoire exhibited premature stop codons in some elderly subjects, indicating that aging may negatively affect the ability of B cells to discriminate between functional and nonfunctional receptors. Finally, we observed a decreased incidence of radical mutations compared with conservative mutations in elderly subjects’ vaccine responses, which suggests that accumulating original antigenic sin may be limiting the accessible space for paratope evolution. Our findings shed light on the complex interplay of environmental and gerontological factors affecting immune senescence, and provide direct molecular characterization of the effects of senescence on the immune repertoire.
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Forgacs, David, Rodrigo B. Abreu, Giuseppe A. Sautto, Greg A. Kirchenbaum, Elliott Drabek, Kevin S. Williamson, Dongkyoon Kim, Daniel E. Emerling, and Ted M. Ross. "Convergent antibody evolution and clonotype expansion following influenza virus vaccination." PLOS ONE 16, no. 2 (February 22, 2021): e0247253. http://dx.doi.org/10.1371/journal.pone.0247253.
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Recent advances in high-throughput single cell sequencing have opened up new avenues into the investigation of B cell receptor (BCR) repertoires. In this study, PBMCs were collected from 17 human participants vaccinated with the split-inactivated influenza virus vaccine during the 2016–2017 influenza season. A combination of Immune Repertoire Capture (IRCTM) technology and IgG sequencing was performed on ~7,800 plasmablast (PB) cells and preferential IgG heavy-light chain pairings were investigated. In some participants, a single expanded clonotype accounted for ~22% of their PB BCR repertoire. Approximately 60% (10/17) of participants experienced convergent evolution, possessing public PBs that were elicited independently in multiple participants. Binding profiles of one private and three public PBs confirmed they were all subtype-specific, cross-reactive hemagglutinin (HA) head-directed antibodies. Collectively, this high-resolution antibody repertoire analysis demonstrated the impact evolution can have on BCRs in response to influenza virus vaccination, which can guide future universal influenza prophylactic approaches.
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Zhang, Li, Harini Kandadi, Alan Paciorek, Nancy N. Chang, Nadeem Anwar Sheikh, and Lawrence Fong. "Tracking of long-term B-cell memory responses using B-cell receptor (BCR) sequencing in prostate cancer (PC) patients (pts) treated with sipuleucel-T (sip-T)." Journal of Clinical Oncology 36, no. 6_suppl (February 20, 2018): 309. http://dx.doi.org/10.1200/jco.2018.36.6_suppl.309.
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309 Background: Sip-T is an autologous cellular immunotherapy for asymptomatic or minimally symptomatic metastatic castration-resistant prostate cancer (mCRPC). Sip-T’s induction of B-cell responses to PAP and other antigens correlates with improved survival (Sheikh 2013, GuhaThakurta 2015). Cancer vaccine-induced changes to BCR repertoire are unknown. To assess changes in BCR repertoire upon booster treatment, we compared patients retreated with sip-T (P10-1) to treatment-naïve patients (STRIDE). Methods: STRIDE pts (N = 52) received sip-T with concurrent or sequential enzalutamide for mCRPC (Petrylak 2015). P10-1 pts (N = 8) previously treated with sip-T for androgen-dependent PC were retreated with a booster course for mCRPC, after a median of 8.9 years (Beer 2017). Blood samples were collected at baseline (wk 0) and during (wk 2, 4)/post-sip-T (wk 6, 26, 52). Deep sequencing was performed using the ImmunoSEQ assay (Adaptive Biotechnologies). BCR diversity was assessed by clonality, and BCR dynamics by fold-change analysis (Zhang 2017). Results: BCR repertoire had significantly higher clonality in P10-1 vs STRIDE (wk 0: p = 0.003, wk 2: p < 0.001, wk 4: p < 0.001), suggestive of a more focused BCR repertoire. P10-1 also showed increased clonality from wk 0 to 4 (p = 0.063), whereas STRIDE showed a significant increase in clonality from wk 0 to 6 (p = 0.039), suggesting that BCR repertoire focused earlier in P10-1. Starting at wk 2, more clones remained in the repertoire in P10-1, indicating that sip-T stimulated immunologic memory early, after 1st retreatment (p < 0.05). There was less change over time (clonal shuffling) within the 100 most abundant baseline clones in P10-1 (p = 0.080), suggesting more relevant clones preexisted at baseline and enriched over time. After the first two sip-T infusions, more clones contracted in P10-1 (p = 0.027, p = 0.014), whereas more new clones were generated in STRIDE (p = 0.083, p = 0.003). Conclusions: Sip-T induces long-lasting changes in the BCR repertoire. Sip-T retreatment leads to quicker focusing of BCR repertoire than initial treatment. These results are consistent with sip-T inducing durable immunologic memory.
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Zhang, Ruoxi, Junling Zhuang, and Bing HAN. "Exploration of B and T Cell Receptor Repertoires Reveals Distinct Mechanisms in Pure Red Cell Aplasia, Autoimmune Hemolytic Anemia, and Aplastic Anemia." Blood 142, Supplement 1 (November 28, 2023): 5196. http://dx.doi.org/10.1182/blood-2023-174862.
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Background: Pure red cell aplasia (PRCA), autoimmune hemolytic anemia (AIHA) and aplastic anemia (AA) are immune-mediated diseases that mainly attack erythrocyte or erythroid progenitor cells. This study aimed to investigate changes related to autoimmunity in the B cell receptor (BCR) and T cell receptor (TCR) repertoires in these diseases. Methods: Patients with newly diagnosed primary PRCA, AIHA, and AA and healthy individuals with matched age and sex (normal controls, NC) were recruited. Peripheral blood was collected, and BCR and TCR repertoires were sequenced by next-generation immunosequencing. The diversity and clonal expansion of the repertoires, gene and gene combination usage, somatic hypermutation (SHM) of BCR, and the sequence length, hydrophobicity, motifs of the most diverse epitope encoded (complementary-determining region 3, CDR3), T cell clustering, and BCR light chain and TCRα chain were analyzed. Results: 10 PRCA, 10 AIHA, 10 AA, and 7 NC were finally enrolled. For the broad repertoire metrics analysis, only the TCR repertoire of the AA group was more diverse compared with NC (p＜0.05). For BCR and TCR repertoires, PRCA, AIHA, and AA showed uniform characteristics in gene usage. The preferential gene usage of PRCA immunoglobulin heavy (IGH) chains such as IGHV3-23, IGHV3-74 were correlated with memory cells and red blood cell antigen recognition; the higher usage of IGHV4-31 in the AIHA group was associated with secretion of auto-antibodies; whereas AA patients had more gene usage of neutralizing antibodies (p＜0.05). In the gene usage in T cell receptor β (TRB) chains, PRCA patients had skewed usage of genes associated with T cell dysregulation and hyperinflammation such as TRBV1 and TRBV11-2; the increased gene usage in AIHA and AA were also found in other autoimmune diseases, which correlated with abnormal antigen recognition. PRCA and AA also had specific TRBV-J gene combination usage. Only PRCA had higher BCR SHM compared with NC (p＜0.05). The length of CDR3 was similar but the hydrophobicity was different among different disease groups. 12, 28, and 22 motifs were found respectively in BCR of PRCA, AIHA, and AA; and 13, 6, and 6 motifs were found in TCR. Compared with NC, PRCA, AIHA, and AA showed a considerable lack of physiology T cell clusters, and 147, 98, and 141 disease-specific T cell clusters in each disease was found, respectively. For light chain of BCR, PRCA and AIHA had the tendency of more usage of κ chains, whereas AA had more usage of λ chains. For TCRα chains, the three diseases used more genes related to hypersensitivity reactions (p＜0.05). Conclusion: PRCA, AIHA, and AA had different BCR and TCR repertoire characteristics in gene and gene combination usage, hydrophobicity and motifs of CDR3, and T cell clustering, which might contribute to autoimmune antigen recognition. The abnormalities were mainly T cell-related for PRCA, B cell-related for AIHA and both for AA. Keywords: pure red cell aplasia; autoimmune hemolytic anemia; aplastic anemia; BCR; TCR
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Wu, Yu-Chang, David Kipling, Hui Sun Leong, Victoria Martin, Alexander A. Ademokun, and Deborah K. Dunn-Walters. "High-throughput immunoglobulin repertoire analysis distinguishes between human IgM memory and switched memory B-cell populations." Blood 116, no. 7 (August 19, 2010): 1070–78. http://dx.doi.org/10.1182/blood-2010-03-275859.
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Abstract B-cell receptor (BCR) diversity is achieved centrally by rearrangement of Variable, Diversity, and Joining genes, and peripherally by somatic hypermutation and class-switching of the rearranged genes. Peripheral B-cell populations are subject to both negative and positive selection events in the course of their development that have the potential to shape the BCR repertoire. The origin of IgM+IgD+CD27+ (IgM memory) cells is controversial. It has been suggested that they may be a prediversified, antigen-independent, population of cells or that they are a population of cells that develop in response to T-independent antigens. Most recently, it was suggested that the majority of IgM memory cells are directly related to switched memory cells and are early emigrants from the germinal center reaction. Advances in sequencing technology have enabled us to undertake large scale IGH repertoire analysis of transitional, naive, IgM memory and switched memory B-cell populations. We find that the memory B-cell repertoires differ from the transitional and naive repertoires, and that the IgM memory repertoire is distinct from that of class-switched memory. Thus we conclude that a large proportion of IgM memory cells develop in response to different stimuli than for class-switched memory cell development.
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Song, Chen, Ariel Erijman, Sean Lund, Bradley W. Langhorst, and Pingfang Liu. "Abstract 3304: Immune repertoire sequencing enables accurate clonality determination." Cancer Research 83, no. 7_Supplement (April 4, 2023): 3304. http://dx.doi.org/10.1158/1538-7445.am2023-3304.
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Abstract The study of complex immunological diseases and tumor microenvironments has advanced through recent developments in sequencing of the immune repertoire. Using this approach, the interrogation of disease progression is facilitated through analysis of millions of V(D)J combinations from B cell receptors (BCRs) and T cell receptors (TCRs). One major challenge of immune repertoire sequencing is to accurately capture the structural and sequence complexities of antibodies and TCR genes during both library preparation and bioinformatic analysis. In addition, the lack of standard reference controls makes assessing sequencing accuracy and sensitivity challenging. Here, we present a method for accurate sequencing of full-length immune gene repertoires of B cells and T cells. RNA extracted from peripheral blood mononuclear cells (PBMCs) or tissues were used for reverse transcription, during which unique molecular identifiers (UMIs) were added to discretely barcode each mRNA molecule. BCR- and TCR-specific PCR primers were used to enrich full-length BCR and TCR sequences. We have implemented a data analysis pipeline to assemble the full length BCR/TCR transcripts and to collapse PCR copies of each mRNA fragment into a single consensus sequence using UMIs. UMI incorporation enables the absolute quantification of input RNA molecules and accurate ranking of antibody/TCR clone abundance. Furthermore, we developed an RNA control pool that contains a large dynamic range of BCR and TCR sequences with a variety of V(D)J combinations, which enables sensitivity and accuracy evaluation of BCR and TCR sequencing. Our immune repertoire sequencing approach allows accurate clonal determination for both BCR and TCR. This technique is applicable for various applications including design of antibody chains for in vitro synthesis, investigation of T cell infiltration of tumor microenvironments, and monitoring of minimal residual disease in cancer patients. Citation Format: Chen Song, Ariel Erijman, Sean Lund, Bradley W. Langhorst, Pingfang Liu. Immune repertoire sequencing enables accurate clonality determination [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3304.
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Maheshwari, Shamoni, Carolyn Morrison, and Sarah Taylor. "Abstract 313: Benchmarking immune repertoire sequencing technologies." Cancer Research 84, no. 6_Supplement (March 22, 2024): 313. http://dx.doi.org/10.1158/1538-7445.am2024-313.
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Abstract In the past decade, single cell gene expression technologies have transformed basic and translational research and are more frequently found in the toolbox of immunologists alongside the standard bulk B-cell receptor (BCR) sequencing and transcriptome profiling assays. While conventional bulk BCR assays capture the frequency of single chains, single-cell immune profiling reveals the frequency of paired heavy and light chains along with the cells transcriptional profile. Here we present the results of a single-blind benchmarking experiment conducted in collaboration with the U.S. Food and Drug Administration (FDA) that compares the performance of standard bulk DNA and RNA BCR repertoire sequencing assays, with the 10x Genomics Single Cell Immune Profiling solution. The FDA selected nine unambiguously monoclonal BCR-expressing cell lines to generate a reference mixture that was shared with each study participant, without revealing the input proportions. This reference cell line mixture was also diluted to 10% and 1% with B-cells sorted from peripheral blood mononuclear cells (PBMC) to further test the limits of clonotype detection. For each experiment, we targeted ~10,000 cells and in aggregate generated a paired gene expression and V(D)J dataset from ~100,000 cells. BCR clonotypes were detected in 78% of the captured cells across all samples profiled. Each of the 9 top unique clonotypes observed in our dataset corresponded to a cell line in the reference mixture, allowing us to decode the mixing percentages of each BCR-expression cell line. In addition, we were able to identify rare cell line clonotypes to as low as 0.01% in the dilution experiments. Furthermore, the multimodal nature of the single cell data also resolved the transcriptional profile of each cell line into its own cluster thus enabling independent verification of clonotype proportions. In summary, we have generated a resource for the immunology community to evaluate and compare data generated with different repertoire sequencing technologies. Citation Format: Shamoni Maheshwari, Carolyn Morrison, Sarah Taylor, BCR-SEQC consortium. Benchmarking immune repertoire sequencing technologies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 313.
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Marks, Claire, and Charlotte M. Deane. "How repertoire data are changing antibody science." Journal of Biological Chemistry 295, no. 29 (May 14, 2020): 9823–37. http://dx.doi.org/10.1074/jbc.rev120.010181.
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Antibodies are vital proteins of the immune system that recognize potentially harmful molecules and initiate their removal. Mammals can efficiently create vast numbers of antibodies with different sequences capable of binding to any antigen with high affinity and specificity. Because they can be developed to bind to many disease agents, antibodies can be used as therapeutics. In an organism, after antigen exposure, antibodies specific to that antigen are enriched through clonal selection, expansion, and somatic hypermutation. The antibodies present in an organism therefore report on its immune status, describe its innate ability to deal with harmful substances, and reveal how it has previously responded. Next-generation sequencing technologies are being increasingly used to query the antibody, or B-cell receptor (BCR), sequence repertoire, and the amount of BCR data in public repositories is growing. The Observed Antibody Space database, for example, currently contains over a billion sequences from 68 different studies. Repertoires are available that represent both the naive state (i.e. antigen-inexperienced) and that after immunization. This wealth of data has created opportunities to learn more about our immune system. In this review, we discuss the many ways in which BCR repertoire data have been or could be exploited. We highlight its utility for providing insights into how the naive immune repertoire is generated and how it responds to antigens. We also consider how structural information can be used to enhance these data and may lead to more accurate depictions of the sequence space and to applications in the discovery of new therapeutics.
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Sherwood, Anna, Harlan Robins, Jonathan Fromm, Harvey Greisman, Daniel Sabath, Ryan Emerson, Mark Rieder, Brent Wood, and David Wu. "Identifying lymphoma antigen receptor sequences by immune repertoire profiling (TUM2P.915)." Journal of Immunology 192, no. 1_Supplement (May 1, 2014): 71.39. http://dx.doi.org/10.4049/jimmunol.192.supp.71.39.
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Abstract Neoplastic T and B cells rapidly expand, leading to T and B-cell receptor (TCR and BCR) repertoires dominated by one rearrangement. However clones reacting to an antigen also expand. Differentiating high-frequency reactive clones from lymphomas is important for both diagnosing and monitoring lymphomas. To document the diverse repertoire of TRB and IGH CDR3 chains, we developed a method to deeply sequence the CDR3 rearrangements using high-throughput sequencing (HTS). This technology provides a potential opportunity to identify and monitor neoplastic clones during the course of therapy. To demonstrate the potential of HTS of T and B-cell receptors to contribute to diagnosing and monitoring neoplasia in mature lymphomas; we amplify the TCR repertoire of 98 and the BCR repertoire of 60 index samples to identify high-frequency TRB or IGH rearrangements. Concurrently, we sequence the TRB and IGH repertoire in control blood, bone marrow, and reactive lymph tissue. Clones are classified as neoplastic if occurring at a proportion greater than 7 standard deviations above the mean frequency of the most abundant rearranged TRB or IGH in control samples. Using these criteria, Eighty-four percent of index samples have a tractable rearrangement. We find that for most disease diagnoses, high-throughput sequencing identifies a tractable clone and T-cell and B-cell receptor repertoire analysis may be useful for clinical laboratory evaluation of patients with T and B-cell neoplasms.
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Xiao, Wenming. "Abstract 80: Toward best practices for B-cell receptor repertoire profiling." Cancer Research 84, no. 6_Supplement (March 22, 2024): 80. http://dx.doi.org/10.1158/1538-7445.am2024-80.
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Abstract FDA anticipates that immune cell receptor profiling will become crucial to the Agency’s evaluation of the efficacy and safety of emerging cancer immunotherapies and immunomodulatory drugs by advancing precision medicine and providing orthogonal, functional evidence. High-throughput sequencing of B-cell receptor sequencing (BCR) gene rearrangements and downstream analysis empowers researchers to define the B-cell clonal landscape, as well as identify biomarkers for minimal residual disease (MRD). Although this field has made vast strides over the last decade, it lacks standardized assay controls, adequate sensitivity and specificity in gene mapping/alignment, and appropriately qualified reference data sets, materials, and validation methods. Moreover, detailed comparisons of wet-bench analytical methods or bioinformatic procedures have not appeared. The FDA led B-cell receptor sequencing quality control (BCR-SEQC) consortium is establishing protocols for the development of reference materials and data sets for the evaluation of NGS-based BCR repertoire reconstruction. The consortium is also developing materials for performance bench-marking studies. To this end, we performed whole-genome sequencing, RNAseq, and BCR-seq on 50 B-cell lines to determine the clonotype, full-length transcripts, and expression abundance of BCR genes in each cell line. Two batches of reference materials (DNA, RNA, and cells) were generated from each of nine cell lines that were determined unambiguously to be monoclonal in BCR expression of both heavy and light chains. These materials were distributed to ten companies to test up to 15 BCR-seq products. Our benchmarking studies include six widely-used sequencing technologies. The overarching objectives of this research were to elucidate current capabilities and limitations, address fundamental technical needs, provide reference materials and data sets, and establish actionable best practices for reconstructing B cell receptor repertoires from NGS data. Citation Format: Wenming Xiao, BCR-SEQC consortium. Toward best practices for B-cell receptor repertoire profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 80.
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Raybould, Matthew I. J., Claire Marks, Aleksandr Kovaltsuk, Alan P. Lewis, Jiye Shi, and Charlotte M. Deane. "Public Baseline and shared response structures support the theory of antibody repertoire functional commonality." PLOS Computational Biology 17, no. 3 (March 1, 2021): e1008781. http://dx.doi.org/10.1371/journal.pcbi.1008781.
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The naïve antibody/B-cell receptor (BCR) repertoires of different individuals ought to exhibit significant functional commonality, given that most pathogens trigger an effective antibody response to immunodominant epitopes. Sequence-based repertoire analysis has so far offered little evidence for this phenomenon. For example, a recent study estimated the number of shared (‘public’) antibody clonotypes in circulating baseline repertoires to be around 0.02% across ten unrelated individuals. However, to engage the same epitope, antibodies only require a similar binding site structure and the presence of key paratope interactions, which can occur even when their sequences are dissimilar. Here, we search for evidence of geometric similarity/convergence across human antibody repertoires. We first structurally profile naïve (‘baseline’) antibody diversity using snapshots from 41 unrelated individuals, predicting all modellable distinct structures within each repertoire. This analysis uncovers a high (much greater than random) degree of structural commonality. For instance, around 3% of distinct structures are common to the ten most diverse individual samples (‘Public Baseline’ structures). Our approach is the first computational method to find levels of BCR commonality commensurate with epitope immunodominance and could therefore be harnessed to find more genetically distant antibodies with same-epitope complementarity. We then apply the same structural profiling approach to repertoire snapshots from three individuals before and after flu vaccination, detecting a convergent structural drift indicative of recognising similar epitopes (‘Public Response’ structures). We show that Antibody Model Libraries derived from Public Baseline and Public Response structures represent a powerful geometric basis set of low-immunogenicity candidates exploitable for general or target-focused therapeutic antibody screening.
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Waltari, Eric, Saba Nafees, Krista M. McCutcheon, Joan Wong, and John E. Pak. "AIRRscape: An interactive tool for exploring B-cell receptor repertoires and antibody responses." PLOS Computational Biology 18, no. 9 (September 20, 2022): e1010052. http://dx.doi.org/10.1371/journal.pcbi.1010052.
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The sequencing of antibody repertoires of B-cells at increasing coverage and depth has led to the identification of vast numbers of immunoglobulin heavy and light chains. However, the size and complexity of these Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) datasets makes it difficult to perform exploratory analyses. To aid in data exploration, we have developed AIRRscape, an R Shiny-based interactive web browser application that enables B-cell receptor (BCR) and antibody feature discovery through comparisons among multiple repertoires. Using AIRR-seq data as input, AIRRscape starts by aggregating and sorting repertoires into interactive and explorable bins of germline V-gene, germline J-gene, and CDR3 length, providing a high-level view of the entire repertoire. Interesting subsets of repertoires can be quickly identified and selected, and then network topologies of CDR3 motifs can be generated for further exploration. Here we demonstrate AIRRscape using patient BCR repertoires and sequences of published monoclonal antibodies to investigate patterns of humoral immunity to three viral pathogens: SARS-CoV-2, HIV-1, and DENV (dengue virus). AIRRscape reveals convergent antibody sequences among datasets for all three pathogens, although HIV-1 antibody datasets display limited convergence and idiosyncratic responses. We have made AIRRscape available as a web-based Shiny application, along with code on GitHub to encourage its open development and use by immuno-informaticians, virologists, immunologists, vaccine developers, and other scientists that are interested in exploring and comparing multiple immune receptor repertoires.
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Shin, Junyoung, Baknoon Ham, Jeong-Han Seo, Sae Byul Lee, In Ah Park, Gyungyub Gong, Sung-Bae Kim, and Hee Jin Lee. "Immune repertoire and responses to neoadjuvant TCHP therapy in HER2-positive breast cancer." Therapeutic Advances in Medical Oncology 15 (January 2023): 175883592311576. http://dx.doi.org/10.1177/17588359231157654.
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Background: Despite the introduction of trastuzumab, pathologic complete response (pCR) is not attained in approximately 30–40% of Human epithelial growth factor receptor-2-positive breast cancer. Tumor-infiltrating lymphocytes (TIL) have been suggested as a predictive marker of treatment response, albeit not always effective. We investigated the relationship between trastuzumab, docetaxel, carboplatin, and pertuzumab (TCHP) treatment and immune repertoire as a treatment response predictor. Design: In all, 35 cases were divided into two experimental groups: 10 and 25 cases in the preliminary and main experiments, respectively. In the preliminary experiment, the biopsy tissues before TCHP treatment and the surgical tissues after TCHP treatment were compared. In the main experiment, the biopsy tissues before TCHP treatment were compared according to the TCHP treatment response. Methods: The T-cell repertoire for TRA, TRB, TRG, and TRD, and B-cell repertoire for immunoglobulin heavy, immunoglobulin kappa, and immunoglobulin lambda were evaluated. Whole transcriptome sequencing was also performed. Results: In the preliminary experiment, the density and richness of the T-cell receptor (TCR) and B-cell receptor (BCR) repertoires decreased after treatment, regardless of TCHP response. In the main experiment, the Shannon’s entropy index, density, and length of CDR3 of the TCR and BCR repertoires did not differ significantly in patients who did and did not achieve pCR. The pCR and non-pCR subgroups according to the level of TILs revealed that the non-pCR/lowTIL group had a higher proportion of low-frequency clones than the pCR/lowTIL group in TRA (non-pCR/lowTIL versus pCR/lowTIL, 0.01–0.1%, 63% versus 45.3%; <0.01%, 32.9% versus 51.8%, p < 0.001) and TRB (non-pCR/lowTIL versus pCR/lowTIL, 0.01–0.1%, 26.5% versus 14.7%; <0.01%, 72.0% versus 84.1%, p < 0.001). Conclusions: The role of the diversity, richness, and density of the TCR and BCR repertoires as predictive markers for TCHP response was not identified. Compositions of low-frequency clones could be candidates for predictive factors of TCHP response; however, validation studies and further research are necessary.
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Lai, Meimei, Qiongdan Wang, Yutian Lu, Xi Xu, Ying Xia, Mengyun Tu, Yanqing Liu, Qi Zhang, Ying Peng, and Xiaoqun Zheng. "Signatures of B-cell receptor diversity in B lymphocytes following Epstein-Barr virus transformation." Physiological Genomics 51, no. 6 (June 1, 2019): 197–207. http://dx.doi.org/10.1152/physiolgenomics.00124.2018.
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Epstein-Barr virus (EBV) is a widespread human virus that establishes latent infection, potentially leading to tumors, hematological disorders, and other severe diseases. EBV infections are associated with diverse symptoms and affect various organs; therefore, early diagnosis and treatment are crucial. B cell receptor (BCR) repertoires of B cell surface immunoglobulins have been widely studied for their association with various infectious diseases. However, the specific genetic changes that modulate the BCR repertoires after an EBV infection are still poorly understood. In this study, we employed high-throughput sequencing (HTS) to investigate the diversity of BCR repertoires in an EBV-transformed lymphoblastic cell line (LCL). Compared with the noninfected control B cell line, the LCL exhibited a decrease in overall BCR diversity but displayed an increase in the expansion of some dominant rearrangements such as IGHV4-31/IGHJ4, IGHV4-59/IGHJ4, IGHV5-51/IGHJ3, and IGHV3-74/IGHJ3. A higher frequency of occurrence of these rearrangement types was confirmed in patients with EBV infection. Interestingly, the IGHV3-74 rearrangement was only detected in EBV-infected children, suggesting that our experimental observations were not coincidental. In addition, we identified a highly dominant consensus motif, CAR(xRx)YGSG(xYx)FD, in complementarity-determining region 3 (CDR3) sequences of the heavy chain in the LCL. Our findings demonstrated the utility of HTS technology for studying the variations in signature motifs of the BCR repertoires after EBV infection. We propose that the analysis of BCR repertoire sequences represents a promising method for diagnosing early EBV infections and developing novel antibody- and vaccine-based therapies against such infections.
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Pollastro, Sabrina, Paul L. Klarenbeek, Marieke E. Doorenspleet, Barbera D. C. van Schaik, Rebecca E. E. Esveldt, Rogier M. Thurlings, Maria J. H. Boumans, et al. "Non-response to rituximab therapy in rheumatoid arthritis is associated with incomplete disruption of the B cell receptor repertoire." Annals of the Rheumatic Diseases 78, no. 10 (June 19, 2019): 1339–45. http://dx.doi.org/10.1136/annrheumdis-2018-214898.
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ObjectiveTo gain more insight into the dynamics of lymphocyte depletion and develop new predictors of clinical response to rituximab in rheumatoid arthritis (RA).MethodsRNA-based next-generation sequencing was used to analyse the B cell receptor (BCR) repertoire in peripheral blood and synovial tissue samples collected from 24 seropositive patients with RA treated with rituximab. Clonal expansion, mutation load and clonal overlap were assessed in samples collected before, at week 4 and at week 16 or 24 after treatment and correlated to the patients’ clinical response.ResultsAfter 4 weeks of rituximab-induced B cell depletion, the peripheral blood BCR repertoire of treated patients consisted of fewer, more dominant and more mutated BCR clones. No significant changes in the synovial tissue BCR repertoire were detected until week 16 post-treatment, when a reduced clonal overlap with baseline and an increased mutation load were observed. In patients who were non-responders at month 3 (n=5) using the European League Against Rheumatism response criteria, peripheral blood samples taken at week 4 after rituximab treatment showed more dominant clones compared with moderate responders (n=9) (median (IQR): 36 (27–52) vs 18 (16–26); p<0.01) and more clonal overlap with the baseline (median (IQR): 5% (2%–20%) vs 0% (0%–0%); p≤0.01).ConclusionSignificant changes in BCR clonality are observed in peripheral blood of patients 4 weeks after rituximab treatment, while changes in synovial tissue were observed at later time points. Incomplete depletion of the dominant baseline peripheral blood BCR repertoire in the first month of treatment might predict clinical non-response at 3 months.
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Li, Jie, Sam A. Bazzi, Florian Schmitz, Hidetaka Tanno, Jonathan R. McDaniel, Chang-Han Lee, Chaitanya Joshi, et al. "Molecular Level Characterization of Circulating Aquaporin-4 Antibodies in Neuromyelitis Optica Spectrum Disorder." Neurology - Neuroimmunology Neuroinflammation 8, no. 5 (June 24, 2021): e1034. http://dx.doi.org/10.1212/nxi.0000000000001034.
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ObjectiveTo determine whether distinct aquaporin-4 (AQP4)-IgG lineages play a role in neuromyelitis optica spectrum disorder (NMOSD) pathogenesis, we profiled the AQP4-IgG polyclonal serum repertoire and identified, quantified, and functionally characterized distinct AQP4-IgG lineages circulating in 2 patients with NMOSD.MethodsWe combined high-throughput sequencing and quantitative immunoproteomics to simultaneously determine the constituents of both the B-cell receptor (BCR) and the serologic (IgG) anti-AQP4 antibody repertoires in the peripheral blood of patients with NMOSD. The monoclonal antibodies identified by this platform were recombinantly expressed and functionally characterized in vitro.ResultsMultiple antibody lineages comprise serum AQP4-IgG repertoires. Their distribution, however, can be strikingly different in polarization (polyclonal vs pauciclonal). Among the 4 serum AQP4-IgG monoclonal antibodies we identified in 2 patients, 3 induced complement-dependent cytotoxicity in a model mammalian cell line (p < 0.01).ConclusionsThe composition and polarization of AQP4-IgG antibody repertoires may play an important role in NMOSD pathogenesis and clinical presentation. Here, we present a means of coupling both cellular (BCR) and serologic (IgG) antibody repertoire analysis, which has not previously been performed in NMOSD. Our analysis could be applied in the future to clinical management of patients with NMOSD to monitor disease activity over time as well as applied to other autoimmune diseases to facilitate a deeper understanding of disease pathogenesis relative to autoantibody clones.
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Schina, Aimilia, Zsofia Sztupinszki, Inge Marie Svane, Zoltan Szallasi, Göran Jönsson, and Marco Donia. "Intratumoral T-cell and B-cell receptor architecture associates with distinct immune tumor microenvironment features and clinical outcomes of anti-PD-1/L1 immunotherapy." Journal for ImmunoTherapy of Cancer 11, no. 8 (August 2023): e006941. http://dx.doi.org/10.1136/jitc-2023-006941.
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BackgroundEffective cooperation between B-cells and T-cells within the tumor microenvironment may lead to the regression of established tumors. B-cells and T-cells can recognize tumor antigens with exquisite specificity via their receptor complexes. Nevertheless, whether a diverse intratumoral B-cells and T-cell receptor (BCR, TCR) repertoire affects the tumor immune microenvironment (TIME) and clinical outcomes in patients treated with immunotherapy is unclear.MethodsWe extracted information on BCR and TCR repertoire diversity from large clinical datasets and measured the association between immune receptor diversity features, the TIME, and clinical outcomes of patients treated with anti-PD-1/PD-L1 immunotherapy.ResultsIn multiple tumor types, an increasingly diverse TCR repertoire was strongly associated with a highly activated TIME, while BCR diversity was more associated with antibody responses but not with the overall B-cell infiltration nor with measures related to intratumoral CD8+T cell activity. Neither TCR nor BCR diversity was independent prognostic biomarkers of survival across multiple cancer types. However, both TCR and BCR diversity improved the performance of predictive models combined with established biomarkers of response to immunotherapy.ConclusionOverall, these data indicate a currently unexplored immunological role of intratumoral B-cells associated with BCR diversity and antibody responses but independent of classical anticancer T-cells intratumoral activities.
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Ramadoss, Nitya S., and William H. Robinson. "Characterizing the BCR repertoire in immune-mediated diseases." Nature Reviews Rheumatology 16, no. 1 (November 28, 2019): 7–8. http://dx.doi.org/10.1038/s41584-019-0339-y.
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Rediti, Mattia, David Venet, Francoise Rothe, Tao Qing, Marion Maetens, Ian Bradbury, Miguel A. Izquierdo, et al. "Association of T- and B-cell receptor repertoires with molecular subtypes and outcome in HER2+ breast cancer: An analysis of the NeoALTTO clinical trial." Journal of Clinical Oncology 38, no. 15_suppl (May 20, 2020): 511. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.511.
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511 Background: Clinicopathological and molecular features, including estrogen receptor (ER) status and PAM50 subtypes, have shown an association with immunogenicity and tumor-infiltrating lymphocyte (TIL) levels in breast cancer (BC). To investigate the complexity of the immune response in HER2+ BC, we explored the association of T- and B-cell receptor (TCR and BCR) repertoires with clinicopathological characteristics, PAM50 subtypes and outcome in the NeoALTTO phase III trial. Methods: RNA sequencing (RNAseq) data from baseline tumor biopsies were available for 254 out of the 455 patients enrolled. TCR and BCR repertoires were extracted from RNAseq data using the MiXCR software. Repertoire and diversity measures (read counts, number of clones, evenness, Gini index, Shannon entropy, length of the complementarity-determining region 3 [CDR3], top and second top clone proportions) were estimated. PAM50 subtypes were computed from RNAseq data. Univariate and multivariate (adjusted for clinicopathological characteristics, TIL levels dichotomized using the median value of 12.5% and treatment arm) Cox proportional hazard models were used for survival analysis, while logistic regressions were used for pathological complete response (pCR), defined as ypT0/is. All results reported had a false discovery rate (FDR) <0.05. Results: Higher BCR read counts, number of clones and Gini index were significantly associated with ER-negative as well as grade 3 tumors. Among the PAM50 subtypes, HER2-enriched (HER2-E) showed significantly higher BCR read counts, number of clones and Gini index along with lower evenness compared to luminal A and B, as well as higher length of CDR3 than luminal A. Of note, basal-like showed similar BCR diversity measures to HER2-E. No significant differences were noted for TCR diversity measures. In multivariate analyses, neither TCR nor BCR features were associated with pCR, while BCR evenness (HR 1.5; 95%CI 1.1-2.1) and Gini index (HR 0.66; 95%CI 0.5-0.88) were associated with event-free survival. Conclusions: BCR repertoire measures suggest a clonal expansion in HER2-E and basal-like PAM50 subtypes. Furthermore, the implementation of BCR-derived biomarkers can help to identify patients with an improved clinical outcome after neoadjuvant anti-HER2 treatment. Our findings highlight the heterogeneity of the immune response within HER2+ BC and provide support for biomarker-driven treatment strategies including immunotherapy in this BC subtype. Further validation is required. Clinical trial information: NCT00553358 .
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dos Santos Araújo, Ronny Petterson, Renato Kaylan Alves França, Napoleão Fonseca Valadares, Andrea Queiroz Maranhão, and Marcelo Macedo Brigido. "A Germline-Encoded Structural Arginine Trap Underlies the Anti-DNA Reactivity of a Murine V Gene Segment." International Journal of Molecular Sciences 22, no. 9 (April 26, 2021): 4541. http://dx.doi.org/10.3390/ijms22094541.
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Autoimmunity may have its origins of early repertoire selection in developmental B cells. Such a primary repertoire is probably shaped by selecting B cells that can efficiently perform productive signaling, stimulated by self-antigens in the bone marrow, such as DNA. In support of that idea, we previously found a V segment from VH10 family that can form antibodies that bind to DNA independent of CDR3 usage. In this paper we designed four antibody fragments in a novel single-chain pre-BCR (scpre-BCR) format containing germinal V gene segments from families known to bind DNA (VH10) or not (VH4) connected to a murine surrogate light chain (SLC), lacking the highly charged unique region (UR), by a hydrophilic peptide linker. We also tested the influence of CDR2 on DNA reactivity by shuffling the CDR2 loop. The scpre-BCRs were expressed in bacteria. VH10 bearing scpre-BCR could bind DNA, while scpre-BCR carrying the VH4 segment did not. The CDR2 loop shuffling hampered VH10 reactivity while displaying a gain-of-function in the nonbinding VH4 germline. We modeled the binding sites demonstrating the conservation of a positivity charged pocket in the VH10 CDR2 as the possible cross-reactive structural element. We presented evidence of DNA reactivity hardwired in a V gene, suggesting a structural mechanism for innate autoreactivity. Therefore, while autoreactivity to DNA can lead to autoimmunity, efficiently signaling for B cell development is likely a trade-off mechanism leading to the selection of potentially autoreactive repertoires.
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Bone, Jennifer, Newell Washburn, Jian Han, Miranda Steele, Pranav Murthy, and Michael Lotze. "348 Interleukin 2(IL-2) systems immunology modeling: machine learning for cancer immunotherapy." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A375. http://dx.doi.org/10.1136/jitc-2021-sitc2021.348.
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BackgroundClinical outcomes are correlated with aggregate B (BCR) and T cell receptor (TCR) diversity (the adaptome) in several infectious diseases and cancers. Advances in dimer avoidance multiplexed PCR (DAM-PCR) followed by next-generation sequencing (NGS) enable measurements of immune repertoire diversity and clonality, allowing prediction of cancer states and response to treatment. Clonotype-mediated predictions collapse a space of up to 1025 possible CDR3 variable region sequences into descriptors such as whole-sequence diversity. Broad descriptors, however, mask cancer-specific information embedded within clonotype sequences. Deep learning algorithms typically need large patient cohorts to make accurate predictions. We present a statistical model predicting response to IL-2 immunotherapy for small cohorts based on natural language processing (NLP) of CDR3 TCR and BCR clonotypes.MethodsIn a completed Phase 2 trial (NCT01550367), the adaptome of 29 patients with metastatic clear cell renal carcinoma (RCC) treated with high dose (HD) IL-2 and the autophagy inhibitor, hydroxychloroquine (HCQ) were measured from peripheral blood samples by DAM-PCR. All seven TCR and BCR chains were measured at three treatment points (pre-treatment, 14D after HCQ initiation, and following recovery from the first cycle of IL-2 on D15). Outcomes were assessed by assigning two states (responder or non-responder) one year following treatment based on radiographic changes in tumor size. Cancer-specific amino acid motifs from TCR and BCR CDR3 sequences on D15 were mined by counting amino acid pairs and calculating a 400-feature transition probability matrix, scoring the likelihood of a motif belonging to the responder or non-responder cohort.ResultsSeven-chain NLP analysis of CDR3 amino acid motifs at > 90% accuracy for each chain independently predicted patient response to IL-2 by D15 (figure 1). Furthermore, longitudinal monitoring of patient CDR3s across the three timepoints revealed a dichotomy in repertoire orchestration. Responding patients, convincingly, were more likely to demonstrate either a TCR-driven (p<0.01) or a BCR-driven (p<0.001) entropy bias while non-responding patients unanimously showed no significant bias.Abstract 348 Figure 1Classification of nonresponding (Non-Res) and responding (Res) patients based on scoring from Feature Selection Filter and Analysis. ****, p<0.0001; ***, p<0.001; **, p<0.01ConclusionsNLP of both TCR and BCR repertoires can provide early predictions of cancer response to treatment. Furthermore, seven-chain longitudinal monitoring across treatment revealed a surprisingly robust repertoire orchestration in responders that was not observed in non-responders, suggesting that the methodology proposed here can be used to gain new mechanistic insight into the role of repertoire evaluation in cancer immunotherapy.
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Abdollahi, Nika, Lucile Jeusset, Anne Langlois De Septenville, Hugues Ripoche, Frédéric Davi, and Juliana Silva Bernardes. "A multi-objective based clustering for inferring BCR clonal lineages from high-throughput B cell repertoire data." PLOS Computational Biology 18, no. 8 (August 29, 2022): e1010411. http://dx.doi.org/10.1371/journal.pcbi.1010411.
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The adaptive B cell response is driven by the expansion, somatic hypermutation, and selection of B cell clonal lineages. A high number of clonal lineages in a B cell population indicates a highly diverse repertoire, while clonal size distribution and sequence diversity antigen selective pressure. Identifying clonal lineages is fundamental to many repertoire studies, including repertoire comparisons, clonal tracking, and statistical analysis. Several methods have been developed to group sequences from high-throughput B cell repertoire data. Current methods use clustering algorithms to group clonally-related sequences based on their similarities or distances. Such approaches create groups by optimizing a single objective that typically minimizes intra-clonal distances. However, optimizing several objective functions can be advantageous and boost the algorithm convergence rate. Here we propose a new method based on multi-objective clustering. Our approach requires V(D)J annotations to obtain the initial groups and iteratively applies two objective functions that optimize cohesion and separation within clonal lineages simultaneously. We show that our method greatly improves clonal lineage grouping on simulated benchmarks with varied mutation rates compared to other tools. When applied to experimental repertoires generated from high-throughput sequencing, its clustering results are comparable to the most performing tools and can reproduce the results of previous publications. The method based on multi-objective clustering can accurately identify clonally-related antibody sequences and presents the lowest running time among state-of-art tools. All these features constitute an attractive option for repertoire analysis, particularly in the clinical context. MobiLLe can potentially help unravel the mechanisms involved in developing and evolving B cell malignancies.
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Chenchik, Alex, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Paul Diehl, and Lester Kobzik. "T-cell and B-cell receptor repertoire profiling for biomarker discovery." Journal of Immunology 210, no. 1_Supplement (May 1, 2023): 246.01. http://dx.doi.org/10.4049/jimmunol.210.supp.246.01.
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Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B-cells. We developed a new technology for combined profiling of all human TCR and BCR variable regions and phenotypic characterization of immune cells in the same workflow. The TCR and BCR immunophenotyping method involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, as well as determining the expression levels of the most informative T- and B-cell phenotyping genes. Results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data allows profiling of the TCR and BCR clonotypes and identifcation of major immune cell subtypes and their activation status. Preliminary data from rheumatoid arthritis blood samples and others will be presented.
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Hou, Xiaohong, Wenjing Pan, Mollye Depinet, Brittany E. Brown, Mary Eisenhower, Miranda Byrne-Steele, Michele Weber, Meyer Dworsky, Sharon Callison, and Jian Han. "Complete all-in-one immune repertoire profiling of newborn babies via novel dimer-avoided multiplex PCR (dam-PCR)." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 131.28. http://dx.doi.org/10.4049/jimmunol.202.supp.131.28.
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Abstract There is an increased demand for immune repertoire profiling in both basic research as well as in various clinical settings, such as during vaccine development, lymphocyte tracking in minimal residual disease (MRD), hematopoietic stem cell transplant recovery monitoring, immunotherapy development, and immunological monitoring for prognosis purposes during treatment. Although next generation sequencing of the immune repertoire allows detailed, sequence-specific insight into the adaptive immune response, it usually focuses on only one or two receptor chains. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. We therefore developed a novel PCR technique, dimer-avoided multiplex PCR, that allows amplification of both BCRs and TCRs in a single, quantitative multiplex reaction. Our method generates next generation sequencing libraries for all TCR and BCR loci including TCR-beta, TCR-alpha, TCR-delta, TCR-gamma, BCR-IgH, and BCR-IgK and IgL. Here, we report the application of this method to investigate the immune repertoire of cord blood from newborn babies. Our study revealed the vast diversity and abundance of babies’ immune repertoire, the composition of TCR and BCR loci, and the sharing status of unique CDR3s among newborns. This single reaction, cost effective, all-inclusive, and quantitative immune-profiling analysis method shows promise for future applications in both basic research and clinical settings.
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Aysola, Varun, Christina Labib, and Neetu Gupta. "Ezrin promotes antigen receptor diversity during B cell development by supporting immunoglobulin heavy chain variable gene recombination." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 107.14. http://dx.doi.org/10.4049/jimmunol.208.supp.107.14.
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Abstract Genome-level rearrangements of immunoglobulin genes during B cell development are critical for generation of a diverse repertoire of B cell antigen receptors (BCRs) that bind to a multitude of foreign antigens and some self-antigens. Bone marrow B cell development involves a variety of cell-cell interactions, cell migration and receptor signaling that likely benefit from the activity of membrane-cytoskeletal reorganizing proteins. However, the specific contribution of such proteins towards BCR repertoire diversification is poorly understood. Ezrin is a membrane-cytoskeletal linker protein that regulates mature B cell activation through spatial organization of the BCR. We employed next generation sequencing to investigate if Ezrin plays a role in immunoglobulin heavy chain rearrangements and generation of BCR diversity in developing bone marrow B cells. BCR repertoire development occurred stochastically in B cell progenitors from both control and B cell conditional Ezrin-deficient mice. However, the loss of Ezrin resulted in fewer unique CDR3s in the BCRs and reduced Shannon entropy. Ezrin-deficient pro-, pre- and immature B cells utilized similar number of joining (J) genes but significantly fewer variable (V) genes, thereby decreasing V-J combinatorial diversity. V-J junctional diversity, measured by CDR3 length, nucleotide additions and deletions, was also altered in Ezrin-deficient pro-, pre- and immature B cells. Mechanistically, Ezrin-deficient bone marrow B cells showed a marked decrease in RAG1 gene expression, indicating a less efficient DNA recombination machinery. Overall, our results demonstrate a novel role for Ezrin in shaping the BCR repertoire through combinatorial and junctional diversification. Supported by grants from NIH (R01 AR067705) to N.G.
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Navarro, Fabio, Eric Levy, Pamela Milani, Qiang Li, Shruti Bhide, Upasana Dutta, Charles W. Abbott, et al. "Abstract 5021: Accurate quantification of infiltrating B cell composition and clone diversity in tumor samples." Cancer Research 82, no. 12_Supplement (June 15, 2022): 5021. http://dx.doi.org/10.1158/1538-7445.am2022-5021.
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Abstract Tumors harbor a complex ecosystem of malignant, immune, and stromal cells. While malignant cells dictate much of the tumor biology, there is evidence that the tumor microenvironment (TME) also plays a major role in disease etiology. Given the complexity and abundance of the TME cellular composition, investigating the role of immune cell types will yield novel biomarkers for tumor progression and response to therapies. The role of B cells as a prognostic biomarker remains elusive. For instance, infiltrating B cells in CRC have both positive and negative prognostic value. Thus, a scalable approach to quantify B cells and the B-cell receptor (BCR) repertoire could yield novel insights into the role of B cells in tumor biology. To address this, we have developed immune cell quantification (InfiltrateID࣪) and immune receptor repertoire profiling (RepertoireID࣪) methods as part of the ImmunoID NeXT Platform®, an augmented, immuno-oncology-optimized exome/transcriptome platform. We estimate B cell abundance and BCR repertoire by profiling FFPE and PBMC samples using ImmunoID NeXT࣪. In expanding upon InfiltrateID to further estimate B cell abundance, here we regress the bulk RNA-seq readout from a reference signature from purified immune cell types. We also generate orthogonal quantifications of B cell abundance by profiling samples with cytometry by time of flight, single-cell RNA-seq, flow cytometry, and immunohistochemistry (IHC). We compare BCR results from ImmunoID NeXT to a standalone sequencing approach to evaluate the concordance of top clones. We then utilize BCR profiling from ImmunoID NeXT to analyze clonality and isotype composition in tumor samples. We first use InfiltrateID to estimate absolute B cell fractions in over 50 samples. Overall, we observe a high correlation between InfiltrateID results and orthogonal data sets in both PBMC and tumor FFPE samples (R2=0.90). When comparing BCR results from RepertoireID to a standalone BCR sequencing method that profiles IgM and IgG, we identify 475 and 387 of the top 500 clones in IgG and IgM, respectively, with highly concordant abundances across all clones (R2>0.72 and R2>0.82 in IgM and IgG, respectively). Next, we use InfiltrateID to estimate absolute B cell fractions in over 650 samples from 14 tumor types. On average, samples display B cell fractions in agreement with the literature and IHC quantifications, with higher B cell fractions in lung, breast, and cervical tumors. We also observe a range of BCR clonality values across tumor types. Finally, we observe differences in B cell composition and repertoire diversity in tumor samples from patients who underwent checkpoint blockade therapy. We show that InfiltrateID and RepertoireID accurately capture the composition and clone diversity of infiltrating B cells in tumor samples. Citation Format: Fabio Navarro, Eric Levy, Pamela Milani, Qiang Li, Shruti Bhide, Upasana Dutta, Charles W. Abbott, Jose Jacob, Rena McClory, John West, John Lyle, Sean Boyle, Richard O. Chen. Accurate quantification of infiltrating B cell composition and clone diversity in tumor samples [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5021.
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Tak, Paul P., Marieke E. Doorenspleet, Maria J. H. de Hair, Paul L. Klarenbeek, Marian H. van Beers-Tas, Antoine H. C. van Kampen, Dirkjan van Schaardenburg, Danielle M. Gerlag, Frank Baas, and Niek de Vries. "Dominant B cell receptor clones in peripheral blood predict onset of arthritis in individuals at risk for rheumatoid arthritis." Annals of the Rheumatic Diseases 76, no. 11 (August 8, 2017): 1924–30. http://dx.doi.org/10.1136/annrheumdis-2017-211351.
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BackgroundThe onset of seropositive rheumatoid arthritis (RA) is preceded by the presence of specific autoantibodies in the absence of synovial inflammation. Only a subset of these at-risk individuals will develop clinical disease. This impedes efforts to implement early interventions that may prevent onset of clinically manifest disease. Here we analyse whether clonal changes in the B cell receptor (BCR) repertoire can reliably predict onset of signs and symptoms.MethodsIn a prospective cohort study in 21 individuals at risk for RA based on the presence of autoantibodies, the BCR repertoire of paired peripheral blood and synovial tissue samples was analysed using next-generation BCR sequencing. BCR clones that were expanded beyond 0.5% of the total repertoire were labelled dominant. The relative risk (RR) for onset of arthritis was assessed using the presence of ≥5 dominant BCR clones as cut-off. Findings in peripheral blood were validated in an independent prospective cohort of 50 at-risk individuals. Based on the test cohort, individuals in the validation cohort were considered positive if peripheral blood at study entry showed ≥5 dominant BCR clones.FindingsBoth in the test and validation cohort, the presence of ≥5 dominant BCR clones in peripheral blood was significantly associated with arthritis development after follow-up (validation cohort RR 6.3, 95% CI 2.7 to 15, p<1×10−4). Even when adjusted for a recently described clinical prediction rule the association remained intact (RR 5.0, 95% CI 1.2 to 20, p=0.024). When individuals developed arthritis, dominant BCR clones disappeared from peripheral blood and appeared in synovial tissue, suggesting a direct role of these clones in disease pathogenesis.InterpretationDominant BCR clones in peripheral blood predict onset of clinical signs and symptoms of RA in at-risk individuals with high accuracy. Our data suggest that during onset of RA these clones shift from peripheral blood to the target tissue.
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Sakurai, Keiichi, Ken Tsumiyama, and Shunichi Shiozawa. "The contribution of autoantibody-inducing CD4+ T cell (aiCD4 T cell) help to the B cell maturation and possible autoantibody in Systemic Lupus Erythematosus." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 163.17. http://dx.doi.org/10.4049/jimmunol.200.supp.163.17.
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Abstract INTRODUCTION Autoantibody-inducing CD4 T (aiCD4 T) cell seems indispensable for the self-organized criticality theory that induces Systemic Lupus Erythematosus (SLE). We previously found that the aiCD4 T cell belongs to CD45RBlo122lo CD4 T cell subpopulation. We here analyze the B cell population and CD19+ splenic B cell receptor (BCR) repertoire, and test our contention whether aiCD4 T cell helps B cell maturation. METHODS BALB/c mice were immunized with OVA twelve times and SLE was induced. Cell surface markers of B cell were detected with flow cytometry. CD19+ splenic B cells were sorted and BCR repertoires of these cells were analyzed on next generation sequencer. RESULTS After twelve times immunization of mice with OVA, follicular B (Fo B) cell and germinal center B (GC B) cell were significantly increased(P<0.05), whereas marginal zone B cell was decreased. Activation markers of the B cell were increased and in particular, CD80 was significantly increased (P<0.05). In addition, the number of clonotypes of CD19+ splenic B cells in OVA immunized mice was increased. Among these repertoires, the same pairs of V and J usage were frequently found in OVA immunized mice. CONCLUSION Fo B cell and GC B cell and the activation markers of B cell, as well, were significantly increased in OVA immunized mice, and the result of BCR repertoire analysis suggested that somatic hyper mutation occurred frequently in OVA-stimulated mice, which is compatible with the contention that aiCD4 T cells helps the maturation of B cells into germinal center B cells to enhance autoantibody formation thereby inducing SLE.
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Iwabuchi, Kumiko A., Noriko Sasakawa, and Akitsu Hotta. "Comprehensive profiling of TCR and BCR repertoire of infiltrated lymphocytes in the mouse injured skeletal muscle." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 86.6. http://dx.doi.org/10.4049/jimmunol.204.supp.86.6.
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Abstract Lymphocytes infiltrating to the injured skeletal muscle have been shown to play an important role in muscle regeneration. Site-specific infiltrating/expanding lymphocytes and its molecular features are potential resources for bioengineering such as a drug delivery system. Although some previous studies showed the accumulation of lymphocytes to the injured skeletal muscle, the actual frequency of distinct lymphocyte clones in situ is still unclear. Hence, a comprehensive picture of the lymphocyte repertoire has yet to be described in detail. To this end, we conducted a comprehensive profiling of TCR and BCR repertoire of infiltrating lymphocytes in the mouse injured skeletal muscle model by next-generation sequencing. In the TCR repertoire profiling, we found that the expression level of TCR transcripts contains unique CDR3 α and distinct population of CDR3 β were increased (7.6–17% and 6.7–15%) in infiltrating T cells in both of cardiotoxin- or cryo-injured skeletal muscle, whereas no clonal expansion of specific TCR α/β transcripts observed in control splenic T cells (<1% respectively). While in the BCR repertoire profiling, various transcripts of immunoglobulin kappa chains showed expansion in infiltrated fraction, despite the proportion of immunoglobulin heavy and lambda chain were remained similar to control splenic B cells. Together these data have allowed us to identify TCR/BCR candidates targeting injured skeletal muscles. Furthermore, we aim to utilize the T cells/B cells expressing high-affinity TCR/BCR for the drug delivery system, especially for large protein complexes.
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Wu, Xiuli, Qifa Liu, Yangqiu Li, Shaohua Chen, Xuan Du, Xianfeng Zha, and Shuyun Zhou. "Analysis of T Cell Cloanlity of Ph+ Acute Lymphoblastic Leukemia with Chronic Gvhd in Continuous Remission after Allogeneic Hematopoietic Stem Cell Transplantation." Blood 112, no. 11 (November 16, 2008): 3941. http://dx.doi.org/10.1182/blood.v112.11.3941.3941.
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Abstract Chronic graft-versus-host disease (cGVHD) is one of the major complications following allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, recent study suggested that the presence of cGVHD might reduce the risk of relapse in Ph+ acute lymphoblastic leukemia (ALL) after allo-HSCT. GVHD is mediated by proliferation of special T-cell clones, we analyzed the T cell receptor (TCR) Vβ repertoire and cloanlity in Ph+ ALL patients undergone cGVHD after allo-HSCT in order to find the special T-cell clones associated with continuous remission. Allogeneic transplantations were performed in the first complete remission (CR) (6 ALLP190+ patients). The numbers of BCR-ABL copies in peripheral blood samples were assessed with the real-time quantitative RT-PCR (RQ-PCR) test at diagnosis, and every month after HSCT. The quantity of BCR-ABL transcript was normalized for normal ABL expression and the result was expressed as the ratio of BCR-ABL copies to ABL copies. With monitoring BCR-ABL copies of 6 BCR-ABLP190+ ALL patients after HSCT (the mean ratio of BCR-ABLP190/ABL at diagnose was 0.41 ± 0.39%), we found that 4 patients with extensive cutancous cGVHD had no evidence of leukemia recurrence, and the BCRABL transcripts of patients with cGVHD were remaining 0 copy, while 2 patients without cGVHD relapsed (the mean ratio of BCR-ABLP190/ABL was 7.56 ± 2.49%) and even dead within six months of HSCT. The expression and cloanlity analysis of TCR Vβ repertoire was detected in peripheral blood samples from patients with cGVHD by RT-PCR and genescan technique. TCR Vβ repertoires with polyclonal pattern were identified in normal controls and donor groups. However, the skew expression pattern of TCR Vβ repertoire could be detected in patients with cGVHD even more than 1 year after allo-HSCT. Among the 24 Vβ subfamilies, there were only 11~17 Vβ subfamilies expressed in cGVHD patients. Oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 1, 2, 3, 7, 8, 12, 14, 16~20, 22 and 23 subfamilies in 4 ALL patients (BCR-ABLP190 remission) with cGVHD. As a contrast, oligoclonal or monoclonal expanded T cells were identified in TCR Vβ 2, 7, 8, 14, 17, 20, and 22 subfamilies in 2 chronic myeloid leukemia patients (BCR-ABLP210 remission) with cGVHD. In conclusion, the predominant usage and clonal expansion of TCR Vβ subfamily T cells could be found in Ph+ ALL patients with cGVHD and these groups of specific T-cell clones (Vβ 1, 3, 12, 16, 18, 19 and 23) were potentially associated with cGVHD in ALL and might be responsible for maintaining CR in Ph+ ALL patients after allo-HSCT. Research Funding.
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Levy, Eric, Pamela Milani, Gabor Bartha, Charles Abbott, Robert Power, Rena McClory, Robin Li, et al. "67 B-cell receptor heavy chain repertoire profiling using an augmented transcriptome." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A72. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0067.
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BackgroundComprehensive profiling of the tumor and tumor microenvironment (TME) is a critical tool for furthering our understanding of tumor progression and response to treatment, including immunotherapies. To address this challenge, we developed an augmented, immuno-oncology-optimized exome/transcriptome platform, ImmunoID NeXTTM, which provides a more comprehensive view of the tumor and TME from limited FFPE tumor biopsies. We have recently added the ability to profile the B-cell receptor (BCR) heavy chain. Here, we show that ImmunoID NeXT is now able to accurately and reproducibly profile abundant B-cell clones and provide information on the diversity of B-cells in tumor samples.MethodsWe analyzed multiple replicates of PBMCs to examine the reproducibility of BCR sequence identification using ImmunoID NeXT. Utilizing a standalone BCR sequencing approach, we further evaluated the concordance of top clones to those identified by ImmunoID NeXT. In addition, we analyzed the reproducibility of BCR sequences in patient-derived FFPE samples. Finally, we used ImmunoID NeXT to profile the B-cell clonal diversity across over 500 solid tumor samples.ResultsReproducibility in PBMC samples was very high, with abundances of clones shared between replicates being very concordant (R2>0.92, R2>0.86, and R2>0.97 for IgG, IgM, and IgA, respectively). When comparing to a standalone BCR sequencing method that profiles IgM and IgG, we observed highly concordant abundances (R2>0.72 and R2>0.82 in IgM and IgG, respectively), as well as strong overlaps of top clones. When comparing subsequent curls of a tumor FFPE sample, we also achieved a high concordance of clonal abundances (R2>0.92, R2>0.93, and R2>0.76 for IgG, IgM, and IgA, respectively). Finally, we observed differences in clonal diversity of B-cell repertoires across over 500 solid tumor samples.ConclusionsWe demonstrate that ImmunoID NeXT can be used to reproducibly, sensitively, and accurately profile high-abundance BCR heavy chain clones, including coverage of all major isotypes. In addition, we show how ImmunoID NeXT can profile the diversity of the BCR repertoire across a variety of tumor samples. Combined with the platform’s TCR profiling capabilities, ImmunoID NeXT can provide insight into the diversity of the immune repertoire, contributing to its ability to provide comprehensive analysis of both the tumor and TME from a single FFPE sample.
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Wesemann, Duane R., Akritee Shrestha, Jennifer Magee, Yuezhou Chen, Jared Silver, and Alessandra Granato. "Role of Microbes in B-Cell Lymphopoiesis and Early Ig Repertoire Development." Blood 124, no. 21 (December 6, 2014): SCI—47—SCI—47. http://dx.doi.org/10.1182/blood.v124.21.sci-47.sci-47.
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Abstract Progenitor (pro-) and precursor (pre-) B cells express the recombination-activating gene (RAG1/RAG2) endonuclease, which initiates the V(D)J recombination reaction that assembles Immunoglobulin heavy (IgH) and light (IgL) chain variable region exons from germline gene segments. Expression of productively assembled IgH μ chains and IgL (Igκ or Igλ) chains generates IgM molecules that form the B-cell antigen binding receptor (BCR) with a diverse primary repertoire of binding specificities. As the newly assembled and expressed IgM interacts with local antigens, RAG expression can continue, allowing continued Igκ V(D)J recombination that can replace the previously assembled VκJκ exon with one that generates a new specificity. This "receptor editing" process provides an antigen-mediated regulatory checkpoint that helps shape the pre-immune BCR repertoire. As the principal location of early B-cell development, the bone marrow antigenic environment is the main force affecting receptor editing. However, the extent to which self-antigens outside of the bone marrow — as well as environmental antigens — may shape the primary Ig repertoire is not known. Here we show that early B-cell development takes place in peripheral sites, including the small intestinal mucosa, in young mice. Cells in these sites co-express RAG with cytoplasmic Igκ and Igμ, indicating active receptor editing; and, correspondingly, they have significant differences in Vκ usage compared to RAG2-expressing bone marrow B-lineage cells and other RAG2-expressing B-lineage cells from other peripheral sites. In addition, the luminal microbial environment appears to also affect Igλ/Igκ ratios. We conclude that early B-cell development can occur in the periphery, including the intestinal mucosa, where commensal microbes may influence BCR diversification and gut pre-immune Ig repertoires. Disclosures No relevant conflicts of interest to declare.
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Mingos, Antonios, Nikolaos Pechlivanis, Georgios Karakatsoulis, Glykeria Gkoliou, Nikolaos Vastarouchas, Smaragdi Marinaki, Paraskevi Tsoutsoura, et al. "Antigen Receptor Gene Repertoire Profiling in Kidney Transplant Recipients Post-Sars-Cov-2 Vaccination." Blood 142, Supplement 1 (November 28, 2023): 5372. http://dx.doi.org/10.1182/blood-2023-188494.
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Kidney transplant recipients (KTRs) are highly susceptible to viral infections, while also exhibiting a higher risk of developing severe COVID-19. Recent studies have demonstrated a suboptimal humoral response to current COVID-19 vaccines in this cohort, necessitating multiple vaccine doses. Nevertheless, low rates of seroconversion have been observed even after booster vaccinations, highlighting the need for the in-depth characterization of both humoral and cellular immune responses towards the development of innovative and effective immunoprophylactic strategies tailored for KTRs. Here, we investigated the immunogenetic features of the T cell receptor (TR) and B cell receptor immunoglobulin (BcR IG) gene repertoires in KTRs using targeted Next Generation Sequencing (NGS). In more detail, we studied 19 KTRs at two consecutive timepoints, after the 2 nd (T1) and multiple vaccination doses (T2), respectively. We included as a control group 19 healthy individuals (HIs) sampled either after two mRNA vaccination doses or after a documented SARS-CoV-2 infection. Seroconversion (>1300 AU/ml of anti-RBD IgG) was measured by the Abbott SARS-CoV-2 IgG assay: all HIs tested positive, in sharp contrast to KTRs who failed to exhibit humoral responses after the 2 nd dose (T1). Immunogenetic characterization of TR/BcR IG gene repertoires was performed by paired-end NGS; resultant data were processed through the TRIP software. Only productive V(D)J gene rearrangements were considered for the computation of clonotypes (i.e. TR/BcR IG gene rearrangements with identical V gene usage and complementarity-determining region 3 amino acid sequence-CDR3 aa). Starting with the TR gene repertoire, diversity significantly increased at T2 (average Shannon diversity: KTR-T1=638 vs KTR-T2=3212, p<0.0005| KTR-T2=3212 vs HI=2146, p<0.05). TR clonality (mean cumulative frequency of the 10 dominant TR clonotypes/sample) was significantly increased at T1 in KTRs vs HIs (KTR-T1=37% vs HI=25%, p<0.05), eventually decreasing at T2, reaching comparable levels to those measured in HIs (KTR-T2=27%). At T1, a median of 7.6% of TR clonotypes/sample in KTRs were SARS-specific (high-affinity TR clonotypes to SARS-specific peptide-MHC complexes, >0.9 binding score determined by the ERGO-II algorithm), almost identical to what we found for HIs (7.7%). Although the TR gene repertoire was renewed in T2, the percentage of SARS-specific clonotypes remained essentially identical (median of 7.8%). Turning to the BcR IG gene repertoire, despite the absence of a serological response at T1, the percentage of the predicted SARS-specific BcR IG clonotypes (high-homology with SARS-specific CDR3 aa sequences published at CoV-AbDab, >0.9 identity score) did not differ between KTRs (0.72% at T1 and 0.65% at T2) vs HIs (0.63%), raising the intriguing possibility of B cell anergy in the former. Overtime assessment revealed that BcR IG clonotype diversity significantly increased at T2 vs T1 in KTRs (average Shannon diversity: KTR-T1=12006 vs KTR-T2=38576 p<0.05) and strongly correlated with the presence of SARS-specific BcR IG clonotypes (r=0.65, p<0.001). Nevertheless, the cumulative fraction of SARS-specific BcR IG clonotypes at KTR-T2 decreased (median cumulative frequency of SARS-specific clonotypes: KTR-T2=0.45% vs HI=0.67%, p<0.05). Of note, multiple immunizations resulted to SARS-specific BcR IG clonotypes with a significantly increased number of somatic hypermutations (SHM) compared to either HIs (p<0.0005) or T1 (p<0.05). In conclusion, our findings suggest that the lack of seroconversion at T1 in KTRs is potentially counteracted by cellular sensitization, likely mediated by SARS-specific T cells, that is sustained by booster vaccination. The latter also drives the accumulation of SHM in BcR IG clonotypes, suggesting distinct differentiation trajectories (germinal center-independent at T1 vs germinal center-dependent at T2) that could give rise to new B cell clones with increased neutralizing potency.
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Sun, Amy, Tatiana I. Novobrantseva, Maryaline Coffre, Susannah L. Hewitt, Kari Jensen, Jane A. Skok, Klaus Rajewsky, and Sergei B. Koralov. "VH replacement in primary immunoglobulin repertoire diversification." Proceedings of the National Academy of Sciences 112, no. 5 (January 21, 2015): E458—E466. http://dx.doi.org/10.1073/pnas.1418001112.
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The genes encoding the variable (V) region of the B-cell antigen receptor (BCR) are assembled from V, D (diversity), and J (joining) elements through a RAG-mediated recombination process that relies on the recognition of recombination signal sequences (RSSs) flanking the individual elements. Secondary V(D)J rearrangement modifies the original Ig rearrangement if a nonproductive original joint is formed, as a response to inappropriate signaling from a self-reactive BCR, or as part of a stochastic mechanism to further diversify the Ig repertoire. VH replacement represents a RAG-mediated secondary rearrangement in which an upstream VH element recombines with a rearranged VHDHJH joint to generate a new BCR specificity. The rearrangement occurs between the cryptic RSS of the original VH element and the conventional RSS of the invading VH gene, leaving behind a footprint of up to five base pairs (bps) of the original VH gene that is often further obscured by exonuclease activity and N-nucleotide addition. We have previously demonstrated that VH replacement can efficiently rescue the development of B cells that have acquired two nonproductive heavy chain (IgH) rearrangements. Here we describe a novel knock-in mouse model in which the prerearranged IgH locus resembles an endogenously rearranged productive VHDHJH allele. Using this mouse model, we characterized the role of VH replacement in the diversification of the primary Ig repertoire through the modification of productive VHDHJH rearrangements. Our results indicate that VH replacement occurs before Ig light chain rearrangement and thus is not involved in the editing of self-reactive antibodies.
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Zhang, Yuntian, and Tzong-Yi Lee. "Revealing the Immune Heterogeneity between Systemic Lupus Erythematosus and Rheumatoid Arthritis Based on Multi-Omics Data Analysis." International Journal of Molecular Sciences 23, no. 9 (May 5, 2022): 5166. http://dx.doi.org/10.3390/ijms23095166.
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The pathogenesis of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) are greatly influenced by different immune cells. Nowadays both T-cell receptor (TCR) and B-cell receptor (BCR) sequencing technology have emerged with the maturity of NGS technology. However, both SLE and RA peripheral blood TCR or BCR repertoire sequencing remains lacking because repertoire sequencing is an expensive assay and consumes valuable tissue samples. This study used computational methods TRUST4 to construct TCR repertoire and BCR repertoire from bulk RNA-seq data of both SLE and RA patients’ peripheral blood and analyzed the clonality and diversity of the immune repertoire between the two diseases. Although the functions of immune cells have been studied, the mechanism is still complicated. Differentially expressed genes in each immune cell type and cell–cell interactions between immune cell clusters have not been covered. In this work, we clustered eight immune cell subsets from original scRNA-seq data and disentangled the characteristic alterations of cell subset proportion under both SLE and RA conditions. The cell–cell communication analysis tool CellChat was also utilized to analyze the influence of MIF family and GALECTIN family cytokines, which were reported to regulate SLE and RA, respectively. Our findings correspond to previous findings that MIF increases in the serum of SLE patients. This work proved that the presence of LGALS9, PTPRC and CD44 in platelets could serve as a clinical indicator of rheumatoid arthritis. Our findings comprehensively illustrate dynamic alterations in immune cells during pathogenesis of SLE and RA. This work identified specific V genes and J genes in TCR and BCR that could be used to expand our understanding of SLE and RA. These findings provide a new insight inti the diagnosis and treatment of the two autoimmune diseases.
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Fraley, Elizabeth Ross, Santosh Khanal, Cas LeMaster, Stephen Pierce, Tomi Pastinen, and Todd Bradley. "B cell receptor repertoire dynamics and convergent evolution following SARS-CoV-2 vaccination." Journal of Immunology 208, no. 1_Supplement (May 1, 2022): 65.10. http://dx.doi.org/10.4049/jimmunol.208.supp.65.10.
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Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus causing Coronavirus disease 2019 (COVID-19). B cell receptors (BCRs) are expressed at the surface of the B cell and are secreted as soluble antibodies. These antibodies can block viral infection by neutralizing the virus, are critical for resolution of SARS-CoV-2 infection, and may be correlates of protection for COVID-19 vaccines. We have previously shown that SARS-CoV-2 immunization elicits a robust antibody response that is significantly higher in individuals who recovered from COVID-19. Moreover, recovered individuals had slower antibody decay in their levels seven months after vaccination. Here, we sequenced the BCR repertoire of individuals undergoing vaccination by SARS-CoV-2 mRNA vaccine (Pfizer, BNT162b2) with (seropositive) or without (seronegative) previous laboratory confirmed COVID-19 infection. We identified genetic differences in the BCR repertoire between groups including V gene usage, CD3R length, percentage of somatic hypermutation, and clonotype diversity. We then focused our analyses to vaccine-expanded clonotypes in both groups to further analyze the differences in BCR repertoire on candidate SARS-CoV-2-specific clonotypes. We determined the frequency of pre-existing clones present after infection that were engaged by the vaccine in the seropositive group. Moreover, in both groups we identified clonotypes that were shared among individuals that could be a result of convergent evolution. Defining the characteristics and evolution of the BCR repertoire during vaccination of individuals with different histories of viral infection will aid in understanding SARS-CoV-2 humoral response dynamics.
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Ahmed, Rizwan, Zahra Omidian, Adebola Giwa, Kagan E. Karakus, Neha Majety, Angela Yang, Hao Zhang, et al. "A newly discovered dual expresser lymphocyte that clonally expanded in Type 1 diabetes (T1D) patients secretes a public antibody that recognize public TCR in T1D." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 142.10. http://dx.doi.org/10.4049/jimmunol.204.supp.142.10.
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Abstract PURPOSE We have recently discovered a new lymphocyte that co-express BCR and TCR (Ahmed et al, Cell, 2019: 177:11583) and referred as X cell to denote its crossover phenotype. Importantly, X cells express a public BCR that also encodes a potent autoantigen in its CDR3 sequence that is 10 fold more potent than native insulin peptide (InsB:9–23) in binding to DQ8 and activating autologous CD4 T cells. The x-autoantigen cross-activate insulin specific CD4 T cells as a peptide in the context of HLA-DQ8 molecules or as a soluble intact mAb (x-mAb). The goal of this study is to characterize autoreactive CD4 T cells that are responsive to x-mAb to determine their phenotype, cytokine profile and TCR repertoire and whether they express public TCRs. METHODS We used EBV-lymphoblastoid X cell clone as a source of x-mAb (IgM) and FACS based protocol to identify IgM reactive CD4 T cells (referred as IgMpos) and their functional properties. ImmunoSEQ assay used to characterize TCR repertoires. RESULTS Preliminary data show that frequency of IgMpos CD4 T cells is significantly higher in T1D as compared to Healthy subjects. In addition, IgMpos CD4 T cells exhibit an activated phenotype as compared to autologous IgMneg CD4 T cells, including expression of CD45RO, CD44, and CD69. Analysis of TCRVβ repertoire shows that IgMpos CD4 T cells are enriched for public clonally expanded TCRs as compared to IgMneg counterparts. CONCLUSIONS X cells in T1D patients are predominated by a single public BCR and that the secreted version of this BCR (x-mAb) is autoreactive against a specific subset of CD4 T cells that predominated by few clonotypes that express public TCRs. Our results are revealing previously unknown mechanism that appears to be a play critical role in pathogenesis of T1D.
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Chenchik, Alex, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Paul Diehl, and Lester Kobzik. "Abstract LB172: Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery." Cancer Research 83, no. 8_Supplement (April 14, 2023): LB172. http://dx.doi.org/10.1158/1538-7445.am2023-lb172.
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Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity, and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B- cells. A new technology we developed that combines profiling of all human TCR and BCR variable regions with phenotypic characterization of immune cells using the same workflow could be particularly useful. The TCR and BCR immunophenotyping method proposed involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, and subsequently determining the expression levels of the most informative T- and B-cell phenotyping genes. Preliminary results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data from the TCR and BCR clonotypes profile combined with RNA expression profiling of the same samples results in a richer data set that includes the identification of major immune cell subtypes and their activation status. Data from human cancer tissue and whole blood samples will be presented. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Paul Diehl, Lester Kobzik. Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB172.
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Chenchik, Alex, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Khadija Ghias, and Lester Kobzik. "Abstract C007: Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery." Molecular Cancer Therapeutics 22, no. 12_Supplement (December 1, 2023): C007. http://dx.doi.org/10.1158/1535-7163.targ-23-c007.
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Abstract T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling, also referred to as adaptive immune receptor repertoire (AIRR) profiling, holds great potential for the understanding of disease mechanisms and for the development of new treatments in infectious disease, autoimmunity, and immuno-oncology. This potential could be greatly improved by combining information about receptor clonotypes with immunophenotypes of T- and B- cells. A new technology we developed that combines profiling of all human TCR and BCR variable regions with phenotypic characterization of immune cells using the same workflow could be particularly useful. The TCR and BCR immunophenotyping method proposed involves RT-PCR amplification and sequencing of the CDR3 regions of the TCR and BCR genes, and subsequently determining the expression levels of the most informative T- and B-cell phenotyping genes. Preliminary results show that this method allows for comprehensive profiling of all seven TCR and BCR chains from a single sample, in a highly reproducible manner, directly from micro-samples including cancer tissue, whole blood, sorted cells and more. Bioinformatic analysis of the next-generation sequencing (NGS) data from the TCR and BCR clonotypes profile combined with RNA expression profiling of the same samples results in a richer data set that includes the identification of major immune cell subtypes and their activation status. Data from human cancer tissues and whole blood samples will be presented. Citation Format: Alex Chenchik, Mikhail Makhanov, Tianbing Liu, Dongfang Hu, Khadija Ghias, Lester Kobzik. Improved T-cell and B-cell receptor repertoire profiling and immunophenotyping for biomarker discovery [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C007.
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Jiang, Roy, Bhaskar Roy, Qian Wu, Subhasis Mohanty, Richard J. Nowak, Albert C. Shaw, Steven H. Kleinstein, and Kevin C. O’Connor. "The Plasma Cell Infiltrate Populating the Muscle Tissue of Patients with Inclusion Body Myositis Features Distinct B Cell Receptor Repertoire Properties." ImmunoHorizons 7, no. 5 (May 1, 2023): 310–22. http://dx.doi.org/10.4049/immunohorizons.2200078.
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Abstract Inclusion body myositis (IBM) is an autoimmune and degenerative disorder of skeletal muscle. The B cell infiltrates in IBM muscle tissue are predominantly fully differentiated Ab-secreting plasma cells, with scarce naive or memory B cells. The role of this infiltrate in the disease pathology is not well understood. To better define the humoral response in IBM, we used adaptive immune receptor repertoire sequencing, of human-derived specimens, to generate large BCR repertoire libraries from IBM muscle biopsies and compared them to those generated from dermatomyositis, polymyositis, and circulating CD27+ memory B cells, derived from healthy controls and Ab-secreting cells collected following vaccination. The repertoire properties of the IBM infiltrate included the following: clones that equaled or exceeded the highly clonal vaccine-associated Ab-secreting cell repertoire in size; reduced somatic mutation selection pressure in the CDRs and framework regions; and usage of class-switched IgG and IgA isotypes, with a minor population of IgM-expressing cells. The IBM IgM-expressing population revealed unique features, including an elevated somatic mutation frequency and distinct CDR3 physicochemical properties. These findings demonstrate that some of IBM muscle BCR repertoire characteristics are distinct from dermatomyositis and polymyositis and circulating Ag-experienced subsets, suggesting that it may form through selection by disease-specific Ags.
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Bagnara, Davide, Andrea Nicola Mazzarello, Fabio Ghiotto, Monica Colombo, Giovanna Cutrona, Franco Fais, and Manlio Ferrarini. "Old and New Facts and Speculations on the Role of the B Cell Receptor in the Origin of Chronic Lymphocytic Leukemia." International Journal of Molecular Sciences 23, no. 22 (November 17, 2022): 14249. http://dx.doi.org/10.3390/ijms232214249.
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The engagement of the B cell receptor (BcR) on the surface of leukemic cells represents a key event in chronic lymphocytic leukemia (CLL) since it can lead to the maintenance and expansion of the neoplastic clone. This notion was initially suggested by observations of the CLL BcR repertoire and of correlations existing between certain BcR features and the clinical outcomes of single patients. Based on these observations, tyrosine kinase inhibitors (TKIs), which block BcR signaling, have been introduced in therapy with the aim of inhibiting CLL cell clonal expansion and of controlling the disease. Indeed, the impressive results obtained with these compounds provided further proof of the role of BcR in CLL. In this article, the key steps that led to the determination of the role of BcR are reviewed, including the features of the CLL cell repertoire and the fine mechanisms causing BcR engagement and cell signaling. Furthermore, we discuss the biological effects of the engagement, which can lead to cell survival/proliferation or apoptosis depending on certain intrinsic cell characteristics and on signals that the micro-environment can deliver to the leukemic cells. In addition, consideration is given to alternative mechanisms promoting cell proliferation in the absence of BcR signaling, which can explain in part the incomplete effectiveness of TKI therapies. The role of the BcR in determining clonal evolution and disease progression is also described. Finally, we discuss possible models to explain the selection of a special BcR set during leukemogenesis. The BcR may deliver activation signals to the cells, which lead to their uncontrolled growth, with the possible collaboration of other still-undefined events which are capable of deregulating the normal physiological response of B cells to BcR-delivered stimuli.
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Ng, Yen-Shing, Hedda Wardemann, James Chelnis, Charlotte Cunningham-Rundles, and Eric Meffre. "Bruton's Tyrosine Kinase Is Essential for Human B Cell Tolerance." Journal of Experimental Medicine 200, no. 7 (October 4, 2004): 927–34. http://dx.doi.org/10.1084/jem.20040920.
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Most polyreactive and antinuclear antibodies are removed from the human antibody repertoire during B cell development. To elucidate how B cell receptor (BCR) signaling may regulate human B cell tolerance, we tested the specificity of recombinant antibodies from single peripheral B cells isolated from patients suffering from X-linked agammaglobulinemia (XLA). These patients carry mutations in the Bruton's tyrosine kinase (BTK) gene that encode an essential BCR signaling component. We find that in the absence of Btk, peripheral B cells show a distinct antibody repertoire consistent with extensive secondary V(D)J recombination. Nevertheless, XLA B cells are enriched in autoreactive clones. Our results demonstrate that Btk is essential in regulating thresholds for human B cell tolerance.
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Kolijn, P. Martijn, Fatemeh Saberi Hosnijeh, Florentin Späth, Paul J. Hengeveld, Andreas Agathangelidis, Manal Saleh, Delphine Casabonne, et al. "High-risk subtypes of chronic lymphocytic leukemia are detectable as early as 16 years prior to diagnosis." Blood 139, no. 10 (March 10, 2022): 1557–63. http://dx.doi.org/10.1182/blood.2021012890.
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Abstract Chronic lymphocytic leukemia (CLL) is preceded by monoclonal B-cell lymphocytosis (MBL), a CLL precursor state with a prevalence of up to 12% in aged individuals; however, the duration of MBL and the mechanisms of its evolution to CLL remain largely unknown. In this study, we sequenced the B-cell receptor (BcR) immunoglobulin heavy chain (IGH) gene repertoire of 124 patients with CLL and 118 matched controls in blood samples taken up to 22 years prior to diagnosis. Significant skewing in the BcR IGH gene repertoire was detected in the majority of patients, even before the occurrence of lymphocytosis and irrespective of the clonotypic IGH variable gene somatic hypermutation status. Furthermore, we identified dominant clonotypes belonging to major stereotyped subsets associated with poor prognosis up to 16 years before diagnosis in 14 patients with CLL. In 22 patients with longitudinal samples, the skewing of the BcR IGH gene repertoire increased significantly over time to diagnosis or remained stable at high levels. For 14 of 16 patients with available samples at diagnosis, the CLL clonotype was already present in the prediagnostic samples. Overall, our data indicate that the preclinical phase of CLL could be longer than previously thought, even in adverse-prognostic cases.
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Depinet, Mollye M., Wenjing Pan, Xiaohong Hou, Brittany E. Brown, Mary Eisenhower, Daniel Weber, Miranda Byrne-Steele, and Jian Han. "Complete multi-chain adaptome amplification from whole blood and PBMC via novel dam-PCR technology." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 159.57. http://dx.doi.org/10.4049/jimmunol.204.supp.159.57.
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Abstract Profiling the immune repertoire through NGS allows detailed, sequence-specific insight into the adaptive immune response. One of the key challenges during immune receptor amplification is the formation of dimers, which can compete with the immune amplicons of interest during library preparation. Therefore, we developed a novel PCR technique, dimer-avoided multiplex PCR (dam-PCR), that effectively avoids primer dimer formation and allows for the all-in-one qualitative amplification of all BCR and TCR receptors in a single, quantitative reaction. Here, we present this technology’s capabilities of bias-free immune repertoire amplification, even for low frequency CDR3s. First, we demonstrate the ability of this method to accurately detect a Jurkat cell line that was spiked into pre-screened sorted donor CD4+ cells at 1%, 0.1%, 0.01%, 0.001%, and 0.0001%. RNA from each spike-in percentage was extracted and amplified in duplicate using our standard amplification protocol to assess repeatability of CDR3 discovery between replicates, as well as establish the limits of detection and quantitation. Then, we demonstrate the method’s ability to amplify a synthetic repertoire, created by spiking RNA from two individuals of known repertoire into each other at 75%, 50%, and 25%. The mixed RNA, as well as pure RNA from each individual, was then amplified to assess correlation between the predicted repertoire and amplified repertoire at each spike-in percentage. For both the Jurkat cell level spike-in test, as well as the RNA level spike-in test, the amplified repertoire was successfully predicted at an R2 value of > 0.99. This single reaction method will allow for a cost effective, all-inclusive, and quantitative analysis of immune repertoires.