Cell Repertoire Sequencing Research Articles

Updated results and biomarker analyses from the phase 2 trial of nab-paclitaxel plus sintilimab as second-line therapy for advanced or metastatic gastric/gastroesophageal junction adenocarcinoma (G/GEJA).

4037 Background: Sintilimab plus Nab-paclitaxel (nab-PTX) has exhibited promising efficacy and safety in the second line (2L) treatment of PD-1/PD-L1 inhibitor naïve advanced G/GEJA (ZC Jiang et al 2022 ASCO GI). This update presents the latest findings on efficacy, survival outcomes and exploration of biological markers. Methods: We collected blood samples from the advanced G/GEJA patients (pts) who were enrolled in our Phase 2 clinical trial and treated with sintilimab plus nab-PTX. Target-capture deep sequencing of 1,021 genes was conducted to identify somatic variants in circulating tumor DNA (ctDNA) from the baseline plasma samples (N=55) and disease progression samples of the pts with PFS >4 months (N=17). And T cell repertoire sequencing was performed on pretreatment samples (N=55) as well as on paired blood samples obtained during the first tumor evaluation (N=45). Results: From Nov 2019 to Sep 2022, 58 pts were enrolled, with a median follow-up of 20.9 months (95% CI 17.04-24.76) until July 10, 2023. The objective response rate (ORR) and disease control rate (DCR) were 44.8% and 79.3%, respectively. The median progression free survival (mPFS) and median overall survival (mOS) were 4.8 months (95%CI 3.06-6.54) and 11.8 months (95%CI 10.43-13.17). Analysis of baseline ctDNA revealed a notably higher prevalence of EPHB1 mutation in non-responders (PD+increased SD) compared to responders (CR+PR+decreased SD) (21.43% vs. 0%, P=0.022). Pts with EPHB1 mutation showed a significantly increased risk of disease progression (HR 3.86, 95% CI 0.43-34.41, p=0.013) and death (HR 4.49, 95% CI 0.43-46.93, p=0.005) compared to wild-type EPHB1 pts. In contrast to high ctDNA level (mean tumor molecules [MTM] per mL of plasma, cutoff value=2999.7), low ctDNA level demonstrated a significantly reduced risk of disease progression (HR 1.88, 95% CI 1.05-3.37, p=0.034). Dynamic T cell receptor (TCR) diversity analysis during baseline and the initial tumor assessment showed a significant post-treatment increase in the Shannon index within the responder group (P=0.045, median Shannon index change: responder 0.43 vs. non-responder -0.49). An increased Shannon index was correlated with significantly prolonged mPFS (HR 0.44, 95% CI 0.22-0.91, P=0.009), while no significant difference was observed in mOS (HR 0.74, 95% CI 0.36-1.54, P=0.399). A negative correlation between the Shannon index and MTM was observed (R=-0.37, P=0.009). Conclusions: 2L-treatment with sintilimab plus nab-PTX maintains favorable efficacy in advanced G/GEJA pts without prior PD-1/PD-L1 inhibitor treatment. EPHB1 mutation and dynamic change of TCR could serve as potential predictive factors. Further investigation is warranted. Clinical trial information: NCT04140318 .

B cell repertoire sequencing of HIV-1 pediatric elite-neutralizers identifies multiple broadly neutralizing antibody clonotypes.

A limited subset of HIV-1 infected adult individuals typically after at least 2-3 years of chronic infection, develop broadly neutralizing antibodies (bnAbs), suggesting that highly conserved neutralizing epitopes on the HIV-1 envelope glycoprotein are difficult for B cell receptors to effectively target, during natural infection. Recent studies have shown the evolution of bnAbs in HIV-1 infected infants. We used bulk BCR sequencing (BCR-seq) to profile the B cell receptors from longitudinal samples (3 time points) collected from a rare pair of antiretroviralnaïve, HIV-1 infected pediatric monozygotic twins (AIIMS_329 and AIIMS_330) who displayed elite plasma neutralizing activity against HIV-1. BCR-seq of both twins revealed convergent antibody characteristics including V-gene use, CDRH3 lengths and somatic hypermutation (SHM). Further, antibody clonotypes with genetic features similar to highly potent bnAbs isolated from adults showed ongoing development in donor AIIMS_330 but not in AIIMS_329, corroborating our earlier findings based on plasma bnAbs responses. An increase in SHM was observed in sequences of the IgA isotype from AIIMS_330. This study suggests that children living with chronic HIV-1 can develop clonotypes of HIV-1 bnAbs against multiple envelope epitopes similar to those isolated from adults, highlighting that such B cells could be steered to elicit bnAbs responses through vaccines aimed to induce bnAbs against HIV-1 in a broad range of people including children.

Isolation of Rare Antigen-Specific Memory B Cells via Antigen Tetramers.

Immunological memory, which sets the foundation for the adaptive immune response, plays a key role in disease protection and prevention. Obtaining a deeper understanding of the mechanisms underlying this phenomenon can aide in research aimed to improve vaccines and therapies. Memory B cells (MBCs) are a fundamental component of immunological memory but can exist in rare populations that prove challenging to study. By combining fluorescent antigen tetramers with multiple enrichment processes, a highly streamlined method for identifying and sorting antigen-specific MBCs from human blood and lymphoid tissues can be achieved. With the output of this process being viable cells, there is a multitude of downstream operations that can be used in conjunction with the antigen-specific cell sorting outlined in this chapter. Single-cell RNA-sequencing paired with B cell repertoire sequencing, which can be linked to distinct antigens in a high-throughput fashion, is a downstream application widely used in disease and vaccination research. Incorporation of this protocol can lead to a variety of applications and a diversity of outcomes aiding in a deeper understanding of how immunological memory not only forms but is recalled and impacted by infection and vaccination.

Fast clonal family inference from large-scale B cell repertoire sequencing data

Advances in high-throughput sequencing technologies have facilitated the large-scale characterization of B cell receptor (BCR) repertoires. However, the vast amount and high diversity of the BCR sequences pose challenges for efficient and biologically meaningful analysis. Here, we introduce fastBCR, an efficient computational approach for inferring B cell clonal families from massive BCR heavy chain sequences. We demonstrate that fastBCR substantially reduces the running time while ensuring high accuracy on simulated datasets with diverse numbers of B cell lineages and varying mutation rates. We apply fastBCR to real BCR sequencing data from peripheral blood samples of COVID-19 patients, showing that the inferred clonal families display disease-associated features, as well as corresponding antigen-binding specificity and affinity. Overall, our results demonstrate the advantages of fastBCR for analyzing BCR repertoire data, which will facilitate the identification of disease-associated antibodies and improve our understanding of the B cell immune response.

Analysis of Immune Intratumor Heterogeneity Highlights Immunoregulatory and Coinhibitory Lymphocytes as Hallmarks of Recurrence in Stage I Non–Small Cell Lung Cancer

Our understanding of the molecular mechanisms underlying postsurgical recurrence of non–small cell lung cancer (NSCLC) is rudimentary. Molecular and T cell repertoire intratumor heterogeneity (ITH) have been reported to be associated with postsurgical relapse; however, how ITH at the cellular level impacts survival is largely unknown. Here we report the analysis of 2880 multispectral images representing 14.2% to 27% of tumor areas from 33 patients with stage I NSCLC, including 17 cases (relapsed within 3 years after surgery) and 16 controls (without recurrence ≥5 years after surgery) using multiplex immunofluorescence. Spatial analysis was conducted to quantify the minimum distance between different cell types and immune cell infiltration around malignant cells. Immune ITH was defined as the variance of immune cells from 3 intratumor regions. We found that tumors from patients having relapsed display different immune biology compared with nonrecurrent tumors, with a higher percentage of tumor cells and macrophages expressing PD-L1 (P =.031 and P =.024, respectively), along with an increase in regulatory T cells (Treg) (P =.018), antigen-experienced T cells (P =.025), and effector-memory T cells (P =.041). Spatial analysis revealed that a higher level of infiltration of PD-L1+ macrophages (CD68+PD-L1+) or antigen-experienced cytotoxic T cells (CD3+CD8+PD-1+) in the tumor was associated with poor overall survival (P =.021 and P =.006, respectively). A higher degree of Treg ITH was associated with inferior recurrence-free survival regardless of tumor mutational burden (P =.022), neoantigen burden (P =.021), genomic ITH (P =.012) and T cell repertoire ITH (P =.001). Using multiregion multiplex immunofluorescence, we characterized ITH at the immune cell level along with whole exome and T cell repertoire sequencing from the same tumor regions. This approach highlights the role of immunoregulatory and coinhibitory signals as well as their spatial distribution and ITH that define the hallmarks of tumor relapse of stage I NSCLC.

The Pre-Existing Human Antibody Repertoire to Computationally Optimized Influenza H1 Hemagglutinin Vaccines.

Computationally optimized broadly reactive Ag (COBRA) hemagglutinin (HA) immunogens have previously been generated for several influenza subtypes to improve vaccine-elicited Ab breadth. As nearly all individuals have pre-existing immunity to influenza viruses, influenza-specific memory B cells will likely be recalled upon COBRA HA vaccination. We determined the epitope specificity and repertoire characteristics of pre-existing human B cells to H1 COBRA HA Ags. Cross-reactivity between wild-type HA and H1 COBRA HA proteins P1, X6, and Y2 were observed for isolated mAbs. The mAbs bound five distinct epitopes on the pandemic A/California/04/2009 HA head and stem domains, and most mAbs had hemagglutination inhibition and neutralizing activity against 2009 pandemic H1 strains. Two head-directed mAbs, CA09-26 and CA09-45, had hemagglutination inhibition and neutralizing activity against a prepandemic H1 strain. One mAb, P1-05, targeted the stem region of H1 HA, but did not compete with a known stem-targeting H1 mAb. We determined that mAb P1-05 recognizes a recently discovered HA epitope, the anchor epitope, and we identified similar mAbs using B cell repertoire sequencing. In addition, the trimerization domain distance from HA was critical to recognition of this epitope by mAb P1-05, suggesting the importance of protein design for vaccine formulations. Overall, these data indicate that seasonally vaccinated individuals possess a population of functional H1 COBRA HA-reactive B cells that target head, central stalk, and anchor epitopes, and they demonstrate the importance of structure-based assessment of subunit protein vaccine candidates to ensure accessibility of optimal protein epitopes.

Abstract 5628: Immune repertoire sequencing facilitates gamma delta T cell clonal determination

Abstract Gamma-delta T cells are a small fraction of T lymphocytes with only 1-5% of the overall T cell population, but they are an important subset that have unique contributions to both innate and adaptive immunity. Gamma-delta T cells can recognize a broad range of antigens to provide rapid responses to pathogens, and can also interact with both immune cells and non-immune tissue cells triggering regulatory and cytotoxic responses. These unique features make them ideal candidates that could be targeted to induce durable immunity in the context of different pathologies. There has been growing interest in understanding the contributions of gamma-delta T cells to immunology and developing efficient gamma-delta T-cell-based therapies for cancer, infectious disease, and autoimmune disease. In this study, we have implemented T cell repertoire sequencing for high throughput characterization. Gamma-delta T cells were enriched from tissues and peripheral blood mononuclear cells (PBMCs). RNA was extracted and used to generate full length TCR libraries. Unique molecular identifiers (UMIs) were incorporated to discretely barcode each mRNA molecule, enabling absolute quantitative ranking of TCR clone abundance. Full length immune repertoire sequencing facilitates the detection of distinct and shared clones in tissue and blood samples, enabling the identification of disease specific clones to evaluate immunotherapy effects. In addition, RNA sequencing was performed for gene expression analysis of gamma-delta T cells to identify cell phenotypes. Citation Format: Chen Song, Ariel Erijman, Bradley W. Langhorst, Pingfang Liu, Eileen T. Dimalanta, Theodore B. Davis. Immune repertoire sequencing facilitates gamma delta T cell clonal determination [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 5628.

Genomic and TCR profiling data reveal the distinct molecular traits in epithelial ovarian cancer histotypes.

Epithelial ovarian cancer (EOC) is classified into five major histotypes: high-grade serous (HGSOC), low-grade serous (LGSOC), clear cell (CCOC), endometrioid (ENOC), and mucinous (MOC). However, the landscape of molecular and immunological alterations in these histotypes, especially LGSOC, CCOC, ENOC, and MOC, is largely uncharacterized. We collected 101 treatment-naive EOC patients. The resected tumor tissues and paired preoperative peripheral blood samples were collected and subjected to target sequencing of 1021 cancer-associated genes and T cell repertoire sequencing. Distinct characteristics of mutations were identified among the five histotypes. Furthermore, tumor mutation burden (TMB) was found to be higher in CCOC and ENOC, but lower in LGSOC and HGSOC. Alterations associated with DNA damage repair (DDR) pathways and homologous recombination deficiencies (HRD) were prevalent in five histotypes. CCOC demonstrated increased level of T cell clonality compared with HSGOC. Interestingly, the proportion of the 100 most common T cell clones was associated with TMB and tumor neoantigen burden in CCOC, highlighting more sensitive anti-tumor responses in this histotype, which was also evidenced by the enhanced convergent recombination of T cell clones. These findings shed light on the molecular traits of genomic alteration and T cell repertoire in the five major EOC histotypes and may help optimize clinical management of EOC with different histotypes.

Thymus-derived B cell clones persist in the circulation after thymectomy in myasthenia gravis

Myasthenia gravis (MG) is a neuromuscular, autoimmune disease caused by autoantibodies that target postsynaptic proteins, primarily the acetylcholine receptor (AChR) and inhibit signaling at the neuromuscular junction. The majority of patients under 50 y with AChR autoantibody MG have thymic lymphofollicular hyperplasia. The MG thymus is a reservoir of plasma cells that secrete disease-causing AChR autoantibodies and although thymectomy improves clinical scores, many patients fail to achieve complete stable remission without additional immunosuppressive treatments. We speculate that thymus-associated B cells and plasma cells persist in the circulation after thymectomy and that their persistence could explain incomplete responses to resection. We studied patients enrolled in a randomized clinical trial and used complementary modalities of B cell repertoire sequencing to characterize the thymus B cell repertoire and identify B cell clones that resided in the thymus and circulation before and 12 mo after thymectomy. Thymus-associated B cell clones were detected in the circulation by both mRNA-based and genomic DNA-based sequencing. These antigen-experienced B cells persisted in the circulation after thymectomy. Many circulating thymus-associated B cell clones were inferred to have originated and initially matured in the thymus before emigration from the thymus to the circulation. The persistence of thymus-associated B cells correlated with less favorable changes in clinical symptom measures, steroid dose required to manage symptoms, and marginal changes in AChR autoantibody titer. This investigation indicates that the diminished clinical response to thymectomy is related to persistent circulating thymus-associated B cell clones.

T-Cell receptor repertoire and genomic analysis of tracheal adenoid cystic carcinoma.

e18576 Background: Tracheal adenoid cystic carcinoma (TACC) is the second type of cancer in bronchial tumors, has a high rate of postoperative recurrence and poor prognosis for which therapeutic targets are unavailable. Currently, studies were mostly about ACC of salivary gland. Our study is aim to elucidate genomic landscape and predict clinical biomarker of TACC. Methods: We performed whole-exome sequencing (WES), T cell repertoire sequencing (TCR) and immunohistochemistry (IHC) on tumor tissue samples and matched peripheral blood (PB) samples from TACC patients. All patients were followed up for their disease-free survival. Results: 13 cases were enrolled from 2010 to 2019. Median age of 13 TACCs was 35 yrs. 7 patients were female. 12 patients were stage I. In 8 WES samples, TCEB3CL (62%, 5/8), LPAR3 (50%, 4/8), ALPI (38%, 3/8), ARID1A (38%, 3/8) and BCOR (38%, 3/8) mutated most frequently. The median TMB was 3.67 (1.37-6.77) mutations/Mb. 1 patient showed microsatellite instability high (MSI-H). 2 patients with the highest TMB both had POLE mutations. We compared frequency of most mutated genes in our 8 samples with three different ACC types, including 935 ACC of salivary gland cancer, 76 ACC of the lung, and 38 ACC of breast cancer. 7 mutated frequently genes were found both in TACC and other types of ACCs, including ARID1A, BCOR, ARID1B, KMT2C, KMT2D, CREBBP and FAT3. 6 of them were more frequent in TACC. 7 FFPE tumors and 13 PB samples from 13 TACCs were used to investigate immune microenvironment. In 13 PB samples, TCR diversity (shannon index) and clonality was negatively associated with age (Pearson, r = -0.53, p = 0.06; r = 0.53, p = 0.06, respectively). There was no significant association between the other clinical characteristics (gender and smoking history) and any TCR parameter (clonality, clonotype, shannon index). Patients with lower clonotype number had longer DFS than those with higher clonotype number (15.0 vs 9.5 months, p < 0.001, 95% CI, 0.23-675.66, HR, 12.46), while shannon index and clonality was not significantly associated with DFS. Conclusions: The genetic mutations associated with TACC are different from those implicated in non-small cell lung cancer. TCR analysis as a useful of indicator of disease development and prognosis in TACC may be utilized to direct further immunotherapy.

1420P - Automated rarefaction analysis for precision B and T cell receptor repertoire profiling from peripheral blood and FFPE-preserved tumour

BackgroundIdentifying the optimal input amount and sequencing depth for B and T cell receptor repertoire profiling is challenging owing to variation in material quality and lymphocyte diversity in blood and FFPE preserved tumor specimens. Rarefaction analysis has emerged as a potential approach for assessing whether immune repertoire libraries have been sequenced to saturation. Here we present a novel automated method for saturation analysis of IGH and TCRB chain libraries derived from sequencing of peripheral blood leukocytes (PBL) and FFPE-preserved tumor RNA and DNA. MethodsHuman TCRB and IGH repertoire libraries were generated using the Oncomine TCRB and IGH assays from: (1) 25ng PBL total RNA (2) 500ng PBL gDNA (3) 150ng RNA from FFPE preserved NSCLC and (4) 200ng gDNA from FFPE preserved brain tissue. Libraries were sequenced on the Ion Torrent Gene Studio S5 then analyzed with Ion Reporter to identify clonotypes, quantify clonal expansion and diversity, and for IGH chain libraries, identify B cell clonal lineages and assess isotype usage. We then repeated clonotyping and analysis of secondary repertoire features using data that had been downsampled to fixed read depths. ResultsWe observed an asymptotic relationship between the sequencing depth and the number of B and T cell clones detected, clone Shannon diversity, and B cell clonal lineage richness and diversity, indicating that libraries had been sequenced to saturation. By contrast, T and B cell normalized Shannon entropy appeared robust to sequencing depth. ConclusionsAutomated downsampling analysis may serve as a convenient tool for optimizing sequencing depth and input amount for B and T cell repertoire sequencing studies. We expect this approach to become a routine component of immune repertoire analysis. Legal entity responsible for the studyThermo Fisher Scientific. FundingThermo Fisher Scientific. DisclosureL. Quagliata: Full / Part-time employment: Thermo Fisher Scientific. T. Looney: Full / Part-time employment: Thermo Fisher Scientific. D. Topacio-Hall: Full / Part-time employment: Thermo Fisher Scientific. G. Lowman: Full / Part-time employment: Thermo Fisher Scientific.

1215P - T cell repertoire sequencing reveals dynamics of response to dendritic cell vaccine plus dasatinib for checkpoint blockade resistant metastatic melanoma

BackgroundWe recently conducted an open- label, single-center, prospective randomized Phase II clinical trial for HLA-A2+ patients with advanced-stage melanoma (NCT01876212) at the University of Pittsburgh Hillman Cancer Center. Patients received ID injections of Type-1-polarized, autologous DC loaded with a mixture of peptides derived from six tumor-associated vascular antigens (DLK1, EPHA2, HBB, NRP1, RGS5, TEM1) combined +/- daily oral administration of dasatinib (70mg BID) as an immune adjuvant/conditioning agent. Here we report an exploratory analysis of T cell repertoire profiling of longitudinally-sampled peripheral blood leukocytes (PBL) to define potential pharmacodynamic and response biomarkers. MethodsTotal RNA was extracted from pre- and post-treatment PBL from 13 therapy recipients (6 responders, 7 non-Responders) including extended longitudinal samples for four responders. TCRB sequencing was performed via the Oncomine TCRB-LR assay using 25ng total RNA as input. We evaluated T cell clonal expansion and TCR convergence as potential biomarkers of response. ResultsTCR convergence values were elevated in pretreatment PBL of responders compared to non-responders (mean frequency .012 vs .006; p=.01, Wilcoxon), and remained elevated in responders up to 25 weeks post treatment. TCR evenness (normalized Shannon entropy) decreased at week 5 compared to baseline (p=.01, one-sided student’s t-test), indicating increased clonal expansion following treatment. ConclusionsThese data suggest that peripheral blood TCRB convergence may serve as a predictive or prognostic biomarker for response to dendritic cell-based immunotherapy. Our finding of increased T cell clonal expansion at week 5 of treatment supports the notion that TCR sequencing may serve as a tool for the measurement of pharmacodynamic markers of therapeutic agent activity. Ongoing and future studies will further clarify the utility of TCR convergence and clonal expansion as immune repertoire biomarkers. Clinical trial identificationNCT01876212. Legal entity responsible for the studyUniversity of Pittsburgh School of Medicine & Hillman Cancer Center. FundingThermo Fisher Scientific. DisclosureL. Quagliata: Full / Part-time employment: Thermo Fisher Scientific. T. Looney: Full / Part-time employment: Thermo Fisher Scientific. D. Topacio-Hall: Full / Part-time employment: Thermo Fisher Scientific. G. Lowman: Full / Part-time employment: Thermo Fisher Scientific. All other authors have declared no conflicts of interest.

Abstract B046: Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment

Abstract The study of complex immunologic diseases and tumor microenvironment has progressed through recent developments that enable the 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 both B cell antibodies (Igs) and T-cell receptors (TCRs). One major challenge of immune repertoire sequencing is to capture the structural and sequence complexities of antibody and TCR genes. We have developed and optimized a method for accurate sequencing of full-length immune gene repertoires of B-cells and T-cells. RNA extracted from tissue and PBMCs were used to generate immune sequencing libraries. Using a unique molecule index (UMI) to discretely barcode each mRNA molecule, PCR copies of each mRNA fragment can be collapsed into a single consensus sequence. The B cell genes were enriched during library preparation by IGH, IGK and IGL primes, including isotype-specific primers (IgA, IgD, IgE, IgG and IgM). The T-cell genes were enriched by TCRα and TCRβ specific primers. To investigate the applicability of minimal residual disease assessment, we obtained Jurkat RNA, a homogenous population of leukemic Jurkat T-cells and spiked it into a PBMC RNA sample at varying proportions of Jurkat RNA (10%, 1%, 0.1%, 0.01% and 0.001%). The RNA mixtures were made into TCR libraries, sequenced on both Illumina MiSeq and Oxford Nanopore MinION, and analyzed to assess the TCR repertoire. Utilization of UMIs enabled absolute quantification of the B cell antibody/TCR clones and accurate ranking of their abundance. For B cell repertoire sequencing, the use of isotype-specific primers enabled measurement of the heavy chain isotype proportions within the samples. Full-length heavy chain antibody analysis enabled measurement of the mutation level of each antibody sequence, providing information on the overall maturity and mutational profile of the sample repertoire. For TCR repertoire sequencing, distinct and shared clonal sequences were quantitatively detected in PBMC samples. The method also accurately and sensitively detected the control TCR clone spiked into both Illumina and Oxford Nanopore libraries at levels appropriate for minimal residual disease assessment.This immune repertoire sequencing approach allows accurate clonal determination for both Igs and TCRs. This technique is applicable for a variety of applications including design of antibody chains for in vitro synthesis, investigation of T-cell infiltration of tumor microenvironments, and monitoring of minimal residual disease. Citation Format: Chen Song, Luo Sun, Pingfang Liu, Bradley Langhorst, Andrew Barry, Theodore B. Davis, Eileen T. Dimalanta. Immune repertoire sequencing enables complete B-cell and T-cell clonality determination and minimal residual disease assessment [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B046.

Optimized Threshold Inference for Partitioning of Clones From High-Throughput B Cell Repertoire Sequencing Data.

During adaptive immune responses, activated B cells expand and undergo somatic hypermutation of their B cell receptor (BCR), forming a clone of diversified cells that can be related back to a common ancestor. Identification of B cell clones from high-throughput Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) data relies on computational analysis. Recently, we proposed an automated method to partition sequences into clonal groups based on single-linkage hierarchical clustering of the BCR junction region with length-normalized Hamming distance metric. This method could identify clonal sequences with high confidence on several benchmark experimental and simulated data sets. However, determining the threshold to cut the hierarchy, a key step in the method, is computationally expensive for large-scale repertoire sequencing data sets. Moreover, the methodology was unable to provide estimates of accuracy for new data. Here, a new method is presented that addresses this computational bottleneck and also provides a study-specific estimation of performance, including sensitivity and specificity. The method uses a finite mixture model fitting procedure for learning the parameters of two univariate curves which fit the bimodal distribution of the distance vector between pairs of sequences. These distributions are used to estimate the performance of different threshold choices for partitioning sequences into clones. These performance estimates are validated using simulated and experimental data sets. With this method, clones can be identified from AIRR-seq data with sensitivity and specificity profiles that are user-defined based on the overall goals of the study.

A spectral clustering-based method for identifying clones from high-throughput B cell repertoire sequencing data.

MotivationB cells derive their antigen-specificity through the expression of Immunoglobulin (Ig) receptors on their surface. These receptors are initially generated stochastically by somatic re-arrangement of the DNA and further diversified following antigen-activation by a process of somatic hypermutation, which introduces mainly point substitutions into the receptor DNA at a high rate. Recent advances in next-generation sequencing have enabled large-scale profiling of the B cell Ig repertoire from blood and tissue samples. A key computational challenge in the analysis of these data is partitioning the sequences to identify descendants of a common B cell (i.e. a clone). Current methods group sequences using a fixed distance threshold, or a likelihood calculation that is computationally-intensive. Here, we propose a new method based on spectral clustering with an adaptive threshold to determine the local sequence neighborhood. Validation using simulated and experimental datasets demonstrates that this method has high sensitivity and specificity compared to a fixed threshold that is optimized for these measures. In addition, this method works on datasets where choosing an optimal fixed threshold is difficult and is more computationally efficient in all cases. The ability to quickly and accurately identify members of a clone from repertoire sequencing data will greatly improve downstream analyses. Clonally-related sequences cannot be treated independently in statistical models, and clonal partitions are used as the basis for the calculation of diversity metrics, lineage reconstruction and selection analysis. Thus, the spectral clustering-based method here represents an important contribution to repertoire analysis.Availability and implementationSource code for this method is freely available in the SCOPe (Spectral Clustering for clOne Partitioning) R package in the Immcantation framework: www.immcantation.org under the CC BY-SA 4.0 license.Supplementary information Supplementary data are available at Bioinformatics online.

BCR repertoire sequencing: different patterns of B-cell activation after two Meningococcal vaccines.

Next-generation sequencing was used to investigate the B cell receptor heavy chain transcript repertoire of different B cell subsets (naïve, marginal zone, IgM memory and IgG memory) at baseline, and of plasma cells 7 days following administration of serogroup ACWY meningococcal polysaccharide and protein-polysaccharide conjugate vaccines. Baseline B cell subsets could be distinguished from each other using a small number of repertoire properties (clonality, mutation from germ-line and complementarity-determining region 3 length) that were conserved between individuals. However, analyzing the complementarity-determining region 3 amino acid sequence (which is particularly important for antigen binding) of the baseline subsets showed few sequences shared between individuals. In contrast, day 7 plasma cells demonstrated nearly tenfold greater sequence sharing between individuals than the baseline subsets, consistent with the plasma cells being induced by the vaccine antigen, and sharing specificity for a more limited range of epitopes. By annotating plasma cell sequences based on IgG subclass usage and mutation, and also comparing them to the sequences of the baseline cell subsets, we were able to identify different signatures after the polysaccharide and conjugate vaccines. Plasma cells produced after conjugate vaccination were predominantly IgG1, and most related to IgG memory cells. In contrast, after polysaccharide vaccination, the plasma cells were predominantly IgG2, less mutated, and were equally likely to be related to marginal zone, IgM memory or IgG memory cells. High-throughput B cell repertoire sequencing thus provides a unique insight into patterns of B cell activation not possible from more conventional measures of immunogenicity.

The Expansion of Gastrointestinal-associated αβ T Cell Clones in Peripheral Blood Over Time Is a Disease Feature of Severe Acute Graft-Versus-Host Disease

AbstractAbstract 228Donor T cell alloreactivity in hematopoietic stem cell transplantation (HSCT) can result in acute graft-versus-host disease (GVHD) which is a major cause of morbidity and mortality that limits the clinical use of HSCT. Despite the potential large diversity in possible T cells (1015) it is thought that within each individual patient undergoing HSCT, a tractable and defined number of T cell clones mediate GVHD. We hypothesized that the identification of the most frequent T cell clones in the gastrointestinal (GI) tract of human patients with GVHD and subsequent tracking of these clones in the peripheral blood may provide a way to bracket and study disease related clones.To investigate T cell clonal dynamics in human GVHD, we collected endoscopic GI tract biopsy samples and matched blood specimens for twelve patients undergoing myeloblative HSCT with suspected GI GVHID prior to or within one day of corticosteroid therapy. We also collected peripheral blood thirty days after biopsy. Five of these patients were negative for GVHD or had mild grade I/II GI GVHD, whereas seven had severe and fatal grade III/IV GI GVHD. These patients had heterogenous indications, conditioning regimens and GVHD prophylaxis which included cyclosporine, tacrolimus and methotrexate. None of the patients had cytomegolovirus (CMV) colitis or other identifiable GI or significant systemic infectious complications, although four patients had low-level blood CMV positivity by PCR. As a control, we compared endoscopic GI biopsies from patients with inflammatory bowel disease as well as normal healthy screening subjects. We extracted genomic DNA and performed T cell receptor (TCR) β CDR3 repertoire sequencing at Stanford and with GigaGen GigaMune Rep-Seq™, both using the Illumina next generation sequencing MiSeq™ and HiSeq™ platforms. Results:For each tissue sample from each patient, between 1,000–4,000 unique T cell sequences were observed in endoscopic tissue samples (2–6 samples per patient). We sorted the most frequent clones in the GI tissue samples by rank order and fractionated the top 100 clones for analysis. Many of these top clones were found in the matched peripheral blood at the time of diagnosis with most of these occurring at frequencies well below those in the GI tissue. For the patients with negative or mild GVHD at the time of diagnosis, the GI-identified clones that were found in the peripheral blood had a mean frequency and standard deviation of 0.001369 +/− 0.0016. This is indistinguishable from peripheral blood samples from patients with severe GVHD which showed a mean frequency and standard deviation of 0.0013 +/− 0.0009 (P=0.36, 2 tailed t-test).We next tracked the GI-identified clones in the peripheral blood to the obtain fold change of the clones at thirty days after diagnosis to the time of diagnosis. The patients with negative or mild GVHD had a mean, median, and standard deviation in fold change of 1.9, 1.7, and 0.9 respectively. The patient with severe GVHD patients had a mean, median and standard deviation in fold change of 32.2, 30.7, and 17.3. A two tailed t-test comparison of the two groups showed they were statistically distinguishable with p-values of 0.0003 for examination of the mean fold change and 0.0005 for the median fold change.For patients with severe GVHD, the peripheral blood at 30 days after diagnosis showed a highly oligoclonal expansion of individually private GI-identified clones. Many of these clones were identified in their respective donor T cell pool. For those patients evaluated with upper and lower endoscopy, many clones were shared between the colon and duodenum or gastrum, however, the dominant clones between these sites appeared to be different. Conclusion:We found that a comparison of the fold change in the peripheral blood frequencies of the top one hundred clones identified in the GI tract biopsies can statistically distinguish five negative or mild GVHD patients from seven severe and fatal cases. These results support the use of T cell repertoire sequencing and associated approaches in human patients to both clarify the pathophysiology of the disease and eventually perhaps to provide an independent immune biomarker that could guide therapy. A major challenge will be to further phenotype and examine the T cell clones identified in the GI tissue and to investigate ways to determine which clones are highly associated or even causal to GVHD. Disclosures:Meyer:GigaGen Inc: Consultancy, Equity Ownership. Löhr:GigaGen Inc.: Employment, Equity Ownership. Hsu:GigaGen Inc.: Employment, Equity Ownership. Johnson:GigaGen Inc.: Employment, Equity Ownership.

Deep B and T Cell Repertoire Sequencing to Evaluate Minimal Residual Disease and T Cell Responses in a Therapeutic Vaccine Trial for Mantle Cell Lymphoma

AbstractAbstract 582 Background:A clinical trial is being conducted at Stanford University for patients with mantle cell lymphoma (MCL) in first remission that interdigitates an autologous CpG-stimulated tumor cell vaccine with high dose therapy and autologous peripheral blood stem cell transplant (PBST). In this trial, patients receive a combination of PSCT and infustion of vaccine primed autologous T cells. Blood samples are collected before and after the priming vaccination and at regular time points following transplant, and they are assayed for minimal residual disease (MRD) and T cell repertoire by high-throughput sequencing of rearranged immunoglobulin heavy chain (IgH) and T cell receptor (TCR) genes. Methods:We developed a sequencing-based platform, LymphoSIGHT, for MRD quantification in lymphoid malignancies. Using universal primer sets, we amplify rearranged IgH variable (V), diversity, and joining (J) gene segments from genomic DNA. To minimize the chance of somatic hypermutation interfering with detection of a cancer sequence, each IgH sequence is amplified by different sets of PCR primers in the three framework regions of the V segments and a common J segment primer. Amplified products can be sequenced to obtain >1 million reads and are analyzed using algorithms for clonotype determination. Tumor-specific clonotypes are identified for each patient based on their high frequency in the original tumor specimen. Quantitative MRD levels are then determined in serial samples of peripheral blood using spiked-in reference sequences. Our test has a sensitivity of 1 tumor cell per million leukocytes. This technology has also been extended to immunoglobulin light chain and T cell receptors. To quantify T cell responses to tumor vaccinations, the same samples are used for amplification, sequencing and analysis of the entire TCRB repertoire allowing for the assessment of T cell immune responses to the vaccine. Results:To date we have analyzed serial samples from 13 MCL patients who received the protocol of induction chemotherapy, vaccination, and PBST followed by vaccine primed T cell infusions and booster vaccinations. We have a pre-planned landmark analysis of MRD at 12 months post PBST. MRD was detected in blood samples immediately post-transplant in 3 patients, 2 of whom ultimately relapsed (Fig 1). In these two cases, disease was detected by sequencing 14 months and 4.5 months prior to detection by radiologic techniques. Clinical relapse in the second patient was originally restricted to the CNS; however, at a later point lymph node relapse occurred. The third patient has not shown signs of clinical relapse. The remaining 10 patients were MRD negative at 1 year following transplant, and these patients have not shown signs of clinical relapse with a median follow up of >24 months.To identify T cells specific to the vaccination, we searched for clonotypes that were highly enriched (>10X) in an in vitro tumor-stimulated culture. In 2 of 3 patients assayed, we identified such clonotypes. The enrichment of these same clonotypes was also seen in the patients after vaccinations and boosters, adding to the evidence that they are directed against the vaccine. An example for one such patient is shown in Fig. 2. These clonotypes increased in frequency significantly after the boost by comparison to a set of frequency-matched unrelated clonotypes (p<2.5x 10−6). Figure 2 also shows the dynamics of these clonotypes through the course of treatment demonstrating frequency increases upon initial and booster vaccinations. Conclusions:Our high-throughput sequencing method for MRD quantification shows promise for predicting clinical relapse in MCL patients after hematopoietic cell transplantation. This method provides detection lead-time that may be superior to clinical methods such as PET and MRI scanning but further follow up and greater numbers of patients will be required. Using the sequencing method, 77% of patients (10/13) were MRD negative at the landmark of 1 year post-transplant. Continued follow-up for molecular and clinical relapse is ongoing. T cell repertoire analysis identified clonotypes responding to the vaccination in some patients and follow up analyses will determine whether the presence of these clonotypes correlates with clinical outcomes in MCL patients. [Display omitted] [Display omitted] Disclosures:Faham:Sequenta, Inc.: Employment, Equity Ownership, Research Funding. Carlton:Sequenta, Inc.: Employment, Equity Ownership, Research Funding.