scholarly journals Single B Cell Immunoglobulin Sequencing Identifies Distinct Features of Monoclonal Antibodies in Patients with Immmune Thrombotic Thrombocytoepenic Purpura

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2081-2081
Author(s):  
Szumam Liu ◽  
Mohammad Abdelgawwad ◽  
Shanrun Liu ◽  
X. Long Zheng
Keyword(s):  
Gene Expression ◽  
Monoclonal Antibodies ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Hek293 Cells ◽  
Adamts13 Activity ◽  
Igg Antibodies ◽  
Human Monoclonal Antibodies ◽  
Blood Disorder

Abstract Introduction. Immune thrombotic thrombocytopenic purpura (iTTP) is a potentially fatal blood disorder, resulting from autoantibodies against ADAMTS13, a plasma metalloprotease that cleaves von Willebrand factor. However, the structural feature, binding epitope, and the mechanism of action of these autoantibodies in patients with acute iTTP are not fully understood. Methods. To further understand the pathogenesis of iTTP, single B cell immunoglobulin (Ig) sequencing using 10xChromium in 4 patients experiencing an acute episode of iTTP was performed; the expression and preliminary functional characterizations of selected clones were also carried out. Results. Approximately 2,631 viable and fluoresceinated ADAMTS13 labeled B cells (e.g., 7AAD -CD19 +CD20 +ADAMTS13 +) were sorted out from peripheral blood mononuclear cells of four patients with acute iTTP. These enriched ADAMTS13 antibody-producing B cells were then used for single cell analysis using 10xGenomics 5'-VDJ kit following the manufacturer's instruction. The single-cell gene expression libraries and VDJ libraries were constructed and sequenced by Hiseq at 20,000 reads/cell for gene expression and 5,000 reads/cell for VDJ sequences. Sequencing FASTQ files were mapped and counted by running through the Cell Ranger pipeline, and the final data were then further analyzed by the Loupe browser. We showed for the first time that the most frequent VJ combinations in the anti-ADAMTS13 IgG were: IGHV4-39:ILGJ4, IGHV3-48:ILGJ4, IGLV1-44:ILGLJ2, GLV5-45:ILGLJ3, IGLV2-14:ILGJ2, and IGLV3-21:ILGJ3 as shown in Figure 1. Of the top ten clones, the most frequently observed CDR3 (complementarity-determining region-3) sequences of these antibodies were CARDQLGISETQGSDLW on the heavy chain and CVIWHNSAWVF on the light chain as shown in Figure 2 and Table 1. The variable region sequences from the heavy and the light chains of Ig molecules were cloned into a human IgHG1 and a human IgL vector, respectively, which was then cotransfected in HEK293 cells. Western blotting, ELISA, immunoprecipitation, and functional assays were used to determine the expression and the function of human monoclonal IgG antibodies. Our preliminary results demonstrated the human monoclonal IgG antibodies bound and/or inhibited plasma ADAMTS13 activity. Conclusions. We conclude that there is clonal expansion of ADAMTS13 antibody producing B cells in acute iTTP and the cloned human monoclonal antibodies using the single B cell sequencing approach are functional. Our ongoing analysis on the structural and functional relationship of a large number of isolated human monoclonal antibodies may shed new light on the pathogenesis of iTTP. These antibodies may be useful to explore structural elements required for allosteric regulation of ADAMTS13 activity. Figure 1 Figure 1. Disclosures Zheng: AJMC: Honoraria; Clotsolution: Other: Co-founder; Takeda: Consultancy, Honoraria; Sanofi-Genzyme: Honoraria, Speakers Bureau; Alexion: Speakers Bureau.

scholarly journals Single-cell analysis of human B cell maturation predicts how antibody class switching shapes selection dynamics

2021 ◽  
Vol 6 (56) ◽  
pp. eabe6291 ◽  
Author(s):  
Hamish W. King ◽  
Nara Orban ◽  
John C. Riches ◽  
Andrew J. Clear ◽  
Gary Warnes ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Germinal Center ◽  
Cell Maturation ◽  
B Cell Maturation ◽  
Antibody Repertoires ◽  
Antibody Class ◽  
Human B Cell

Protective humoral memory forms in secondary lymphoid organs where B cells undergo affinity maturation and differentiation into memory or plasma cells. Here, we provide a comprehensive roadmap of human B cell maturation with single-cell transcriptomics matched with bulk and single-cell antibody repertoires to define gene expression, antibody repertoires, and clonal sharing of B cell states at single-cell resolution, including memory B cell heterogeneity that reflects diverse functional and signaling states. We reconstruct gene expression dynamics during B cell activation to reveal a pre–germinal center state primed to undergo class switch recombination and dissect how antibody class–dependent gene expression in germinal center and memory B cells is linked with a distinct transcriptional wiring with potential to influence their fate and function. Our analyses reveal the dynamic cellular states that shape human B cell–mediated immunity and highlight how antibody isotype may play a role during their antibody-based selection.

scholarly journals Desynchronization of the Germinal Center Dynamics and Remodeling of the Tumor Microenvironment Characterize KMT2D-Driven Lymphomagenesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 670-670
Author(s):  
Gabriel Brisou ◽  
Manon Zala ◽  
Laurine Gil ◽  
Giulia Pagano ◽  
Agnès Bru ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Germinal Center ◽  
Research Funding ◽  
Double Mutant ◽  
Advisory Board ◽  
Memory B Cells ◽  
Expression Program

Abstract Follicular lymphoma (FL) is a prototypical example of germinal center (GC) derived B-cell lymphoma. Using a mouse model recapitulating the sporadic occurrence of the FL hallmark BCL2/IGH translocation in healthy individuals, our previous work demonstrated that FL genesis is a dynamic process that requires multiple re-entries of BCL2+ memory B-cells into the GC to ultimately accumulate in lymphoid organs. In line with this, using single-cell gene expression analysis of human FL vs normal GC B-cells, we recently discovered that FL cells are not 'frozen' at a particular GC maturation stage but instead exhibit a major desynchronization of the GC-specific expression program (Milpied et al. Nat Immunol in press). Since KMT2D loss-of-function mutations and BCL2 translocations are the 2 main alterations in FL, we hypothesized these 2 genetic events might explain the GC program desynchronization we observe in humans. To explore the in vivo consequences of Kmt2d inactivation with Bcl2 overexpression in regulating GC/memory dynamics, we transduced bone marrow progenitors carrying B-cell-specific conditional Cd19-Cre Kmt2dflox/flox alleles with a retrovirus encoding human BCL2 or reporter alone, followed by iv transplantation into lethally irradiated WT recipients. Only double-mutant Kmt2d-/-Bcl2+ mice manifested with GC-derived lymphomas in chronically challenged animals, recapitulating histological and phenotypic features associated with human FL progression from early preneoplastic lesions to overt FL-like tumors. We used integrative single-cell analysis of surface phenotype (10-color panel), gene expression (88-gene panel by microfluidic RT-qPCR) and IGH clonality to deconvolute cellular heterogeneity of flow-sorted GC and memory B-cells after acute (day 10) or chronic T-cell dependent immunization in double-mutant vs. single Bcl2+, Kmt2d-/- or WT mice, retaining >4000 cells for downstream analysis. Populations of WT GC B-cells were molecularly heterogeneous and spanned a cyclic continuum of transitional B-cell states polarized between the light and dark zone where synchronized expression of gene modules characterized mouse GC functional identity. In acute responses, single and double-mutant mice formed GC similar to control chimera mice, and single GC B-cells from all genotypes clustered together with WT GCs, suggesting unperturbed GC transcriptional dynamics upon first antigen encounter. However, preneoplastic/tumoral Kmt2d-/-Bcl2+ mice after chronic challenge showed massive GC hyperplasia and single B-cells expressed a distinct transcriptional signature that clustered separately from WT GC or memory cells. Specifically, murine lymphoma B-cells sorted with a GC-like phenotype accumulated in transitional cell states where the synchrony of most GC-specific co-expression patterns was progressively lost whereas expression of cytokines (Lta) or surface markers linked to GC to memory transition (Gpr183, Cxcr3) became markedly expressed, indicating that Kmt2d-deficient lymphomas were not blocked at a particular GC stage. Given the importance of T-cell help for the fate 'decisions' of GC-to-memory B-cells, we further explored whether tumor cell-intrinsic factors may affect immune cell phenotypes thereby supporting the GC gene expression desynchronization. Using flow cytometry, we found that Kmt2d inactivation instructed a progressive remodeling of the tumor microenvironment with an increased recruitment of CD4+ T-follicular helper (TFH) (n=21, p<0.01). Using droplet-based single-cell RNA-seq to profile total spleens from 2 WT mice and 2 lymphomas (>9000 cells), we revealed a TFH cluster with an activation signature distinct from normal TFH and a concomitant expansion of exhausted CD8+ T-cells strongly co-expressing inhibitory receptors (Lag3, Tim3, Pdcd1), thereby indicating that Kmt2d inactivation in B-cells may favor lymphoma formation by shaping the FL tumor supportive niche and contributing to immune evasion. In summary, our integrative single-cell analyses in murine lymphomas revealed that Kmt2d mutations in FL not only instruct B-cell intrinsic effects involving the desynchronization of the normal GC expression program, but also trigger a concomitant re-education of a tumor-supportive immune microenvironment, establishing for the first time a key link between the most frequent epigenetic alteration and the FL microenvironment. Disclosures Salles: F. Hoffmann-La Roche Ltd: Consultancy, Honoraria, Research Funding; Celgene: Honoraria, Other: Advisory Board, Research Funding; Janssen: Honoraria, Other: Advisory Board; Novartis: Consultancy, Honoraria; Takeda: Honoraria; Servier: Honoraria, Other: Advisory Board; Epizyme: Honoraria; Merck: Honoraria; BMS: Honoraria, Other: Advisory Board; Morphosys: Honoraria; Acerta: Honoraria; Amgen: Honoraria; Gilead: Honoraria, Other: Advisory Board; Pfizer: Honoraria; Servier: Honoraria; Abbvie: Honoraria.

scholarly journals Single-cell RNA-Seq of follicular lymphoma reveals malignant B-cell types and coexpression of T-cell immune checkpoints

Blood ◽  
2019 ◽  
Vol 133 (10) ◽  
pp. 1119-1129 ◽  
Author(s):  
Noemi Andor ◽  
Erin F. Simonds ◽  
Debra K. Czerwinski ◽  
Jiamin Chen ◽  
Susan M. Grimes ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Immune Checkpoint ◽  
Gene Networks ◽  
Immune Checkpoints ◽  
Transcriptional Networks ◽  
Low Grade

Abstract Follicular lymphoma (FL) is a low-grade B-cell malignancy that transforms into a highly aggressive and lethal disease at a rate of 2% per year. Perfect isolation of the malignant B-cell population from a surgical biopsy is a significant challenge, masking important FL biology, such as immune checkpoint coexpression patterns. To resolve the underlying transcriptional networks of follicular B-cell lymphomas, we analyzed the transcriptomes of 34 188 cells derived from 6 primary FL tumors. For each tumor, we identified normal immune subpopulations and malignant B cells, based on gene expression. We used multicolor flow cytometry analysis of the same tumors to confirm our assignments of cellular lineages and validate our predictions of expressed proteins. Comparison of gene expression between matched malignant and normal B cells from the same patient revealed tumor-specific features. Malignant B cells exhibited restricted immunoglobulin (Ig) light chain expression (either Igκ or Igλ), as well the expected upregulation of the BCL2 gene, but also downregulation of the FCER2, CD52, and major histocompatibility complex class II genes. By analyzing thousands of individual cells per patient tumor, we identified the mosaic of malignant B-cell subclones that coexist within a FL and examined the characteristics of tumor-infiltrating T cells. We identified genes coexpressed with immune checkpoint molecules, such as CEBPA and B2M in regulatory T (Treg) cells, providing a better understanding of the gene networks involved in immune regulation. In summary, parallel measurement of single-cell expression in thousands of tumor cells and tumor-infiltrating lymphocytes can be used to obtain a systems-level view of the tumor microenvironment and identify new avenues for therapeutic development.

scholarly journals Single-Cell Transcriptome Reveals the Role of Sting Pathway in Acute B Lymphoblastic Leukemia Bone Marrow Cells

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 47-48
Author(s):  
Yiqing Cai ◽  
Xiangxiang Zhou ◽  
Juan Yang ◽  
Jiarui Liu ◽  
Yi Zhao ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Lymphoblastic Leukemia ◽  
Marker Genes ◽  
Type I ◽  
Development Trajectory ◽  
Sting Pathway ◽  
Cell Transcriptome

Introduction Cancer immunotherapy and targeted therapy have yielded impressive clinical efficacy in acute B lymphoblastic leukemia (B-ALL). Despite the initial high complete remission (CR) rate following first-line therapy, treatment refractoriness and disease relapse remain are correlated with dismal survival. By the time the malignant cells generate, they are accompanied by a rich network of stromal cells and cytokines in bone marrow (BM). This tumor microenvironment (TME) represents an important feature of the biology of B-ALL but also shapes the clinical behavior of the disease. It remains to be confirmed whether the cellular composition and transcriptional heterogeneity impacts the clinical effects of B-ALL. Herein, we analyzed the immune cell infiltration features and related marker genes for B-ALL based on single cell RNA sequencing (scRNA-seq) data, which would be of significance for the development of novel immunotherapies. Methods ScRNA-seq data of 11373 BM cells from 3 B-ALL patients were obtained from the Gene Expression Omnibus (GEO, GSE153358). After quality control and data normalization, cell filtration and marker genes extraction were performed by the Seurat package. Principal component analysis (PCA) and t-distributed stochastic neighbor embedding (t-SNE) were then applied to cluster cells, following with cell types' definition and gene expression profiles in total subsets. Cell clustering was demonstrated using t-SNE-1 and t-SNE-2. In order to determine the cellular characteristics of TME cells mainly mediated by STING pathway, dendritic cell (DC) and B cell were extracted and further plotted gene expression including immunosuppressive molecules and STING pathway, respectively. The pseudo-time analysis was finally performed by Monocle package to display B cell development trajectory and gene expressions over time. Results 9 cell subsets in B-ALL BM, including naïve and memory CD4+ T cell, CD14+ monocyte, B cell, CD8+ T cell, FCGRA3+monocyte, nature killer (NK) cell, DC, and platelet, were identified based on t-SNE analysis (Fig.1A). Top 10 marker genes in each cell cluster were presented in heat map (Fig.1B). Through analyzing differentially expressed genes, we found that BM B cells hardly expressed PD-L1 (CD274), but partially carried TMEM173 (STING), NFKB1 (NF-κB) and GSDMD. In addition, immune cells in BM TME broadly distributed and highly expressed STING and NF-κB, indicating the potential response to type I innate immune response and higher sensitivity to STING agonists than PD-1 antibody (Fig.2A and B). Previous studies had revealed that STING pathway participated in the activation of DC following with production of type I IFNs. We further isolated DC from 9 subsets and profiled the gene expression features. T-SNE analysis revealed 3 subtypes of DC in BM, marker genes comparison further identified as monocyte derived DC, CD1C-CD14-DC and myeloid conventional DC. Cyclic GMP-AMP synthase (cGAS), STING and NF-κB were highly expressed in each type in compared with PDCD1 (PD-1) and highest existed on myeloid conventional DC (Fig.3). We then explored B cell subsets to determine whether STING pathway could induce cell pyroptosis in BM B cells. Different subgroup of B cells shared similar marker genes, companying with higher expression of NF-κB and GDSMD (Fig.4). Furthermore, pseudo-time analysis plotted the development trajectory of malignant B cells. The results showed that GSDMD gradually increased along with cell development, suggesting that STING agonist would be sensitive to mature B cells (Fig.5). Subsets analysis shown that anti-tumor immune response of DC and pyroptosis of B cell might be triggered through STING pathway activation. Conclusion Our study profiled for the first time the expression of STING pathway in BM DC and B cell from B-ALL patients based on single-cell transcriptome. Combination of STING agonist and conventional immunotherapy had been shown prospects in antitumor therapy. STING agonist is expected to be an adjuvant drug for B-ALL immunotherapies in the future. Keywords: Single-cell RNA sequencing; acute B lymphoblastic leukemia; STING; PD-1; immunotherapy. Disclosures No relevant conflicts of interest to declare.

scholarly journals Increased Activated Circulating T Follicular Helper Cells and Changes in B Cell Subsets in Immune TTP (iTTP) and in Response to Rituximab Treatment

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 23-24
Author(s):  
Jin-Sup Shin ◽  
Geraldine Cambridge ◽  
Yanping Guo ◽  
Marie Scully ◽  
Mari Thomas
Keyword(s):  
B Cells ◽  
B Cell ◽  
Advisory Board ◽  
Memory B Cells ◽  
Adamts13 Activity ◽  
Igg Antibodies ◽  
Follicular Helper ◽  
B Cell Subsets ◽  
T Cell Help

Background: T follicular helper cells (Tfh), characterised by surface expression of CXCR5, PD1 and ICOS, regulate development of antigen-specific B cell immunity through germinal centre (GC) formation, generation of long-lived memory B cells and high-affinity plasma cells. Methods: In this prospective study of peripheral blood B and circulating Tfh (cTfh) cell subsets, iTTP patients at 32 acute presentations, 23 elective rituximab (ER) episodes and 27 age & sex-matched healthy controls (HC) were studied using flow cytometry. All acute cases received PEX, steroids and RTX. All ER patients previously received RTX as acute therapy or previous ER at a median of 22 months (range 12-191 months). Serial samples were taken post-rituximab (RTX). B cell return was defined by laboratory CD19 count (&lt;5 x 106/L). Statistical analysis was performed using GraphPad Prism 8. Results: 11/32 (34%) acute cases received potentially immunomodulatory therapy prior to blood sampling and were excluded. Median ADAMTS13 activity was &lt;5 IU/dL (&lt;5-10.4 IU/dL) and anti-ADAMTS13 IgG 46% (2-127%). In 23 ER cases, median ADAMTS13 activity was 9 IU/dL (&lt;5-24IU/dL) and antibody 8% (2-89%). At acute presentation, CD4+CXCR5+ and CD4+CXCR5+PD1+ cTfh were decreased compared to HC (6.1% vs 9.4%; [p=0.003] and 1.2% vs 1.8%; [p=0.003] respectively), whereas activated cTfh (CD4+CXCR5+PD1+ICOS+) were increased (0.95% vs 0.55%; [p=0.01]) (Table 1). B cell subsets in acute iTTP showed decreased pre-switch and switched memory subsets compared to HC: IgD+/CD27+ [p=0.003]; IgD-/CD27+ [p=0.02] and IgD-/CD38+ [p=0.008]. Plasmablasts (IgD-CD38++) were increased [p=0.03] (Figure 1). ER patients pre-RTX had increased transitional and naïve B cells compared to HC [p=0.001; p&lt;0.0001 respectively] and increased percentages of plasmablasts [p&lt;0.0001]. Memory subsets defined by IgD/CD38 were all significantly decreased [p&lt;0.0001] (Figure 1). Activated cTfh were increased in ER pre-RTX compared to HC [p&lt;0.0001], whereas CD4+CXCR5+ICOS+ cells were reduced. There was no difference in CD4+CXCR5+ cells (Table 1). Memory subsets (defined by IgD/CD38) in ER were all significantly reduced compared with acute iTTP cases (Figure 1) likely to be due to previously described (often long term) changes in B cell subsets following B cell return after RTX. Longitudinal analysis: B cell return post-RTX in acute cases occurred mainly with transitional/naïve cells at a median of 8 months (0.5-14) but was not associated with iTTP relapse. Memory B cell subsets (defined by IgD/CD38) were significantly reduced at B cell return (Table 2). In ER patients, B cell subsets at repopulation were generally similar to levels seen prior to re-treatment with RTX. Frequency of cTfh was not significantly altered by RTX therapy in either acute TTP or ER. Two ER patients were followed longitudinally from RTX therapy, through ADAMTS13 normalisation & subsequent fall requiring further RTX re-treatment. Asymptomatic ADAMTS13 relapse (activity &lt;15 IU/dL) was temporally related with an apparent maturation to memory phenotype and increase in % plasmablasts. Conclusions: At acute iTTP presentation and prior to elective re-treatment with RTX, activated (CD4+CXCR5+PD1+ICOS+) cTfh cells are increased, suggesting a role of T cell help in development of anti-ADAMTS13 IgG antibodies. Prior to RTX, B cell phenotype is also altered in acute TTP, with decreased frequency of memory subsets and a trend to increased naïve cells and plasmablasts. Persistent changes in B cell subsets were seen in ER patients who had received previous RTX with naive cells predominating and reduced memory cells. Interestingly, no patient relapsed/ required re-treatment related to B cell return, with relapse occurring at least 4 months after detection of B cells. Resumption of the autoimmune response therefore appeared limited by the rate of maturation of autoantigen(ADAMTS13)-specific B cells, either by selection/differentiation of ADAMTS13-naive B cells and/or expansion of ADAMTS13-specific memory B cells to Ig producing cells. This process, presumably driven by interaction with Tfh cells, suggests a role of T cell help in development of anti-ADAMTS13 IgG antibodies. Longitudinal analysis of the evolution of B and cTfh cells may help in predicting relapse in iTTP. Disclosures Scully: Alexion: Consultancy, Speakers Bureau; Ablynx/Sanofi: Consultancy, Other: Advisory Board, Speakers Bureau; Novartis: Other: Advisory Board, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Sanofi: Consultancy, Speakers Bureau; Takeda: Speakers Bureau; Shire/Takeda: Other: Advisory Board, Research Funding, Speakers Bureau. Thomas:Ablynx: Honoraria, Other: Advisory Board; Sanofi: Honoraria, Other: Advisory Board; Bayer: Honoraria, Speakers Bureau.

Single-Cell RNA Sequencing Identifies a Pseudo-Immune Differentiation Axis As the Main Source of Functional Heterogeneity in Follicular Lymphoma B-Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 548-548
Author(s):  
Noudjoud Attaf ◽  
Inaki Cervera-Marzal ◽  
Laurine Gil ◽  
Chuang Dong ◽  
Jean-Marc Navarro ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lymph Node ◽  
B Cells ◽  
Follicular Lymphoma ◽  
B Cell ◽  
Single Cell ◽  
Research Funding ◽  
Affinity Maturation ◽  
Patient Specific ◽  
Specific Gene

Introduction Follicular Lymphoma (FL), the second most frequent lymphoma in adults, often presents as a disseminated disease at diagnosis. Despite a generally slow progression and a median overall survival of more than 15 years with current chemo-immunotherapies, FL patients often suffer from multiple relapses. Yet, the biological mechanisms promoting FL dissemination, progression and relapse are still poorly understood. FL, like most B-cell lymphomas, originates from germinal centers (GC) where B-cells physiologically undergo clonal expansion, antibody affinity maturation, and differentiation into antibody-producing plasma cells (PC) or recirculating memory (Mem) B-cells. Recently, we provided evidence that FL B-cells are not blocked in a GC B-cell state but might adopt new dynamic modes of functional diversity (Milpied et al., Nature Immunology 2018), yet the main sources of intratumoral heterogeneity within FL remained to be identified. Methods Frozen live cell suspensions were obtained from the CeVi collection of the Institute Carnot/Calym (ANR, France). We initially applied a plate-based 5'-end single-cell RNAseq (scRNAseq) method for deep integrative single-cell analyses of transcriptome, B-cell receptor (BCR) sequence, and surface phenotype on FACS-sorted FL B-cells (4 patients, lymph node biopsies) and their non-malignant counterparts (6 adult healthy donors, spleen and tonsil samples). We confirmed our findings on additional FL samples with high-throughput droplet-based 3'-end scRNAseq (9 patients, lymph node biopsies), and 5'-end scRNAseq paired with BCRseq (5 patients, lymph node biopsies). Custom and existing bioinformatics analysis pipelines were combined for quality control and cell filtering, dimensionality reduction (PCA, t-SNE, UMAP), clustering, pseudo-time analysis, BCR sequence analysis and integrative data analysis. We further validated our transcriptomic data with FACS-based surface and intracellular protein analysis (8 patients, lymph node biopsies). Results Consistent with our previous findings, FL B-cells were transcriptionally diverse, with most cells exhibiting a patient-specific gene expression profile distinct from PC, GC and Mem cells. Challenging the mainstream view of a differentiation blockade in FL, we identified rare FL B-cells carrying a PC-like profile (including low expression of MS4A1/CD20, high expression of XBP1, MZB1, PRDM1). PC-like FL B-cells expressed high levels of the tumor clonal BCR heavy and light chain mRNA, and BCR sequence phylogenetic analysis revealed that those cells did not branch out from a specific tumor subclone. Most importantly, we found that the molecular profiles of the vast majority of FL B-cells spanned a continuum of transitional states between proliferating GC-like and quiescent Mem-like gene expression states. Principal component analysis and pseudo-time reconstruction revealed that pseudo-immune differentiation axis was consistently the main source of intra-sample transcriptional heterogeneity. On top of cell cycle related genes, GC-like FL B-cells notably expressed AICDA, BCL6, RGS13, NANS, CD81, and CD38 genes. By contrast, Mem-like FL B-cells expressed CD44, GPR183, CD69, CXCR4, CCR7, SELL, KLF2, suggesting that those cells may not be confined to the FL follicles. Flow cytometry analysis of dissociated FL tumors confirmed that only the CD38hiCD81hi subset of FL B-cells (GC-like cells), expressed Ki67 and high levels of Bcl6, whereas only CD38negCD81neg FL B-cells (Mem-like cells) consistently contained CD44+ and GPR183+ cells. Conclusions Our study suggests that FL B-cells hijack the physiological GC differentiation process to dynamically alternate between GC-like and Mem-like states that might be responsible for FL progression and dissemination, respectively. We anticipate that such FL-specific clonal dynamics may be orchestrated by extrinsic signals delivered by tumor-infiltrating T cells. Disclosures Milpied: Innate Pharma: Research Funding; Institut Roche: Research Funding.

scholarly journals Antibody repertoire and gene expression dynamics of diverse human B cell states during affinity maturation

2020 ◽  
Author(s):  
Hamish W King ◽  
Nara Orban ◽  
John C Riches ◽  
Andrew J Clear ◽  
Gary Warnes ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Single Cell ◽  
Affinity Maturation ◽  
Germinal Centre ◽  
Integrated Analysis ◽  
Cell Mediated Immunity ◽  
Antibody Repertoires ◽  
B Cell Subsets

AbstractIn response to antigen challenge, B cells clonally expand, undergo selection and differentiate to produce mature B cell subsets and high affinity antibodies. However, the interplay between dynamic B cell states and their antibody-based selection is challenging to decipher in primary human tissue. We have applied an integrated analysis of bulk and single-cell antibody repertoires paired with single-cell transcriptomics of human B cells undergoing affinity maturation. We define unique gene expression and antibody repertoires of known and novel B cell states, including a pre-germinal centre state primed to undergo class switch recombination. We dissect antibody class-dependent gene expression of germinal centre and memory B cells to find that class switching prior to germinal centre entry dictates the capacity of B cells to undergo antibody-based selection and differentiate. Together, our analyses provide unprecedented resolution into the gene expression and selection dynamics that shape B cell-mediated immunity.

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