Integrative single-cell chromatin and transcriptome analysis of human plasma cell differentiation.

Plasma cells (PC) are highly specialized cells representing the end stage of B cell differentiation. We have shown that PC differentiation can be reproduced in vitro using elaborate culture systems. The molecular changes occurring during PC differentiation are recapitulated in this in vitro differentiation model. However, a major challenge exists to decipher the spatiotemporal epigenetic and transcriptional programs that drives the early stages of PC differentiation. We combined single cell (sc) RNA-seq and single cell ATAC-seq to decipher the trajectories involved in PC differentiation. ScRNA-seq experiments revealed a strong heterogeneity of the preplasmablastic and plasmablastic stages. Among genes that were commonly identified using scATAC-seq and scRNA-seq, we identified several transcription factors with significant stage specific potential importance in PC differentiation. Interestingly, differentially accessible peaks characterizing the preplasmablastic stage were enriched in motifs of BATF3, FOS and BATF, belonging to the AP-1 transcription factor family, that may represent key transcriptional nodes involved in PCD. Integration of transcriptomic and epigenetic data at the single cell level revealed that a population of preplasmablasts already undergone epigenetic remodeling related to PC profile together with UPR activation and are committed to differentiate in PC. These results and the supporting data generated with our in vitro PC differentiation model provide a unique resource for the identification of molecular circuits that are crucial for early and mature plasma cell maturation and biological functions. These data thus provide critical insights into epigenetic- and transcriptional-mediated reprogramming events that sustain PC differentiation.

The immunoglobulin heavy chain super enhancer controls class switch recombination in developing B cells.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

NGFR regulates stromal cell activation in germinal centers.

Nerve growth factor receptor (NGFR) is expressed by follicular dendritic cells (FDCs). However, the role of NGFR in the humoral response is not well defined. Here, we study the effect of Ngfr loss on lymph node organization and function, demonstrating that Ngfr depletion leads to spontaneous germinal center (GC) formation and an expansion of the GC B cell compartment. In accordance with this effect, stromal cells are altered in Ngfr-/- mice with a higher frequency of FDCs, characterized by CD21/35, MAdCAM-1, and VCAM-1 overexpression. GCs are located ectopically in Ngfr-/- mice, with lost polarization together with impaired high-affinity antibody production and an increase in circulating autoantibodies. We observe higher levels of autoantibodies in Bcl2 Tg/Ngfr-/- mice, concomitant with a higher incidence of autoimmunity and lower overall survival. Our work shows that NGFR is involved in maintaining GC structure and function, participating in GC activation, antibody production, and immune tolerance.

Unique repetitive nucleic acid structures mirror switch regions in the human IgH locus.

Immunoglobulin (Ig) genes carry the unique ability to be reshaped in peripheral B lymphocytes after these cells encounter a specific antigen. B cells can then further improve their affinity, acquire new functions as memory cells and eventually end up as antibody-secreting cells. Ig class switching is an important change that occurs in this context, thanks to local DNA lesions initiated by the enzyme activation-induced deaminase (AID). Several cis-acting elements of the Ig heavy (IgH) chain locus make it accessible to the AID-mediated lesions that promote class switch recombination (CSR). DNA repeats, with a non-template strand rich in G-quadruplexes (G4)-DNA, are prominent cis-targets of AID and define the so-called "switch" (S) regions specifically targeted for CSR. By analyzing the structure of the human IgH locus, we uncover that abundant DNA repeats, some with a putative G4-rich template strand, are additionally present in downstream portions of the IgH coding genes. These like-S (LS) regions stand as 3' mirror-images of S regions and also show analogies to some previously reported repeats associated with the IgH locus 3' super-enhancer. A regulatory role of LS repeats is strongly suggested by their specific localization close to exons encoding the membrane form of Ig molecules, and by their conservation during mammalian evolution.

Attempts to evaluate locus suicide recombination and its potential role in B cell negative selection in the mouse.

Introduction: In mature B cells, activation-induced deaminase reshapes Ig genes through somatic hypermutation and class switch recombination of the Ig heavy chain (IgH) locus under control of its 3' cis-regulatory region (3'RR). The 3'RR is itself transcribed and can undergo "locus suicide recombination" (LSR), then deleting the constant gene cluster and terminating IgH expression. The relative contribution of LSR to B cell negative selection remains to be determined. Methods: Here, we set up a knock-in mouse reporter model for LSR events with the aim to get clearer insights into the circumstances triggering LSR. In order to explore the consequences of LSR defects, we reciprocally explored the presence of autoantibodies in various mutant mouse lines in which LSR was perturbed by the lack of Sµ or of the 3'RR. Results: Evaluation of LSR events in a dedicated reporter mouse model showed their occurrence in various conditions of B cell activation, notably in antigen-experienced B cells Studies of mice with LSR defects evidenced increased amounts of self-reactive antibodies. Discussion: While the activation pathways associated with LSR are diverse, in vivo as well as in vitro, this study suggests that LSR may contribute to the elimination of self-reactive B cells.

Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19.

To understand the fine differential elements that can lead to or prevent acute respiratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investigate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes associated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expansion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

Roles of G4-DNA and G4-RNA in Class Switch Recombination and Additional Regulations in B-Lymphocytes.

Mature B cells notably diversify immunoglobulin (Ig) production through class switch recombination (CSR), allowing the junction of distant "switch" (S) regions. CSR is initiated by activation-induced deaminase (AID), which targets cytosines adequately exposed within single-stranded DNA of transcribed targeted S regions, with a specific affinity for WRCY motifs. In mammals, G-rich sequences are additionally present in S regions, forming canonical G-quadruplexes (G4s) DNA structures, which favor CSR. Small molecules interacting with G4-DNA (G4 ligands), proved able to regulate CSR in B lymphocytes, either positively (such as for nucleoside diphosphate kinase isoforms) or negatively (such as for RHPS4). G4-DNA is also implicated in the control of transcription, and due to their impact on both CSR and transcriptional regulation, G4-rich sequences likely play a role in the natural history of B cell malignancies. Since G4-DNA stands at multiple locations in the genome, notably within oncogene promoters, it remains to be clarified how it can more specifically promote legitimate CSR in physiology, rather than pathogenic translocation. The specific regulatory role of G4 structures in transcribed DNA and/or in corresponding transcripts and recombination hereby appears as a major issue for understanding immune responses and lymphomagenesis.

Distinct B-Cell Specific Transcriptional Contexts of the BCL2 Oncogene Impact Pre-Malignant Development in Mouse Models.

Upregulated expression of the anti-apoptotic BCL2 oncogene is a common feature of various types of B-cell malignancies, from lymphoma to leukemia or myeloma. It is currently unclear how the various patterns of deregulation observed in pathology eventually impact the phenotype of malignant B cells and their microenvironment. Follicular lymphoma (FL) is the most common non-Hodgkin lymphoma arising from malignant germinal center (GC) B-cells, and its major hallmark is the t(14:18) translocation occurring in B cell progenitors and placing the BCL2 gene under the control of the immunoglobulin heavy chain locus regulatory region (IgH 3'RR), thus exposing it to constitutive expression and hypermutation. Translocation of BCL2 onto Ig light chain genes, BCL2 gene amplification, and other mechanisms yielding BCL2 over-expression are, in contrast, rare in FL and rather promote other types of B-cell lymphoma, leukemia, or multiple myeloma. In order to assess the impact of distinct BCL2 deregulation patterns on B-cell fate, two mouse models were designed that associated BCL2 and its full P1-P2 promoter region to either the IgH 3'RR, within a "3'RR-BCL2" transgene mimicking the situation seen in FL, or an Ig light chain locus context, through knock-in insertion at the Igκ locus ("Igκ-BCL2" model). While linkage to the IgH 3' RR mostly yielded expression in GC B-cells, the Igκ-driven up-regulation culminated in plasmablasts and plasma cells, boosting the plasma cell in-flow and the accumulation of long-lived plasma cells. These data demonstrate that the timing and level of BCL2 deregulation are crucial for the behavior of B cells inside GC, an observation that could strongly impact the lymphomagenesis process triggered by secondary genetic hits.

Anti-FcαRI Monoclonal Antibodies Resolve IgA Autoantibody-Mediated Disease.

Immunoglobulin A (IgA) is generally considered as a non-inflammatory regulator of mucosal immunity, and its importance in diversifying the gut microbiota is increasingly appreciated. IgA autoantibodies have been found in several autoimmune or chronic inflammatory diseases, but their role in pathophysiology is ill-understood. IgA can interact with the Fc receptor FcαRI on immune cells. We now established a novel IgA autoimmune blistering model, which closely resembles the human disease linear IgA bullous disease (LABD) by using genetically modified mice that produce human IgA and express human FcαRI. Intravital microscopy demonstrated that presence of IgA anti-collagen XVII, - the auto-antigen in LABD-, resulted in neutrophil activation and extravasation from blood vessels into skin tissue. Continued exposure to anti-collagen XVII IgA led to massive neutrophil accumulation, severe tissue damage and blister formation. Importantly, treatment with anti-FcαRI monoclonal antibodies not only prevented disease, but was also able to resolve existing inflammation and tissue damage. Collectively, our data reveal a novel role of neutrophil FcαRI in IgA autoantibody-mediated disease and identify FcαRI as promising new therapeutic target to resolve chronic inflammation and tissue damage.

Beneficial effects of citrulline enteral administration on sepsis-induced T cell mitochondrial dysfunction.

Severe sepsis induces a sustained immune dysfunction associated with poor clinical behavior. In particular, lymphopenia along with increased lymphocyte apoptosis and decreased lymphocyte proliferation, enhanced circulating regulatory T cells (Treg), and the emergence of myeloid-derived suppressor cells (MDSCs) have all been associated with persistent organ dysfunction, secondary infections, and late mortality. The mechanisms involved in MDSC-mediated T cell dysfunction during sepsis share some features with those described in malignancies such as arginine deprivation. We hypothesized that increasing arginine availability would restore T cell function and decrease sepsis-induced immunosuppression. Using a mouse model of sepsis based on cecal ligation and puncture and secondary pneumonia triggered by methicillin-resistant Staphylococcus aureus inoculation, we demonstrated that citrulline administration was more efficient than arginine in increasing arginine plasma levels and restoring T cell mitochondrial function and proliferation while reducing sepsis-induced Treg and MDSC expansion. Because there is no specific therapeutic strategy to restore immune function after sepsis, we believe that our study provides evidence for developing citrulline-based clinical studies in sepsis.

Targeting IgE polyadenylation signal with antisense oligonucleotides decreases IgE secretion and plasma cell viability.

BACKGROUND: Allergy regroups numerous complex and various diseases classified as IgE-dependent or non-IgE-dependent hypersensitivities. IgEs are expressed as membrane and secreted forms by B cells and plasma cells, respectively. In IgE-mediated hypersensitivity, IgE secretion and binding to the high-affinity IgE receptor FcεRI on effector cells are responsible for the onset of allergic symptoms; in contrast, surface IgE expression as a B-cell receptor is barely detectable. OBJECTIVE: Our aim was to test an innovative antisense approach to reducing IgE secretion. METHODS: We designed an antisense oligonucleotide (ASO) targeting the polyadenylation signal of human secreted IgE to redirect IgE transcript polyadenylation from the secreted form to the membrane form. ASO treatments were performed on B cells from transgenic mice expressing humanized IgE (InEps mice), as well as on human primary B cells and myeloma cells. In vivo ASO delivery was tested by using an InEps mouse model. RESULTS: We demonstrated that treatment with a morpholino ASO targeting the secreted IgE polyadenylation signal drastically decreased IgE secretion and inversely increased membrane IgE mRNA expression. In addition, ASO treatment induced apoptosis of IgE-expressing U266 myeloma cells, and RNA sequencing revealed attenuation of their plasma cell phenotype. Remarkably, systemic administration of an ASO coupled with Pip6a as an arginine-rich cell-penetrating peptide decreased IgE secretion in vivo. CONCLUSION: Altogether, this ASO strategy could be an effective way to decrease IgE secretion and allergic symptoms in patients with IgE-dependent allergies, and it could also promote allergen tolerance through apoptosis of IgE+ antibody-secreting cells.

RNA-based immunoglobulin repertoire sequencing is a new tool for the management of monoclonal gammopathy of renal (kidney) significance.

The diagnostic approach of monoclonal gammopathy of renal significance is based on the detection of a monoclonal immunoglobulin in the blood and urine, and the identification of the underlying clone through bone marrow and/or peripheral blood cytologic and flow cytometry analysis. However, the monoclonal component and its corresponding clone may be undetectable using these routine techniques. Since clone identification is the cornerstone for guiding therapy and assessing disease response, more sensitive methods are required. We recently developed a high-throughput sequencing assay from bone marrow mRNA encoding immunoglobulins (RACE-RepSeq). This technique provides both full-length V(D)J region (variable, diversity and joining genes that generate unique receptors as antigen receptors) of the monoclonal immunoglobulin and the dominant immunoglobulin repertoire. This allows analysis of mutational patterns, immunoglobulin variable gene frequencies and diversity due to somatic hypermutation. Here, we evaluated the diagnostic performance of RACE-RepSeq in 16 patients with monoclonal-associated kidney lesions, and low serum monoclonal immunoglobulin and free light chain levels at diagnosis. Bone marrow immunohistochemical analysis was negative in all 11 patients so tested and 7 of 12 patients had no detectable clone matching the kidney deposits using flow cytometry analysis. By contrast, RACE-RepSeq detected a dominant clonal light chain sequence of matched isotype with respect to kidney deposits in all patients. Thus, high throughput mRNA sequencing appears highly sensitive to detect subtle clonal disorders in monoclonal gammopathy of renal significance and suggest this novel approach could help improve the management of this kidney disease.

Committed Human CD23-Negative Light-Zone Germinal Center B Cells Delineate Transcriptional Program Supporting Plasma Cell Differentiation.

B cell affinity maturation occurs in the germinal center (GC). Light-zone (LZ) GC B cells (BGC-cells) interact with follicular dendritic cells (FDCs) and compete for the limited, sequential help from T follicular helper cells needed to escape from apoptosis and complete their differentiation. The highest-affinity LZ BGC-cells enter the cell cycle and differentiate into PCs, following a dramatic epigenetic reorganization that induces transcriptome changes in general and the expression of the PRDM1 gene in particular. Human PC precursors are characterized by the loss of IL-4/STAT6 signaling and the absence of CD23 expression. Here, we studied the fate of human LZ BGC-cells as a function of their CD23 expression. We first showed that CD23 expression was restricted to the GC LZ, where it was primarily expressed by FDCs; less than 10% of tonsil LZ BGC-cells were positive. Sorted LZ BGC-cells left in culture and stimulated upregulated CD23 expression but were unable to differentiate into PCs - in contrast to cells that did not upregulate CD23 expression. An in-depth analysis (including single-cell gene expression) showed that stimulated CD23-negative LZ BGC-cells differentiated into plasmablasts and time course of gene expression changes delineates the transcriptional program that sustains PC differentiation. In particular, we identified a B cell proliferation signature supported by a transient MYC gene expression. Overall, the CD23 marker might be of value in answering questions about the differentiation of normal BGC-cells and allowed us to propose an instructive LZ BGC-cells maturation and fate model.

Fam72a enforces error-prone DNA repair during antibody diversification.

Efficient humoral responses rely on DNA damage, mutagenesis and error-prone DNA repair. Diversification of B cell receptors through somatic hypermutation and class-switch recombination are initiated by cytidine deamination in DNA mediated by activation-induced cytidine deaminase (AID)1 and by the subsequent excision of the resulting uracils by uracil DNA glycosylase (UNG) and by mismatch repair proteins1-3. Although uracils arising in DNA are accurately repaired1-4, how these pathways are co-opted to generate mutations and double-strand DNA breaks in the context of somatic hypermutation and class-switch recombination is unknown1-3. Here we performed a genome-wide CRISPR-Cas9 knockout screen for genes involved in class-switch recombination and identified FAM72A, a protein that interacts with the nuclear isoform of UNG (UNG2)5 and is overexpressed in several cancers5. We show that the FAM72A-UNG2 interaction controls the levels of UNG2 and that class-switch recombination is defective in Fam72a-/- B cells due to the upregulation of UNG2. Moreover, we show that somatic hypermutation is reduced in Fam72a-/- B cells and that its pattern is skewed upon upregulation of UNG2. Our results are consistent with a model in which FAM72A interacts with UNG2 to control its physiological level by triggering its degradation, regulating the level of uracil excision and thus the balance between error-prone and error-free DNA repair. Our findings have potential implications for tumorigenesis, as reduced levels of UNG2 mediated by overexpression of Fam72a would shift the balance towards mutagenic DNA repair, rendering cells more prone to acquire mutations.

UnAIDed Class Switching in Activated B-Cells Reveals Intrinsic Features of a Self-Cleaving IgH Locus.

Activation-induced deaminase (AID) is the major actor of immunoglobulin (Ig) gene diversification in germinal center B-cells. From its first description, it was considered as mandatory for class switch recombination (CSR), and this discovery initiated a long quest for all of the AID-interacting factors controlling its activity. The mechanisms focusing AID-mediated DNA lesions to given target sequences remain incompletely understood with regards the detailed characterization of optimal substrates in which cytidine deamination will lead to double strand breaks (DSBs) and chromosomal cleavage. In an effort to reconsider whether such CSR breaks absolutely require AID, we herein provide evidence, based on deep-sequencing approaches, showing that this dogma is not absolute in both human and mouse B lymphocytes. In activated B-cells from either AID-deficient mice or human AID-deficient patients, we report an intrinsic ability of the IgH locus to undergo "on-target" cleavage and subsequent synapsis of broken regions in conditions able to yield low-level CSR. DNA breaks occur in such conditions within the same repetitive S regions usually targeted by AID, but their repair follows a specific pathway with increased usage of microhomology-mediated repair. These data further demonstrate the role of AID machinery as not initiating de novo chromosomal cleavage but rather catalyzing a process which spontaneously initiates at low levels in an appropriately conformed IgH locus.

Preclinical study of 212Pb alpha-radioimmunotherapy targeting CD20 in non-Hodgkin lymphoma.

BACKGROUND: Despite therapeutic advances, Non-Hodgkin lymphoma (NHL) relapses can occur. The development of radioimmunotherapy (RIT) with α-emitters is an attractive alternative. In this study, we investigated the potential of α-RIT in conjunction with 212Pb-rituximab for the treatment of NHL. METHODS: EL4-hCD20-Luc cells (mouse lymphoma cell line) were used for in vitro and in vivo studies. Biodistribution and efficacy studies were performed on C57BL/6 mice injected intravenously with 25 × 103 cells. RESULTS: 212Pb-rituximab (0.925-7.4 kBq/mL) inhibit proliferation of EL4-hCD20-Luc cells in vitro. Biodistribution of 203/212Pb-rituximab in mice showed a significant tumour uptake and suggested that the liver, spleen, and kidneys were the organs at risk. For efficacy studies, mice were treated at either 11 days (early stage) or 20-30 days after injection of tumour cells (late stage). Treatment with 277.5 kBq 212Pb-rituximab significantly prolonged survival. Even at an advanced tumour stage, significant tumour regression occurred, with an increase in the median survival time to 28 days, compared with 9 days in the controls. CONCLUSIONS: These results show the efficacy of 212Pb-rituximab in a murine syngeneic lymphoma model, in terms of significant tumour regression and increased survival, thereby highlighting the potency of α-RIT for the treatment of NHL.

Transcription/Replication Conflicts in Tumorigenesis and Their Potential Role as Novel Therapeutic Targets in Multiple Myeloma.

Plasma cells (PCs) have an essential role in humoral immune response by secretion of antibodies, and represent the final stage of B lymphocytes differentiation. During this differentiation, the pre-plasmablastic stage is characterized by highly proliferative cells that start to secrete immunoglobulins (Igs). Thus, replication and transcription must be tightly regulated in these cells to avoid transcription/replication conflicts (TRCs), which could increase replication stress and lead to genomic instability. In this review, we analyzed expression of genes involved in TRCs resolution during B to PC differentiation and identified 41 genes significantly overexpressed in the pre-plasmablastic stage. This illustrates the importance of mechanisms required for adequate processing of TRCs during PCs differentiation. Furthermore, we identified that several of these factors were also found overexpressed in purified PCs from patients with multiple myeloma (MM) compared to normal PCs. Malignant PCs produce high levels of Igs concomitantly with cell cycle deregulation. Therefore, increasing the TRCs occurring in MM cells could represent a potent therapeutic strategy for MM patients. Here, we describe the potential roles of TRCs resolution factors in myelomagenesis and discuss the therapeutic interest of targeting the TRCs resolution machinery in MM.

History of IgA Nephropathy Mouse Models.

IgA nephropathy (IgAN) is the most common primary glomerulonephritis in the world. It was first described in 1968 by Jean Berger and Nicole Hinglais as the presence of intercapillary deposits of IgA. Despite this simple description, patients with IgAN may present very broad clinical features ranging from the isolated presence of IgA in the mesangium without clinical or biological manifestations to rapidly progressive kidney failure. These features are associated with a variety of histological lesions, from the discrete thickening of the mesangial matrix to diffuse cell proliferation. Immunofluorescence on IgAN kidney specimens shows the isolated presence of IgA or its inconsistent association with IgG and complement components. This clinical heterogeneity of IgAN clearly echoes its complex and multifactorial pathophysiology in humans, inviting further analyses of its various aspects through the use of experimental models. Small-animal models of IgAN provide the most pertinent strategies for studying the multifactorial aspects of IgAN pathogenesis and progression. Although only primates have the IgA1 subclass, several murine models have been developed in which various aspects of immune responses are deregulated and which are useful in the understanding of IgAN physiopathology as well as in the assessment of IgAN therapeutic approaches. In this manuscript, we review all murine IgAN models developed since 1968 and discuss their remarkable contribution to understanding the disease.