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IntroductionFollicular Lymphoma (FL) results from the malignant transformation of germinal center (GC) B cells. FL B cells display recurrent and diverse genetic alterations, some of them favoring their direct interaction with their cell microenvironment, including follicular helper T cells (Tfh). Although FL-Tfh key role is well-documented, the impact of their regulatory counterpart, the follicular regulatory T cell (Tfr) compartment, is still sparse.MethodsThe aim of this study was to characterize FL-Tfr phenotype by cytometry, gene expression profile, FL-Tfr origin by transcriptomic analysis, and functionality by in vitro assays.ResultsCD4+CXCR5+CD25hiICOS+ FL-Tfr displayed a regulatory program that is close to classical regulatory T cell (Treg) program, at the transcriptomic and methylome levels. Accordingly, Tfr imprinting stigmata were found on FL-Tfh and FL-B cells, compared to their physiological counterparts. In addition, FL-Tfr co-culture with autologous FL-Tfh or cytotoxic FL-CD8+ T cells inhibited their proliferation in vitro. Finally, although FL-Tfr shared many characteristics with Treg, TCR sequencing analyses demonstrated that part of them derived from precursors shared with FL-Tfh. DiscussionAltogether, these findings uncover the role and origin of a Tfr subset in FL niche and may be useful for lymphomagenesis knowledge and therapeutic management.

Summary: Autoimmune diseases (ADs) are characterized by loss of immune tolerance, high chronicity, with substantial morbidity and mortality, despite conventional immunosuppression (IS) or targeted disease modifying therapies (DMTs), which usually require repeated administration. Recently, novel cellular therapies (CT), including mesenchymal stromal cells (MSC), Chimeric Antigen Receptors T cells (CART) and regulatory T cells (Tregs), have been successfully adopted in ADs. An international expert panel of the European Society for Blood and Marrow Transplantation and the International Society for the Cell and Gene Therapy, reviewed all available evidence, based on the current literature and expert practices, on use of MSC, CART and Tregs, in AD patients with rheumatological, neurological, and gastroenterological indications. Expert-based consensus and recommendations for best practice and quality of patient care were developed to support clinicians, scientists, and their multidisciplinary teams, as well as patients and care providers and will be regularly updated.

Summary: 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.

B-cell lymphomas are a group of heterogeneous neoplasms resulting from the clonal expansion of mature B cells arrested at various stages of differentiation. Specifically, two lymphoma subtypes arise from germinal centers (GCs), namely follicular lymphoma (FL) and GC B-cell diffuse large B-cell lymphoma (GCB-DLBCL). In addition to recent advances in describing the genetic landscape of FL and GCB-DLBCL, tumor microenvironment (TME) has progressively emerged as a central determinant of early lymphomagenesis, subclonal evolution, and late progression/transformation. The lymphoma-supportive niche integrates a dynamic and coordinated network of immune and stromal cells defining microarchitecture and mechanical constraints and regulating tumor cell migration, survival, proliferation, and immune escape. Several questions are still unsolved regarding the interplay between lymphoma B cells and their TME, including the mechanisms supporting these bidirectional interactions, the impact of the kinetic and spatial heterogeneity of the tumor niche on B-cell heterogeneity, and how individual genetic alterations can trigger both B-cell intrinsic and B-cell extrinsic signals driving the reprogramming of non-malignant cells. Finally, it is not clear whether these interactions might promote resistance to treatment or, conversely, offer valuable therapeutic opportunities. A major challenge in addressing these questions is the lack of relevant models integrating tumor cells with specific genetic hits, non-malignant cells with adequate functional properties and organization, extracellular matrix, and biomechanical forces. We propose here an overview of the 3D in vitro models, xenograft approaches, and genetically-engineered mouse models recently developed to study GC B-cell lymphomas with a specific focus on the pros and cons of each strategy in understanding B-cell lymphomagenesis and evaluating new therapeutic strategies.

Sjögren syndrome (SjS) is an autoimmune disease characterized by the destruction of the exocrine gland epithelia, causing a dryness of mucosa called sicca symptoms, and whose main life-threatening complication is lymphoma. There is a need for new biomarkers in this disease, notably diagnostic biomarkers for patients with genuine sicca symptoms that do not meet current criteria, and prognostic biomarkers for patients at risk of lymphoma. Plasma extracellular vesicles (EVs) are promising biomarker candidates in several diseases, but their potential has not yet been explored in SjS. In this proof-of-concept study, we characterized EVs from primary SjS patients (pSS, n=12) at the phenotypic and proteomic levels, compared to EVs from healthy donor (HD, n=8) and systemic lupus erythematosus patients (SLE, n=12). Specific plasma EVs subpopulations, derived from neutrophils, endothelial, and epithelial cells, were found increased in pSS. We also identified a pSS proteomic signature in plasma EVs, including neutrophil-, epithelial-, and endothelial-related proteins, such as integrin alpha M (ITGAM), olfactomedin-4 (OLFM4), Ras-related protein RAB10, and CD36. Overall, our results support the relevance of plasma EVs as biomarkers in SjS.

Summary: 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.

Background: Nanofat grafting (NG) is a simple and cost-effective method of lipoaspirates with inter-syringe passages, to produce stromal vascular fraction (SVF) and isolate adipose-derived stem cells (ASCs). This represents a tremendous interest in the future clinical needs of tissue engineering. In this study, we optimized the NG technique to increase the yield of ASC extractions.Methods: We analyzed three groups of SVF obtained by 20, 30, and 40 inter-syringe passages. The control group was an SVF obtained by enzymatic digestion with Celase. We studied their cell composition by flow cytometry, observed their architecture by confocal microscopy, and observed immunomodulatory properties of the ASCs from each of the SVFs by measuring inflammatory markers of macrophages obtained by an ASC monocyte co-culture.Results: We have established the first cell mapping of the stromal vascular fraction of adipose tissue. The results showed that SVF obtained by 20 inter-syringe passages contains more statistically significant total cells, more cells expressing the ASC phenotype, more endothelial cells, and produces more CFU-F than the SVF obtained by 30 and 40 passages and by enzymatic digestion. Confocal microscopy showed the presence of residual adipocytes in SVF obtained by inter-syringe passages but not by enzymatic digestion. The functional study indicates an orientation toward a more anti-inflammatory profile and homogenization of their immunomodulatory properties.Conclusion: This study places mechanically dissociated SVF in the center of approaches to easily extract ASCs and a wide variety and number of other progenitor cells, immediately available in a clinical setting to provide both the amount and quality of cells required for decellularized tissues.

Abstract Background Venoarterial extracorporeal membrane oxygenation (VA-ECMO) provides heart mechanical support in critically ill patients with cardiogenic shock. Despite important progresses in the management of patients under VA-ECMO, acquired infections remain extremely frequent and increase mortality rate. Since immune dysfunctions have been described in both critically ill patients and after surgery with cardiopulmonary bypass, VA-ECMO initiation may be responsible for immune alterations that may expose patients to nosocomial infections (NI). Therefore, in this prospective study, we aimed to study immune alterations induced within the first days by VA-ECMO initiation. Methods We studied immune alterations induced by VA-ECMO initiation using cytometry analysis to characterize immune cell changes and enzyme-linked immunosorbent assay (ELISA) to explore plasma cytokine levels. To analyze specific changes induced by VA-ECMO initiation, nine patients under VA-ECMO (VA-ECMO patients) were compared to nine patients with cardiogenic shock (control patients). Results Baseline immune parameters were similar between the two groups. VA-ECMO was associated with a significant increase in circulating immature neutrophils with a significant decrease in C5a receptor expression. Furthermore, we found that VA-ECMO initiation was followed by lymphocyte dysfunction along with myeloid-derived suppressor cells (MDSC) expansion. ELISA analysis revealed that VA-ECMO initiation was followed by an increase in pro-inflammatory cytokines such as IL-6, IL-8 and TNF-α along with IL-10, a highly immunosuppressive cytokine. Conclusion VA-ECMO is associated with early immune changes that may be responsible for innate and adaptive immune alterations that could confer an increased risk of infection.

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.

B-cell non-Hodgkin lymphoma (B-NHL) evolution and treatment are complicated by a high prevalence of relapses primarily due to the ability of malignant B cells to interact with tumor-supportive lymph node (LN) and bone marrow (BM) microenvironments. In particular, progressive alterations of BM stromal cells sustain the survival, proliferation, and drug resistance of tumor B cells during diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and chronic lymphocytic leukemia (CLL). The current review describes how the crosstalk between BM stromal cells and lymphoma tumor cells triggers the establishment of the tumor supportive niche. DLBCL, FL, and CLL display distinct patterns of BM involvement, but in each case tumor-infiltrating stromal cells, corresponding to cancer-associated fibroblasts, exhibit specific phenotypic and functional features promoting the recruitment, adhesion, and survival of tumor cells. Tumor cell-derived extracellular vesicles have been recently proposed as playing a central role in triggering initial induction of tumor-supportive niches, notably within the BM. Finally, the disruption of the BM stroma reprogramming emerges as a promising therapeutic option in B-cell lymphomas. Targeting the crosstalk between BM stromal cells and malignant B cells, either through the inhibition of stroma-derived B-cell growth factors or through the mobilization of clonal B cells outside their supportive BM niche, should in particular be further evaluated as a way to avoid relapses by abrogating resistance niches.

Absolute count of circulating monocytes has been proposed as an independent prognostic factor in diffuse large B-cell lymphoma (DLBCL). However, monocyte nomenclature includes various subsets with pro-, anti-inflammatory, or suppressive functions, and their clinical relevance in DLBCL has been poorly explored. Herein, we broadly assessed circulating monocyte heterogeneity in 91 DLBCL patients. Classical- (cMO, CD14pos CD16neg) and intermediate- (iMO, CD14pos CD16pos) monocytes accumulated in DLBCL peripheral blood and exhibited an inflammatory phenotype. On the opposite, nonclassical monocytes (ncMOSlanpos, CD14low CD16pos Slanneg and ncMOSlanneg, CD14low CD16pos, Slanneg) were decreased in peripheral blood. Tumor-conditioned monocytes presented similarities with ncMO phenotype from DLBCL and were prone to migrate in response to CCL5 and CXCL12, and presented similarities with DLBCL-infiltrated myeloid cells, as defined by mass cytometry. Finally, we demonstrated the adverse value of an accumulation of nonclassical monocytes in 2 independent cohorts of DLBCL.

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.

Summary: Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19−ARDS+, COVID-19+ARDS+, and COVID-19+ARDS−, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19−ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS− patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Multiple sclerosis (MS) is an immune-driven demyelinating disease of the central nervous system. Immune cell features are particularly promising as predictive biomarkers due to their central role in the pathogenesis but also as drug targets, even if nowadays, they have no impact in clinical practice. Recently, high-resolution approaches, such as mass cytometry (CyTOF), helped to better understand the diversity and functions of the immune system. In this study, we performed an exploratory analysis of blood immune response profiles in healthy controls and MS patients sampled at their first neurological relapse, using two large CyTOF panels including 62 markers exploring myeloid and lymphoid cells. An increased abundance of both a T-bet-expressing B cell subset and a CD206+ classical monocyte subset was detected in the blood of early MS patients. Moreover, T-bet-expressing B cells tended to be enriched in aggressive MS patients. This study provides new insights into understanding the pathophysiology of MS and the identification of immunological biomarkers. Further studies will be required to validate these results and to determine the exact role of the identified clusters in neuroinflammation.

Resistance to BRAF inhibitors limits their clinical benefit in melanoma patients. Here, the authors show that the Aryl hydrocarbon Receptor (AhR) is a key mediator of resistant genes and use resveratrol, an AhR antagonist, to revert resistance in melanoma bearing mice.

Fibroblastic reticular cells (FRCs) are the specialized lymphoid stromal cells initially identified as triggering T-cell recruitment and dynamic motion in secondary lymphoid organs. Interestingly, FRCs also display antigen presentation capacities and support lymphocyte survival. CXCR5+CD4+ follicular T cells are important players of B-cell maturation and antibody response. Our study reported that in vitro-differentiated FRC-like cells enhanced the growth of the whole CXCR5+CD4+ T-cell compartment, while enhancing IL-4 secretion specifically by the PD1dimCXCR5+CD4+ cell subset, in a Notch- and ICAM1/LFA1-dependent manner. In addition, we revealed that in follicular lymphoma (FL) tissues, previously identified as enriched for PD1hiCXCR5hiCD4+ mature follicular helper T cells, PD1dimCXCR5+CD4+ T cells displayed an enrichment for Notch and integrin gene signatures, and a Notch and ICAM-1-dependent overexpression of IL-4 compared to their non-malignant counterparts. These findings suggest that the crosstalk between FRCs and CXCR5+PD1dimCD4+ T cells may contribute to the FL IL-4 rich environment, thus providing new insights in FL lymphomagenesis.

Abstract: A better characterization of T-cell subsets in the microenvironment of classical Hodgkin lymphoma (cHL) would help to develop immunotherapies. Using multicolor flow cytometry, we identified in 6 of 43 cHL tissue samples a previously unrecognized subset of CD8 T cells coexpressing CXCR5 and inducible T-cell costimulator (ICOS) molecules (CD8CXCR5+ICOS+). These cells shared phenotypic features with follicular helper T (TFH) cells including low CCR7 expression together with high expression of B-cell lymphoma-6, programmed cell death 1, B and T lymphocyte attenuator, CD200, and OX40. They had deficient cytotoxicity, low interferon-γ secretion, and common functional properties with intratumoral CD4+ TFH cells, such as production of interleukin-4 (IL-4), IL-21, CXCL13, and capacity to sustain B cells. Gene profiling analysis showed a significant similarity between the signatures of CD8CXCR5+ICOS+ T cells and CD4+ TFH cells. Benign lymphadenitis tissues (n = 8) were devoid of CD8CXCR5+ICOS+ cells. Among the 35 B-cell lymphoma tissues analyzed, including follicular lymphomas (n = 13), diffuse large cell lymphomas (n = 12), marginal zone lymphomas (MZLs; n = 3), mantle cell lymphomas (n = 3), and chronic lymphocytic leukemias (n = 4), only 1 MZL sample contained CD8CXCR5+ICOS+ cells. Lymphoma tumors with CD8CXCR5+ICOS+ cells shared common histopathological features including residual germinal centers, and contained high amounts of activated CD8CXCR5−ICOS+ cells. These data demonstrate a CD8 T-cell differentiation pathway leading to the acquisition of some TFH similarities. They suggest a particular immunoediting process with global CD8 activation acting mainly, but not exclusively, in HL tumors.

Using drugs to treat COVID-19 symptoms may induce adverse effects and modify patient outcomes. These adverse events may be further aggravated in obese patients, who often present different illnesses such as metabolic-associated fatty liver disease. In Rennes University Hospital, several drug such as hydroxychloroquine (HCQ) have been used in the clinical trial HARMONICOV to treat COVID-19 patients, including obese patients. The aim of this study is to determine whether HCQ metabolism and hepatotoxicity are worsened in obese patients using an in vivo/in vitro approach. Liquid chromatography high resolution mass spectrometry in combination with untargeted screening and molecular networking were employed to study drug metabolism in vivo (patient’s plasma) and in vitro (HepaRG cells and RPTEC cells). In addition, HepaRG cells model were used to reproduce pathophysiological features of obese patient metabolism, i.e., in the condition of hepatic steatosis. The metabolic signature of HCQ was modified in HepaRG cells cultured under a steatosis condition and a new metabolite was detected (carboxychloroquine). The RPTEC model was found to produce only one metabolite. A higher cytotoxicity of HCQ was observed in HepaRG cells exposed to exogenous fatty acids, while neutral lipid accumulation (steatosis) was further enhanced in these cells. These in vitro data were compared with the biological parameters of 17 COVID-19 patients treated with HCQ included in the HARMONICOV cohort. Overall, our data suggest that steatosis may be a risk factor for altered drug metabolism and possibly toxicity of HCQ.

T cells help B cells to differentiate into antibody-producing plasma cells. Here the authors show that T cells produce interleukin-2 to activate ERK/ELK1 and suppress BACH2 expression by modulating the BACH2 super-enhancer, thereby altering BACH2 downstream transcription programs for plasma cell differentiation.

Follicular lymphoma (FL), the most frequent indolent non-Hodgkin’s B cell lymphoma, is considered as a prototypical centrocyte-derived lymphoma, dependent on a specific microenvironment mimicking the normal germinal center (GC). In agreement, several FL genetic alterations affect the crosstalk between malignant B cells and surrounding cells, including stromal cells and follicular helper T cells (Tfh). In our study, we sought to deconvolute this complex FL supportive synapse by comparing the transcriptomic profiles of GC B cells, Tfh, and stromal cells, isolated from normal versus FL tissues, in order to identify tumor-specific pathways. In particular, we highlighted a high expression of IL-6 and IL-7 in FL B cells that could favor the activation of FL Tfh overexpressing IFNG, able in turn to stimulate FL B cells without triggering MHC (major histocompatibility) class II expression. Moreover, the glycoprotein clusterin was found up-regulated in FL stromal cells and could promote FL B cell adhesion. Finally, besides its expression on Tfh, CD200 was found overexpressed on tumor B cells and could contribute to the induction of the immunosuppressive enzyme indoleamine-2,3 dioxygenase by CD200R-expressing dendritic cells. Altogether our findings led us to outline the contribution of major signals provided by the FL microenvironment and their interactions with malignant FL B cells.

Abstract DCC (Deleted in Colorectal Carcinoma) has been demonstrated to constrain tumor progression by inducing apoptosis unless engaged by its ligand netrin‐1. This has been shown in breast and colorectal cancers; however, this tumor suppressive function in other cancers is not established. Using a transgenic mouse model, we report here that inhibition of DCC‐induced apoptosis is associated with lymphomagenesis. In human diffuse large B‐cell lymphoma (DLBCL), an imbalance of the netrin‐1/DCC ratio suggests a loss of DCC‐induced apoptosis, either via a decrease in DCC expression in germinal center subtype or by up‐regulation of netrin‐1 in activated B‐cell (ABC) one. Such imbalance is also observed in mantle cell lymphoma (MCL). Using a netrin‐1 interfering antibody, we demonstrate both in vitro and in vivo that netrin‐1 acts as a survival factor for ABC‐DLBCL and MCL tumor cells. Together, these data suggest that interference with the netrin‐1/DCC interaction could represent a promising therapeutic strategy in netrin‐1‐positive DLBCL and MCL.

Molecular mechanisms underlying terminal differentiation of B cells into plasma cells are major determinants of adaptive immunity but remain only partially understood. Here we present the transcriptional and epigenomic landscapes of cell subsets arising from activation of human naive B cells and differentiation into plasmablasts. Cell proliferation of activated B cells was linked to a slight decrease in DNA methylation levels, but followed by a committal step in which an S phase-synchronized differentiation switch was associated with an extensive DNA demethylation and local acquisition of 5-hydroxymethylcytosine at enhancers and genes related to plasma cell identity. Downregulation of both TGF-β1/SMAD3 signaling and p53 pathway supported this final step, allowing the emergence of a CD23-negative subpopulation in transition from B cells to plasma cells. Remarkably, hydroxymethylation of PRDM1, a gene essential for plasma cell fate, was coupled to progression in S phase, revealing an intricate connection among cell cycle, DNA (hydroxy)methylation, and cell fate determination.

Fibroblastic reticular cells (FRCs), the T-cell zone stromal cell subtype in the lymph nodes, create a scaffold for adhesion and migration of immune cells, thus allowing them to communicate. Although known to be important for the initiation of immune responses, studies about FRCs and their interactions have been impeded because FRCs are limited in availability and lose their function upon culture expansion. To circumvent these limitations, stromal cell precursors can be mechanotranduced to form mature FRCs. Here, we used a library of designed surface topographies to trigger FRC differentiation from tonsil-derived stromal cells (TSCs). Undifferentiated TSCs were seeded on a TopoChip containing 2176 different topographies in culture medium without differentiation factors, then monitored cell morphology and the levels of ICAM-1, a marker of FRC differentiation. We identified 112 and 72 surfaces that upregulated and downregulated, respectively, ICAM-1 expression. By monitoring cell morphology, and expression of the FRC differentiation marker ICAM-1 via image analysis and machine learning, we discovered correlations between ICAM-1 expression, cell shape and design of surface topographies and confirmed our findings by using flow cytometry. Our findings confirmed that TSCs are mechano-responsive cells and identified particular topographies that can be used to improve FRC differentiation protocols.

Obinutuzumab (OBZ) shows stronger antibody-dependent cell cytotoxicity (ADCC) compared to rituximab and improved clinical activity for treating certain CD20+ neoplasia. However, the efficacy of monoclonal antibody (mAb) as a monotherapy is limited. Natural Killer (NK) cells are mediators of ADCC. Hematological cancer patients possess antitumor NK cells that are unable to control disease, possibly because they are dysfunctional. The immunomodulatory drug lenalidomide (LEN) could be a treatment to restore exhausted NK cell cytotoxic functions. The clinical trial GALEN is a Phase Ib/II study of OBZ combined with LEN for the treatment of relapsed/refractory follicular and aggressive (DLBCL and MCL) B-cell Lymphoma. During treatment, we analyzed specific aspects of NK cell biology. Treatment reversed the immature NK phenotype of patients and increased expression of NK activating receptors. Inhibitory receptors were either unchanged or decreased. There was a strong NK response at the end of the 1st cycle: NK number and intracellular granzyme B (GrzB) expression decreased, degranulation increased and NK responded better to allogeneic target challenge. Moreover, the interaction of NK cells with B cell targets, measured by trogocytosis, decreased during treatment. At the end of treatment, when target cells had been wiped out, the proportion of reactive NK cells (CD69+, CD45RARO+, CD107a+, CD19+) strongly decreased. Because all patients received LEN and OBZ, it was uncertain which drug was responsible of our observations, or even if a combination of both products was necessary for the described effects on this lymphocyte lineage.

During the last decades, considerable efforts have been done to decipher mechanisms supported by microorganisms or viruses involved in the development, differentiation, and function of immune cells. Pathogens and their associated secretome as well as the continuous inflammation observed in chronic infection are shaping both innate and adaptive immunity. Secondary lymphoid organs are functional structures ensuring the mounting of adaptive immune response against microorganisms and viruses. Inside these organs, germinal centers (GCs) are the specialized sites where mature B-cell differentiation occurs leading to the release of high-affinity immunoglobulin (Ig)-secreting cells. Different steps are critical to complete B-cell differentiation process, including proliferation, somatic hypermutations in Ig variable genes, affinity-based selection, and class switch recombination. All these steps require intense interactions with cognate CD4+ helper T cells belonging to follicular helper lineage. Interestingly, pathogens can disturb this subtle machinery affecting the classical adaptive immune response. In this review, we describe how viruses could act directly on GC B cells, either through B-cell infection or by their contribution to B-cell cancer development and maintenance. In addition, we depict the indirect impact of viruses on B-cell response through infection of GC T cells and stromal cells, leading to immune response modulation.

Among immunoglobulins (Igs), IgE can powerfully contribute to antimicrobial immunity and severe allergy despite its low abundance. IgE protein and gene structure resemble other Ig classes, making it unclear what constrains its production to thousand-fold lower levels. Whether class-switched B cell receptors (BCRs) differentially control B cell fate is debated, and study of the membrane (m)IgE class is hampered by its elusive in vivo expression. Here, we demonstrate a self-controlled mIgE+ B cell stage. Primary or transfected mIgE+ cells relocate the BCRs into spontaneously internalized lipid rafts, lose mobility to chemokines, and change morphology. We suggest that combined proapoptotic mechanisms possibly involving Hax1 prevent mIgE+ memory lymphocyte accumulation. By uncoupling in vivo IgE switching from cytokine and antigen stimuli, we show that these features are independent from B cell stimulation and instead result from mIgE expression per se. Consequently, few cells survive IgE class switching, which might ensure minimal long-term IgE memory upon differentiation into plasma cells.

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.

DNA microarrays have considerably helped to improve the understanding of biological processes and diseases. Large amounts of publicly available microarray data are accumulating, but are poorly exploited due to a lack of easy-to-use bioinformatics resources. The aim of this study is to build a free and convenient data-mining web site (www.genomicscape.com). GenomicScape allows mining dataset from various microarray platforms, identifying genes differentially expressed between populations, clustering populations, visualizing expression profiles of large sets of genes, and exporting results and figures. We show how easily GenomicScape makes it possible to construct a molecular atlas of the B cell differentiation using publicly available transcriptome data of naïve B cells, centroblasts, centrocytes, memory B cells, preplasmablasts, plasmablasts, early plasma cells and bone marrow plasma cells. Genes overexpressed in each population and the pathways encoded by these genes are provided as well as how the populations cluster together. All the analyses, tables and figures can be easily done and exported using GenomicScape and this B cell to plasma cell atlas is freely available online. Beyond this B cell to plasma cell atlas, the molecular characteristics of any biological process can be easily and freely investigated by uploading the corresponding transcriptome files into GenomicScape.

Angioimmunoblastic T-cell Lymphoma (AITL) is one of the most frequent T-cell lymphoma entities. Follicular helper T lymphocytes (TFH) are recognized as the normal cellular counterpart of the neoplastic component. Despite a clonal T-cell feature and few described recurrent cytogenetic abnormalities, a driving oncogenic event has not been identified so far. It has been recently reported that in mice, heterozygous inactivation of Roquin/Rc3h1, a RING type E3 ubiquitine ligase, recapitulates many of the clinical, histological, and cellular features associated with human AITL. In this study we explored whether ROQUIN alterations could be an initial event in the human AITL oncogenic process. Using microarray and RT-PCR analyses, we investigated the levels of ROQUIN transcripts in TFH tumor cells purified from AITL (n = 8) and reactive tonsils (n = 12) and found similar levels of ROQUIN expression in both. Moreover, we also demonstrated that ROQUIN protein was expressed by AITL TFH (PD1+) cells. We then analysed ROQUIN coding sequence in 12 tumor cell-rich AITL samples and found no mutation in any of the samples. Finally, we analysed the expression of MiR101, a putative partner of ROQUIN involved in the modulation of ICOS expression and found similar levels of expression in tumor and reactive TFH. Altogether, this study shows that neither alteration of ROQUIN gene nor deregulation of miR101 expression is likely to be a frequent recurrent event in AITL.

Background While most antibody-based therapies use IgG because of their well-known biological properties, some functional limitations of these antibodies call for the development of derivatives with other therapeutic functions. Although less abundant than IgG in serum, IgA is the most abundantly produced Ig class in humans. Besides the specific targeting of its dimeric form to mucosal areas, IgA was shown to recruit polymorphonuclear neutrophils against certain targets more efficiently than does IgG1.Design and Methods In this study, we investigated the various pathways by which anti-tumor effects can be mediated by anti-CD20 IgA against lymphoma cells.Results We found that polymeric human IgA was significantly more effective than human IgG1 in mediating direct killing or growth inhibition of target cells in the absence of complement. We also demonstrated that this direct killing was able to indirectly induce the classical pathway of the complement cascade although to a lesser extent than direct recruitment of complement by IgG. Recruitment of the alternative complement pathway by specific IgA was also observed. In addition to activating complement for lysis of lymphoma cell lines or primary cells from patients with lymphoma, we showed that monomeric anti-CD20 IgA can effectively protect mice against tumor development in a passive immunization strategy and we demonstrated that this protective effect may be enhanced in mice expressing the human FcαRI receptor on their neutrophils.Conclusions We show that anti-CD20 IgA antibodies have original therapeutic properties against lymphoma cells, with strong direct effects, ability to recruit neutrophils for cell cytotoxicity and even recruitment of complement, although largely through an indirect way.